Lymphocyte Signal Transduction (Advances in Experimental Medicine and Biology Vol 584)

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LYMPHOCYTE SIGNAL TRANSDUCTION

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: NATHAN BACK, State University of New York at Buffalo IRUN R. COHEN, The Weizmann Institute of Science DAVID KRITCHEVSKY, Wistar Institute ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research RODOLFO PAOLETTI, University of Milan

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Volume 584 LYMPHOCYTE SIGNAL TRANSDUCTION Edited by Constantine Tsoukas

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LYMPHOCYTE SIGNAL TRANSDUCTION Edited by

Constantine Tsoukas San Diego State University San Diego, California

Editor: Constantine Tsoukas San Diego State University San Diego, CA 92182 USA [email protected]

Library of Congress Control Number: 2006922768 Printed on acid-free paper. ISBN 10: 0-387-31335-4 ISBN-13: 978-0387-31335-1

Proceedings of the 3rd Lymphocyte Signal Transduction Conference, held May 27-June 1, 2005, in Crete, Greece. © 2006 Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed in the United States of America. 9 8 7 6 5 4 3 2 1 springer.com

Foreword

Signal transduction through leukocyte receptors involves a variety of signaling molecules — including kinases, phosphatases, adaptor proteins, small GTPases GTP exchange factors, membrane phospholipids, as well as others. These signal transducers, regulated by inter- and intramolecular interactions, as well as by various post-translational modifications, lead to the activation of transcription factors that mediate cellular differentiation and growth, effector cell functions, and apoptotic cell death. Several investigators from various parts of the world convened at the 3rd Lymphocyte Signal Transduction Workshop in Crete, Greece, from May 27 to June 1, 2005, to discuss their most recent findings in leukocyte signaling. This volume represents a collection of topics discussed during the conference. One of the early signaling events during lymphocyte stimulation is activation of a number of protein kinases. In Chapter 1 Barouch-Bentov and Altman discuss the mechanisms through which PKCθ activates its two major targets — the transcription factors NF-κB and AP-1 — as well as the involvement of PKCθ in different T cell-dependent immune responses and in T cell survival. Garçon and Nunès (Chapter 2) summarize the knowledge on the Tec family kinases and discuss the available techniques to visualize the intracellular fate of this family of kinases during lymphocyte activation. Tsoukas et al. (Chapter 3) discuss the intracellular migration of one of the Tec kinases, the Inducible T cell Kinase (Itk), and the role of its various domains in this process. In addition, these authors present evidence of the ability of Itk to regulate the actin cytoskeleton during T-cell activation. In addition to the important roles protein kinases play in lymphocyte signaling, lipid kinases also play important functions. In Chapter 4 Vigorito and Turner discuss Phosphoinositide 3-Kinase (PI3K), a family of enzymes that generates D3-phosphorylated phosphoinositides with important biological functions. In particular, the authors focus on the role that PI3K subunits play in activation of the MAP Kinase ERK during BCR-induced stimulation. Protein phosphatases play a critical role in regulation of lymphocyte activation. In particular, protein tyrosine phosphatases (PTP) regulate key cellular processes in immune cells and are implicated in numerous human diseases. In Chapter 5, Tomas Mustelin reviews the ways that PTPs can contribute to pathophysiology and discusses the potential of PTPs as drug targets. Adaptor proteins play critical roles in propagating signals from leukocyte receptors. Lapinski and his colleagues (Chapter 6) discuss recent data relating to v

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FOREWORD

the role of the T-Cell Specific Adaptor protein (TSAd) in T-cell signal transduction. The importance of this adaptor molecule is underscored by the breakdown of immunological tolerance and ensuing autoimmune disease in TSAd-deficient mice. Moreno-Garcia et al. (Chapter 7) discuss the significance of another adaptor protein, CARMA1, and new evidence identifying the mechanism(s) that link(s) specific protein kinase C (PKC) isoforms and CARMA1 to the initiation of NF-κB activation induced by the BCR. Deckert and Rottapel (Chapter 8) review the current understanding of how the adaptor 3BP2 regulates leukocyte signaling and discuss the importance of this adaptor in a rare human disease called cherubism, where the 3bp2/sh3bp2 locus has been shown to be mutated. In Chapter 9 Schneider et al. address the issue of costimulation in leukocyte signaling. In particular, these authors discuss the mechanism by which the CD28 family of costimulators generates intracellular signals. The available data suggest that CTLA-4, a member of this family of costimulators, might regulate integrin-mediated adhesion in T cells in a fashion involving the small GTPbinding protein RAP-1. Lipid rafts have been argued to be important conduits for the effective propagation of signals. In Chapter 10, Nunes and colleagues review the knowledge and recent advances in the organization of signalling complexes as protein networks in the plasma membrane and the role of lipid rafts in this process. Larbi et al. (Chapter 11) review the role of lipid rafts in signaling through the Fas receptor and induction of Activation-Induced Cell Death (AICD). Furthermore, these authors discuss the effects of age-related changes in this process. This general topic is further discussed by Fülöp et al. (Chapter 12), with special emphasis on the role of aging and membrane cholesterol on T-cell functions. Susanne Miyamoto (Chapter 13) discusses the current knowledge of the mechanisms of translation in lymphocytes and how these mechanisms might be regulated during the early activation process. Post-translational modifications of signaling molecules is an important regulatory process in signal transduction. Schurter et al. (Chapter 14) review the recent advances implicating protein arginine methylation in various cellular functions including T cell activation. Numerous studies have documented the importance of protein ubiquitination in regulation of the immune system. Venuprasad and colleagues (Chapter 15) discuss the involvement of E3 ligases in aspects of the immune response including T-cell activation, differentiation, migration, and tolerance induction. Recent research indicates that the environment in which cells of the immune system operate plays an important role. In particular Fukamachi et al. (Chapter 16) discusses the immune response to acidic environments and the role of the CTerminus Protein of IκB-β (CTIB) on cellular functions under acidic stress. Many pathogenic microorganisms, such as HIV, have evolved mechanisms for evading the integrity of the immune system, thus causing disease. In Chapter 17, Cloyd and colleagues discuss the various mechanisms that have been proposed for depletion of CD4 T cells by HIV and discuss the possibility of HIV targeting non-permissive resting CD4 lymphocytes through its receptors.

FOREWORD

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In order to understand and appreciate the complexity of signal transduction pathways in a comprehensive and integrated manner, the traditional, hypothesisdriven methodologies will have to be combined with novel technologies. Such technologies will have to incorporate both genomic and proteomic approaches and the power of bioinformatics. In Chapter 18, Huang et al. review the recent advances in functional genomics screens using arrayed cDNA and siRNA/shDNA libraries that have drastically accelerated the pace of in-vitro and tissue culture-based functional gene annotation. Furthermore, Cao and colleagues (Chapter 19) review recent developments in bioinformatics and proteomics methods based on mass spectrometry that provide new capabilities to examine the structure of signaling cascades through global phosphorylation site analysis from complex lysates. Overall, the compendium of chapters that follow provide a selective overview of recent findings and novel approaches in the leukocyte signal transduction field for both the general reader and the specialist in the leukocyte signal transduction field. Constantine Tsoukas

Acknowledgments

The organizing committee of the 3rd Lymphocyte Signal Transduction Workshop (Amnon Altman, Toshi Kawakami, Yun-Cai Liu, Tomas Mustelin, and Constantine Tsoukas) would like to thank the La Jolla Insitute for Allergy and Immunology, Kirin, The College of Sciences at San Diego State University, Upstate Biotechnology, and the Aegean Conferences for their generous financial support that made this conference a success.

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Contents

List of Contributors ........................................................................................... xiii 1.

Protein Kinase C-Theta (PKCθ): New Perspectives on Its Functions in T-Cell Biology

Rina Barouch-Bentov and Amnon Altman.............................................. 2.

Travel Informations on the Tec Kinases during Lymphocyte Activation

Fabien Garçon and Jacques A. Nunès.................................................... 3.

5. 6.

29

Differential Requirements of PI3K Subunits for BCR or BCR/CD19-Induced ERK Activation

Elena Vigorito and Martin Turner..........................................................

43

Protein Tyrosine Phosphatases in Human Disease Tomas Mustelin.......................................................................................

53

The T Cell-Specific Adapter Protein Functions as a Regulator of Peripheral but not Central Immunological Tolerance

Philip E. Lapinski, Jennifer N. MacGregor, Francesc Marti, and Philip D. King.................................................................................. 7.

15

Inducible T-Cell Tyrosine Kinase (ITK): Structural Requirements and Actin Polymerization

Constantine D. Tsoukas, Juris A. Grasis, Cecille D. Browne, and Keith A. Ching ................................................................................. 4.

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Proximal Signals Controlling B-Cell Antigen Receptor (BCR) Mediated NF-κB Activation

Miguel E. Moreno-García, Karen M. Sommer, Ashok D. Bandaranayake, and David J. Rawlings .................................

89

8.

The Adapter 3BP2: How It Plugs into Leukocyte Signaling Marcel Deckert and Robert Rottapel...................................................... 107

9.

CTLA-4 Regulation of T-Cell Function via RAP-1-Mediated Adhesion

Helga Schneider, Elke Valk, Silvy da Rocha Dias, Bin Wei, and Christopher E. Rudd ........................................................................ 115 xi

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10. Protein Crosstalk in Lipid Rafts

Raquel J. Nunes, Mónica A. A. Castro, and Alexandre M. Carmo ....................................................................... 127 11. Role of Lipid Rafts in Activation-Induced Cell Death: The Fas Pathway in Aging

Anis Larbi, Elisa Muti, Roberta Giacconi, Eugenio Mocchegiani, and Tamàs Fülöp ............................................... 137 12. T-Cell Response in Aging: Influence of Cellular Cholesterol Modulation

Tamàs Fülöp, Gilles Dupuis, Carl Fortin, Nadine Douziech, and Anis Larbi ........................................................................................ 157 13. Lymphocyte Signaling and the Translatability of mRNA

Suzanne Miyamoto.................................................................................. 171 14. Protein Arginine Methylation: A New Frontier in T-Cell Signal Transduction

Brandon T. Schurter, Fabien Blanchet, and Oreste Acuto ..................... 189 15. Immune Regulation by Ubiquitin Conjugation

K. Venuprasad, Chun Yang, Yuan Shao, Dmytro Demydenko, Yohsuke Harada, Myung-shin Jeon, and Yun-Cai Liu............................ 207 16. CTIB (C-Terminus Protein of IκB-β): A Novel Factor Required for Acidic Adaptation

Toshihiko Fukamachi, Qizong Lao, Shinya Okamura, Hiromi Saito, and Hiroshi Kobayashi .................................................... 219 17. HIV May Deplete Most CD4 Lymphocytes by a Mechanism Involving Signaling through its Receptors on Non-Permissive Resting Lymphocytes

Miles W. Cloyd, Jiaxiang Ji, Melissa Smith, and Vivian Braciale.......... 229 18. Integrating Traditional and Postgenomic Approaches to Investigate Lymphocyte Development and Function

Yina Hsing Huang, Rina Barouch-Bentov, Ann Herman, John Walker, and Karsten Sauer ............................................................ 245 19. Phosphoproteomic Analysis of Lymphocyte Signaling

Lulu Cao, Kebing Yu, and Arthur R. Salomon........................................ 277 Author Index....................................................................................................... 289 Index.................................................................................................................... 291

List of Contributors Oreste Acuto Molecular Immunology Unit Institut Pasteur Paris, France

Lulu Cao Department of Molecular Biology, Cell Biology and Biochemistry Brown University Providence, Rhode Island, USA

Amnon Altman Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA

Alexandre M. Carmo Group of Cell Activation and Gene Expression Institute for Molecular and Cellular Biology Porto, Portugal

Ashok D. Bandaranayake Department of Immunology Children's Hospital and Regional Medical Center Seattle, Washington, USA

Mónica A. A. Castro Group of Cell Activation and Gene Expression Institute for Molecular and Cellular Biology Porto, Portugal

Rina Barouch-Bentov Department of Immunology Genomics Institute Novartis Research Foundation San Diego, California, USA

Keith A. Ching Genomics Institute Novartis Research Foundation San Diego, California, USA

Fabien Blanchet Molecular Immunology Unit Institut Pasteur Paris, France

Miles W. Cloyd Departments of Microbiology, Immunology and Pathology The University of Texas Medical Branch Galveston, Texas, USA

Vivian Braciale Departments of Microbiology, Immunology and Pathology The University of Texas Medical Branch Galveston, Texas, USA

Silvy da Rocha Dias Department of Immunology Division of Investigative Sciences Imperial College London Hammersmith Campus, London, UK

Cecille D. Browne The Burnham Institute La Jolla, California, USA

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Marcel Deckert INSERM Unit 576 Hôpital de l'Archet Nice, France Dmytro Demydenko Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Nadine Douziech Immunological Graduate Programme Faculty of Medicine University of Sherbrooke Sherbrooke, Québec, Canada Gilles Dupuis Immunological Graduate Programme Faculty of Medicine University of Sherbrooke Sherbrooke, Québec, Canada Carl Fortin Immunological Graduate Programme Faculty of Medicine University of Sherbrooke Sherbrooke, Québec, Canada Toshihiko Fukamachi Graduate School of Pharmaceutical Sciences Chiba University Chiba, Japan Tamàs Fülöp Immunological Graduate Programme Faculty of Medicine University of Sherbrooke Sherbrooke, Québec, Canada Fabien Garçon Laboratory of Lymphocyte Signaling and Development Babraham Institute Babraham Research Campus Cambridge, UK

CONTRIBUTORS

Roberta Giacconi Section on Nutrition, Immunity and Aging Immunology Centre, Research Department

INRCA Ancona, Italy Juris A. Grasis Department of Biology and the Center for Microbial Sciences San Diego State University San Diego, California, USA Yohsuke Harada Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Ann Herman Genomics Institute Novartis Research Foundation San Diego, California, USA Yina Hsing Huang Genomics Institute Novartis Research Foundation San Diego, California, USA Myung-shin Jeon Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Jiaxiang Ji Departments of Microbiology, Immunology and Pathology The University of Texas Medical Branch Galveston, Texas, USA Philip D. King Department of Microbiology and Immunology University of Michigan Medical School

Ann Arbor, Michigan, USA

CONTRIBUTORS

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Hiroshi Kobayashi Graduate School of Pharmaceutical Sciences Chiba University Chiba, Japan

Suzanne Miyamoto Division of Hematology/Oncology Cancer Center University of California Davis Sacramento, California, USA

Qizong Lao Graduate School of Pharmaceutical Sciences Chiba University Chiba, Japan

Eugenio Mocchegiani Section on Nutrition, Immunity and Aging Immunology Centre, Research Department INRCA Ancona, Italy

Philip E. Lapinski Department of Microbiology and Immunology University of Michigan Medical School Ann Arbor, Michigan, USA Anis Larbi Immunological Graduate Programme Faculty of Medicine University of Sherbrooke Sherbrooke, Québec, Canada Yun-Cai Liu Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Jennifer MacGregor Department of Microbiology and Immunology University of Michigan Medical School Ann Arbor, Michigan, USA Francesc Marti Department of Microbiology and Immunology University of Michigan Medical School Ann Arbor, Michigan, USA

Miguel E. Moreno-García Department of Pediatrics University of Washington School of Medicine Seattle, Washington, USA Tomas Mustelin Inflammatory and Infectious Disease Center Program of Signal Transduction Cancer Center, The Burnham Institute La Jolla, California, USA Elisa Muti Section on Nutrition, Immunity and Aging Immunology Centre, Research Department INRCA Ancona, Italy Jacques A. Nunès Centre de Recherche en Cancérologie de Marseille INSERM UMR599, Institut Paoli-Calmettes Université de la Méditerranée Marseilles, France

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Raquel J. Nunes Group of Cell Activation and Gene Expression Institute for Molecular and Cellular Biology Porto, Portugal Shinya Okamura Graduate School of Pharmaceutical Sciences Chiba University Chiba, Japan David J. Rawlings Departments of Pediatrics and Immunology Children's Hospital University of Washington School of Medicine Seattle, Washington, USA Robert Rottapel Princess Margaret Hospital and Ontario Cancer Institute Toronto, Ontario, Canada Christopher E. Rudd Department of Immunology Division of Investigative Sciences Imperial College London Hammersmith Campus London, UK Hiromi Saito Graduate School of Pharmaceutical Sciences Chiba University Chiba, Japan Arthur R. Salomon Department of Molecular Biology, Cell Biology and Biochemistry Brown University Providence, Rhode Island, USA

CONTRIBUTORS

Karsten Sauer Genomics Institute Novartis Research Foundation San Diego, California, USA Helga Schneider Department of Immunology Division of Investigative Sciences Imperial College London Hammersmith Campus London, UK Brandon Schurter Molecular Immunology Unit Institut Pasteur Paris, France Yuan Shao Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Melissa Smith Departments of Microbiology, Immunology and Pathology The University of Texas Medical Branch Galveston, Texas, USA Karen M. Sommer Department of Immunology University of Washington School of Medicine Seattle, Washington, USA Constantine D. Tsoukas Department of Biology and the Center for Microbial Sciences San Diego State University San Diego, California, USA

CONTRIBUTORS

Martin Turner Laboratory of Lymphocyte Signaling and Development The Babraham Institute Babraham Research Campus Cambridge, UK Elke Valk Department of Immunology Division of Investigative Sciences Imperial College London Hammersmith Campus, London, UK K. Venuprasad Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Elena Vigorito Laboratory of Lymphocyte Signaling and Development The Babraham Institute Babraham Research Campus Cambridge, UK

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John Walker Genomics Institute Novartis Research Foundation San Diego, California, USA Bin Wei Department of Immunology Division of Investigative Sciences Imperial College London Hammersmith Campus, London, UK Chun Yang Division of Cell Biology La Jolla Institute for Allergy and Immunology San Diego, California, USA Kebing Yu Department of Molecular Biology, Cell Biology and Biochemistry Brown University Providence, Rhode Island, USA

1 PROTEIN KINASE C-THETA (PKCθ): NEW PERSPECTIVES ON ITS FUNCTIONS IN T CELL BIOLOGY Rina Barouch-Bentov1,2 and Amnon Altman1

1. INTRODUCTION 1-3

Since its isolation in 1992–93 , protein kinase C-theta (PKCθ), a member of the 2+ novel Ca -independent PKC family, has attracted considerable interest among T cell biologists. PKCθ, which is expressed in a relatively selective manner mainly in T cells (but also in skeletal muscle and platelets), has emerged as a key enzyme involved in T cell activation. Consistent with biochemical analysis of cul4-6 –/– tured T cells mature T cells from PKCθ mice display defects in TCR/CD28induced activation of transcription factors essential for cell activation and differ7, 8 entiation, i.e., NF-κB, AP-1, and NFAT . This impaired activation is associated with severe defects in interleukin 2 (IL-2) expression and CD3/CD28-mediated proliferation as measured by thymidine uptake. However, T cell development in 7 the thymus proceeds normally in the absence of PKCθ . Another unique property of PKCθ is its specific recruitment to the central 9 supramolecular activation cluster (cSMAC) of the immunological synapse (IS) following the encounter of antigen-specific T cells with antigen-presenting cells 10,11 (APCs) . Additionally, upon TCR/CD28 stimulation PKCθ translocates to membrane lipid rafts, specialized membrane microdomains that play an impor12-15 2+ tant role in T cell activation and signaling . Like other conventional Ca 2+ dependent and novel Ca -independent members of the PKC family, binding of the second messenger, diacylglycerol (DAG), to the tandem cysteine-rich C1 16 domains of PKCθ recruits it to the inner leaflet of the plasma membrane . However, this mechanism does not explain the localization of PKCθ (but not other 1

Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, California 92121, USA. 2Present address: Department of Immunology, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA.

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PKCs) to specific membrane microdomains, i.e., the cSMAC in the IS or lipid rafts. Our group and others have studied this selective localization pattern, which positively correlates with enzymatic activation of PKCθ. Several signaling proteins were implicated in regulating this process and the effector functions of PKCθ, including Vav1 and its immediate target, the Rac small GTPase, which are essential for TCR/CD28-induced actin cytoskeleton reorganization, phosphatidylinositol 3-kinase (PI3K) and 3-phosphoinositide-dependent kinase 17–20 1 (PDK1) . In addition, PKCθ membrane localization and activation may also be regulated by Lck-mediated phosphorylation of a tyrosine residue (Y90) in its 12, 21 regulatory domain . A growing body of studies has documented the central role of PKCθ in T lymphocyte biology, and we refer the reader to comprehensive reviews pub22–25 lished on this topic in recent years . Intriguingly, however, more recent findings demonstrated that the functions of PKCθ in T cells are far more subtle and complex than initially thought. These studies revealed that PKCθ is selectively required in different arms of the immune response and, furthermore, that in addition to playing a central role in T cell activation PKCθ is also an important survival signal in mature T cells. These recent findings raise interesting and unresolved questions in the basic scientific arena, and also serve to reignite interest in PKCθ as a potential drug target to modulate certain immunological diseases in favor of the host. In this chapter, we focus on recent observations that demonstrated a selective requirement for PKCθ in different T cell responses. First, we discuss recent evidence addressing the mechanisms through which PKCθ activates its two major targets, the transcription factors NF-țB and AP-1. The second part of this review will summarize recent observations on the involvement of PKCθ in different T cell-dependent immune responses and in T cell survival.

2. PKCθ REGULATES NF-κB AND AP-1 THROUGH DIFFERENT INTERMEDIATE PATHWAYS The importance of PKCθ in controlling the activation of mature T cells reflects, for the most part, its central role in activating transcription factors NF-κB, AP-1, 7,8 and NFAT, as evident from the analysis of PKCθ-deficient T cells , and the effects of PKCθ on reporter gene activation in transfected T cell lines. The precise mechanisms through which PKCθ activates these transcription factors are still not entirely clear, but significant progress was recently made in this area. 2.1. NF-κB Activation by PKCθ Proteins of the NF-țB family dimerize in different combinations to form a transactivating complex. NF-κB dimers are normally sequestered in the cytoplasm in an inactive form by interaction with inhibitory κB (IκB) proteins, and they are activated following stimulation of antigen, cytokine, and other receptors by two

NEW PERSPECTIVES ON PKCθ

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pathways known as the canonical (NF-κB1) and non-canonical (NF-κB2) path26, 27 ways . In the canonical pathway, stimulation of the IκB kinase (IKK) complex leads to phosphorylation, ubiquitination, and degradation of IκB, thereby exposing a nuclear localization sequence in NF-κB, which then causes the NF–κB1 complex to translocate to the nucleus and activate gene transcription. In the noncanonical pathway, signaling by surface receptors leads to activation of NF-κBinduced kinase (NIK), a mitogen-activated protein kinase kinase kinase (MAP3K)-like enzyme that induces proteolytic processing of p100/p52 allowing 27, 28 p52-containing NF-κB complexes to translocate to the nucleus . Members of the NF-κB family play an essential role in lymphocyte activation and the gen26 eration of immune response . Notably, stimulation of the TCR/CD3 complex alone is not sufficient to activate NF-κB, and costimulation of CD28 through its 29 ligand, B7, is required for optimal activation of this pathway . PKCθ plays an essential role in the activation of the canonical NF-κB1 pathway downstream of TCR/CD28 signaling in T cells. Studies using overexpression of wild-type PKCθ or its kinase-dead mutants first placed PKCθ downstream of TCR/CD28 signaling in the activation of an NF-κB reporter gene and demonstrated that PKCθ activates IKKβ (but not IKKα) to phosphorylate IκB 5,6 proteins . Similarly, PKCθ-deficient mice displayed defects in NF-κB1 activa8,30,31 –/– . In addition, PKCθ T cells display deficiencies in TCR/CD28-induced tion p50 and RelA/p65 nuclear mobilization, the former resulting from impairments in IKKβ activation and a subsequent defect in phosphorylation and degradation 5,31,7 of IκB isomers (IκBα and IκΒγ, but not IκBβ) . Upon TCR/CD28 costimulation, PKCθ physically interacts with the IKK 14 complexes, and the complex is found in lipid rafts . However, IKKβ kinase is not a direct substrate for PKCθ, as evident from in vitro studies. Therefore, some intermediate kinase(s), which phosphorylates and activates IKKβ downstream of PKCθ, must exist. While the identity of this kinase is not known, recent studies identified several adaptor proteins that play a crucial role in mediating TCR/CD28-induced NF-κB activation by functionally cooperating with 32–34 PKCθ signaling: CARMA1, Bcl10 and MALT1 . CARMA1 (caspase recruitment domain, CARD, membrane-associated guanylate kinase, MAGUK, protein 1) is constitutively associated with lipid rafts, and becomes further en35 riched in these rafts after TCR stimulation . Upon TCR triggering, interaction of CARMA1 with BCL10 (B-cell lymphoma-10) and functional interaction of 35–37 BCL10 with MALT1 is essential for further activation of NF-κB . It has been suggested that, following TCR ligation, CARMA1 recruits BCL10 to the TCR– CD3 complex and to lipid rafts, but the molecular details of this recruitment are unknown, as is the basis for the linkage of CARMA1, BCL10, and MALT1 to 34 the IKK complex . A recent report provided some insight into this process by suggesting that the Bcl10-MALT1 complex can activate IKK by inducing ubiq38 uitination of NEMO (IKKγ), the regulatory subunit of the IKK complex .

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A functional link between the CARM1/Bcl10 complex and PKCθ was demonstrated by findings that a constitutively active mutant of PKCθ (PKCθ39 A/E) cooperates with CARMA1 and/or Bcl10 to activate NF-κB in T cells . A model placing PKCθ upstream of CARMA1 in the pathway leading to NF-κB 34 activation was proposed based on several observations . First, phorbol ester (PMA)-mediated activation of NF-κB, which involves activation of a PMAsensitive PKC family member (most likely PKCθ), is abrogated in CARMA1-, 34 Bcl10-, or MALT1-deficient T lymphocytes . Second, PKCθ-A/E failed to re39 constitute NF-κB activation in CARMA1-deficient Jurkat T cells . Third, re40, 41 cruitment of PKCθ to the TCR is unaffected in CARMA1-deficient T cells . A very recent study suggested that PDK1 plays an essential role in linking the PKCθ-IKK and CARMA1–MALT1–Bcl10 cascades by activating PKCθ and through signal-dependent recruitment of both PKCθ and CARMA1 to lipid 20 rafts . However, neither has direct association of PKCθ with Bcl10 or CARMA1 been demonstrated in T cells, nor has either protein been shown to be a direct substrate of PKCθ; therefore, the physiological relevance of this pathway has yet to be conclusively demonstrated. Although TCR/CD28 costimulation can activate components of the NF-kB2 pathway such as IKKα and NIK, the non-canonical NF-κB2 pathway is not op31 erative downstream of TCR/CD28 signaling . NIK- and IKKα-dependent activation of the NF-κB2 pathway is associated with p100/p52 processing, resulting in reduced p100 expression and a corresponding increase in p52 expression. However, we found that, although TCR/CD28 costimulation increased the level of p52 in T cells, this increase was paralleled by a similar increase in the expres31 sion of its precursor, p100 . Analysis of metabolically labeled cells confirmed that the increased p52 expression was secondary to enhanced p100 synthesis in TCR/CD28-stimulated T cells. This effect most likely reflects the fact that p100 transcription itself is positively regulated by NF-κB1. Consistent with this explanation, we found that the TCR/CD28-stimulated upregulation of p100 ex–/– 31 pression was impaired in PKCθ T cells . Because both PKCθ-dependent NFκB activation and NIK activation occur downstream of TCR/CD28 signaling, it was important to determine whether these two proteins cooperate in the activation of NF-κB signaling, or whether they act in separate pathways. We found that a dominant negative NIK mutant could block activation of an NF-κB reporter construct following anti-CD3/CD28 stimulation, but it failed to block IKKβ or NF-κB activation induced by expression of a constitutively active PKCθ mutant, indicating that NIK does not act downstream of PKCθ in PKCθmediated activation of NF-κB. Indeed TCR/CD28-induced NIK activation was –/– 31 intact in PKCθ T cells . Thus, PKCθ plays an indirect role in activation of the NF-κB2 pathway.

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2.2. AP-1 Activation by PKCθ The nuclear transcription factor AP-1, which is composed of dimers of Jun and Fos proteins, has emerged as an important factor involved in many cellular events — including proliferation, differentiation, apoptosis, and transforma42 tion . Regulation of AP-1, which occurs at both the transcriptional and posttranslational levels, is complex, and mitogen-activated protein kinases 42 (MAPKs, ERK, JNK, and p38) all play an important role in AP-1 activation . Studies using overexpression of constitutively active PKCθ or its catalytically inactive mutant in Jurkat T cells first placed PKCθ downstream of TCR/CD28 4 signaling in the activation of an AP-1 reporter gene . Subsequently, PKCθ7,8 deficient mice were found to display defects in AP-1 activation . Although the mechanism by which PKCθ mediates activation of AP-1 is unclear, several recent studies have provided some insight into this process. Activation of JNK and ERK by PKCθ may be part of the mechanism by which PKCθ mediates activation of AP-1. Several studies reported that PKCθ specifically mediates activation of JNK in Jurkat cells in response to TCR/CD28 costimulation. However, other PKC family members, in addition to PKCθ, can activate 43,45 ERK . In addition, it was demonstrated that PKCθ synergizes with calcineurin 44 to activate JNK and subsequent IL-2 production . Nevertheless, despite these findings, which were obtained by analyzing activation responses in transformed Jurkat T cells, the activation of MAPKs was found to be intact in primary -/7,8,46 PKCθ T cells stimulated with anti-CD3 plus anti-CD28 antibodies . How-/+ ever, our more recent analysis of TCR-transgenic PKCθ CD8 T cells revealed impaired ERK and JNK (but not p38) activation when the cells were stimulated 46 with antigen . The apparent contradiction between these two sets of findings perhaps reflects the fact that very strong (less physiological) stimulation with saturating amounts of anti-CD3/CD28 antibodies somehow bypasses the requirement for PKCθ in MAPK activation (see further discussion below). In a search for PKCθ-interacting proteins, we recently isolated a poorly characterized Ste20-related MAP3K, termed SPAK, that couples PKCθ to AP-1, 47 but not NF-κB, activation . Specifically, the C-terminal domain of SPAK directly bound PKCθ (but not PKCα), and this association was substantially enhanced by CD3/CD28 costimulation. Moreover, PKCθ directly phosphorylated SPAK, and SPAK phosphorylation and activation were largely impaired in T -/cells from PKCθ mice. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1 but not NF-κB, and siRNA-mediated depletion of SPAK 47 inhibited PKCθ-mediated AP-1 but not NF-κB activation . Another potential intermediate step in the pathway leading from TCR/CD28-stimulated PKCθ to AP-1 activation was revealed by our recent study, which demonstrated that preactivated and restimulated T cells from -/2+ PKCθ mice display impaired Ca response and NFAT activation, which corre48 lated with reduced activation of PLCγ1 and Tec kinase . Furthermore, we observed a constitutive association between Tec and PKCθ in T cells. Importantly,

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a dominant negative Tec mutant blocked the PKCθ-induced activation of an AP1 reporter gene, but had no effect on PKCθ-induced NF-κB activation. These findings somewhat parallel an earlier report of reduced NFAT activation in -/8 2+ PKCθ naive T cells . At present, it is unclear how PKCθ can regulate Ca sig48 naling, but one possibility suggested by our study is that PKCθ, once residing stably in the IS, recruits Tec kinase and promotes its activation, which would, in 2+ turn, sustain PLCγ1 activation and the resulting increase in intracellular Ca concentration.

3. NOVEL FUNCTIONS OF PKCθ IN THE IMMUNE SYSTEM -/-

The early findings that PKCθ peripheral T cell are grossly impaired in their 7, 8 CD3/CD28-induced proliferation and IL-2 production implied that PKCθ is required for all aspects of T cell activation and proliferation, raising a potential concern that selective inhibition of PKCθ function, e.g., by pharmacological means, would induce an undesired global immunosuppression in the host. Surprisingly, however, recent studies characterizing in vivo immune responses of -/PKCθ T cells have challenged this view by clearly demonstrating that certain T cell responses are relatively intact in PKCθ-deficient mice and, in addition, that besides its role in T cell activation, PKCθ also constitutes an important survival signal in mature T cells. 3.1. The Role of PKCθ in CTL Differentiation and Survival Two recent studies investigated the role of PKCθ in the activation and differen+ tiation of CD8 effector T cells by using OT-I or P14 mice expressing an MHC class I-restricted TCR transgene specific for an ovalbumin (Ova) peptide or an LCMV viral peptide, respectively, which were crossed to PKCθ-deficient 46,49 mice . Somewhat unexpectedly, both studies reported that the antigen-induced -/proliferation of PKCθ T cells assessed by CFSE dilution was intact. Berg49 Brown et al. also demonstrated that PKCθ-deficient mice generated intact LCMV-specific primary and recall CTL responses as well as an anti-viral (VSV) antibody response following virus infection. However, when the mice were immunized with gp33, a peptide representing an immunodominant CTL epitope of the LCMV glycoprotein, late peptide-specific proliferative and CTL responses were reduced but could be rescued by adding exogenous IL-2. Based on the deficient late expansion and its rescue by IL-2, and on the similarity between the -/-/functional phenotype of PKCθ and CD28 T cells, the authors concluded that PKCθ is important for preventing induction of T cell anergy. The intact CTL -/and proliferative response of PKCθ mice to LCMV infection (but not against the viral gp33 peptide) may reflect compensatory innate immune signals that are 49 generated by immunization with live virus .

NEW PERSPECTIVES ON PKCθ 46

7

Our very recent study extended the study of Berg-Brown et al. by demon-/strating that the major defect displayed by PKCθ OT-I TCR-transgenic cells upon specific Ova peptide stimulation is poor survival of the antigen-stimulated T cells, rather than defective early proliferation, which was assessed by CFSE -/dilution and found to be intact. The accelerated death of PKCθ CD8 T cells, which resulted from caspase mediated-apoptosis, was associated with dysregulation of Bcl-2, Bcl-xL, and BimEL expression, largely indicative of a death by -/neglect pathway. Retroviral transduction of PKCθ T cells with anti-apoptotic Bcl-xL or Bcl-2 proteins enhanced, but did not fully rescue, their survival, indicating that the lower expression levels of endogenous Bcl-xL and Bcl-2 in these cells contributed, at least in part, to their accelerated and increased death. Overall, these findings suggest that PKCθ promotes cell survival in a complex manner by regulating both cytokine-dependent and independent pathways. Some -/defects in the function of PKCθ OT-I T cells, including reduced Bcl-2 and BclxL expression, CTL activity, and IFNγ expression, were partially or fully restored by coculture with wild-type cells or by addition of exogenous IL-2, while others, i.e., increased expression of the proapoptotic protein BimEL and TNFα production were not. Additionally, our study revealed novel, previously unre-/ported, defects in antigen-induced ERK and JNK activation in PKCθ primary T cells. These defects are potentially linked to impaired survival and dysregulated Bcl-2 family protein expression based on our finding that selective pharmacological ERK or JNK inhibition in wild-type T cells reduced their survival and modulated expression of Bcl-2, Bcl-xL, and BimEL. Altogether, this analysis reveals that PKCθ is, most likely, an important early signal in a genetic program required for the transition of T cells through a checkpoint critical for their differentiation into functional, cytokine-producing CTLs. One possible explanation for the difference between the reported require46,49 ments for PKCθ in mediating survival vs. anergy in the two related studies could be the use of different TCR-transgenic mouse lines and/or different modes of stimulation. Therefore, it is possible that the requirement of PKCθ in different types of T cell responses depends on the overall strength of signaling by the TCR and costimulatory receptors. Consistent with this hypothesis, the defects in -/antigen-induced ERK and JNK activation in PKCθ OT-I cells upon specific 46 -/antigen/APC stimulation cannot be detected in primary PKCθ T cells stimu7,8,46 lated with anti-CD3/CD28 antibodies . How important is PKCθ in antimicrobial defense? The answer to this ques+ tion is not simple. Although the role of CD8 T cells in providing protective immunity against many pathogens is well established, and PKCθ plays an important role in regulating activation/anergy/survival of these cells, it is still possible that in the absence of functional CD8 T cells other adaptive and innate immune effector mechanisms can provide protection against pathogens. Indeed, individuals with defects in the MHC class I antigen processing pathway are still + resistant to infection with some pathogens that are known to induce CD8 effec50 tor T cell responses . Notably, the finding that PKCθ is not required for initial T

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cell proliferation, at least in the CD8 T cell compartment, suggests that pharmacological inhibition of this enzyme may induce selective immunosuppression and apoptotic elimination of effector T cells, e.g., in autoimmune disease, without interfering with the desired activation of naive T cells against, e.g., pathogens. 3.2. A Selective Role for PKCθ in Th2 Cell Responses? 51

52

One group and we reported that PKCθ is required for developing Th2dependent immunity in vivo, including in a mouse model of asthma, but is less critical for Th1 responses. The first study documented severely impaired airway hyperresponsiveness (AHR), eosinophilia, and IgE and Th2 cytokine responses to inhaled antigen or to Nippostongylus brasiliensis infection, but intact Th1mediated protection against Leishmania major infection. Reduction in IL-4, but not IFNγ, expression was also observed in antigen-primed and restimulated -/TCR–Tg PKCθ T cells, and exogenous IL-2 partially restored IL-4 produc51 -/tion . Similarly, we found that PKCθ mice were strongly compromised in generating Th2 cells, and exhibited reduced airway eosinophilia, AHR, mucus production, and Th2 cytokines in the lungs. However, leukocyte infiltration and Th1 cytokine production were largely intact in a Th1-mediated lung inflamma52 tion model . Importantly, adoptive transfer experiments revealed that the Th2 -/-/defect in PKCθ mice is T cell-autonomous, since PKCθ mice receiving Ovaspecific wild-type effector Th2 cells developed intact lung inflammation upon Ova challenge. Still, PKCθ was required for optimal Th1 development after antigen priming, with these cells exhibiting delayed kinetics of differentiation and accumulation. Exogenous IL-2 restored the partially impaired primary IFNγ 52 response, but had no effect on IL-4 production . The finding that the early Th1 defect was eventually overcome with recall antigen challenge via the airways suggests that mechanism(s) capable of compensating for the lack of PKCθ, e.g., costimulatory signals provided by innate immune cells, operate during Th1, but not Th2, development and in vivo expansion. However, at this point it is premature to conclude that PKCθ is not required for certain Th1 responses. Indeed, in a more recent study, we found that PKCθdeficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) failed to develop cell infiltrates and Th1 cytokines in the central nervous system, and were resistant to the development of clinical experimental autoimmune en53 + cephalitis (EAE) . CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKCθ but had severely diminished IFNγ, TNFα, and IL-17 production. Increasing antigen exposure and inflammatory conditions failed to induce EAE in PKCθ-deficient mice, showing a profound defect in the MOG-reactive T cell population. These data provide evidence of a pivotal role for PKCθ in generation and effector function of autoimmune Th1 cells in at least some diseases. It is not clear why PKCθ may be more critical for the development of certain Th1-dependent diseases but not others, but the

NEW PERSPECTIVES ON PKCθ

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strength and duration of alternative disease-promoting signals, e.g., innate immunity signals, may affect this differential requirement for PKCθ. At any rate, the fact that deletion of a single PKC gene (PKCθ) out of 6–7 PKC genes expressed in T cells, has such a pronounced effect on development of Th2mediated allergic lung inflammation suggests that PKCθ might represent an attractive drug target for Th2-mediated allergic diseases. Therefore, it would be important to define the pathway(s) through which PKCθ exerts its essential function in Th2-mediated immune reactions. More recently, we found that cul-/turing PKCθ naive T cells under standard Th2-polarizing conditions overcomes, at least partially, the Th2 differentiation defect and allows for the development of largely “normal” effector Th2 cells as determined by their ability to produce Th2 cytokines upon restimulation (unpublished observations). This and -/other observations suggest that PKCθ is an early component of TCR/CD28induced instructive signals that are essential for IL-4 priming in naive T cells. This and some other important and unresolved questions are currently under study in our laboratory.

4. CONCLUSIONS -/-

Early functional characterization of peripheral PKCθ T cells led to the conclu7,8 sion that PKCθ is required for T cell activation and proliferation , leading to the general belief that PKCθ would be globally required for all T cell-dependent immune responses. However, more recent studies assessing in vivo responses of -/PKCθ mice to various antigens revealed a more complex picture. Thus, certain T cell-dependent immune responses were severely impaired, whereas other responses were largely intact. Therefore, it now appears that there is a differential -/requirement for PKCθ in various arms of the immune response. Further studies are needed in order to understand the molecular basis for this differential requirement. Progress in understanding the biological functions of PKCθ and its role in various immunological disease will require a multi-pronged approach but, at a minimum, improved understanding of the signal transduction pathways controlled by this enzyme at the molecular and biochemical level. Such analysis might benefit from the use of phosphoproteomic- and gene profiling-based approaches to identify the physiological substrates of PKCθ and the genes that are -/regulated by it, respectively. Second, further analysis of PKCθ mice in terms of their susceptibility to various immunological disease models would be informative, including the potential role of innate immunity signals operating in vivo and their ability to compensate for the lack of PKCθ. The results of these future studies are likely to establish a rational basis for using PKCθ as a drug target for selective inhibition of a subset of T cell responses, e.g., Th2-dependent allergic disease.

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5. REFERENCES 1. S. Osada, K. Mizuno, T. C. Saido, K. Suzuki, T. Kuroki and S. Ohno, A new member of the protein kinase C family, nPKCθ, predominantly expressed in skeletal muscle, Mol Cell Biol 12, 3930–3938 (1992). 2. J. D. Chang, Y. Xu, M. K. Raychowdhury and J. A. Ware, Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC). A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets, J Biol Chem 268, 14208–14214 (1993). 3. G. Baier, D. Telford, L. Giampa, K. M. Coggeshall, G. Baier-Bitterlich, N. Isakov and A. Altman, Molecular cloning and characterization of PKCθ, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells, J Biol Chem 268, 4997–5004 (1993). 4. G. Baier-Bitterlich, F. Uberall, B. Bauer, F. Fresser, H. Wachter, H. Grunicke, G. Utermann, A. Altman and G. Baier, Protein kinase C-θ isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes, Mol Cell Biol 16, 1842–1850 (1996). 5. N. Coudronniere, M. Villalba, N. Englund and A. Altman, NF-κB activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-θ, Proc Natl Acad Sci USA 97, 3394–3399 (2000). 6. X. Lin, A. O'Mahony, Y. Mu, R. Geleziunas and W. C. Greene, Protein kinase C-θ participates in NF-κB activation induced by CD3–CD28 costimulation through selective activation of IκB kinase β, Mol Cell Biol 20, 2933–2940 (2000). 7. Z. Sun, C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg and D. R. Littman, PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes, Nature 404, 402–407 (2000). 8. C. Pfeifhofer, K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges and 2+ G. Baier, Protein kinase Cθ affects Ca mobilization and NFAT cell activation in primary mouse T cells, J Exp Med 197, 1525–1535 (2003). 9. W. R. Burack, K. H. Lee, A. D. Holdorf, M. L. Dustin and A. S. Shaw, Cutting edge: quantitative imaging of raft accumulation in the immunological synapse, J Immunol 169, 2837–2841 (2002). 10. C. R. Monks, H. Kupfer, I. Tamir, A. Barlow and A. Kupfer, Selective modulation of protein kinase C-θ during T-cell activation, Nature 385, 83–86 (1997). 11. C. R. Monks, B. A. Freiberg, H. Kupfer, N. Sciaky and A. Kupfer, Three-dimensional segregation of supramolecular activation clusters in T cells, Nature 395, 82– 86 (1998). 12. K. Bi, Y. Tanaka, N. Coudronniere, K. Sugie, S. Hong, M. J. van Stipdonk and A. Altman, Antigen-induced translocation of PKCθ to membrane rafts is required for T cell activation, Nat Immunol 2, 556–563 (2001). 13. J. Huang, P. F. Lo, T. Zal, N. R. Gascoigne, B. A. Smith, S. D. Levin and H. M. Grey, CD28 plays a critical role in the segregation of PKCθ within the immunologic synapse, Proc Natl Acad Sci USA 99, 9369–9373 (2002). 14. A. Khoshnan, D. Bae, C. A. Tindell and A. E. Nel, The physical association of protein kinase Cθ with a lipid raft-associated inhibitor of κB factor kinase (IKK) complex plays a role in the activation of the NF-κB cascade by TCR and CD28, J Immunol 165, 6933–6940 (2000).

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15. C. E. Sedwick and A. Altman, Ordered just so: lipid rafts and lymphocyte function, Sci STKE 2002, RE2 (2002). 16. E. Oancea and T. Meyer, Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals, Cell 95, 307–318 (1998). 17. M. Villalba, N. Coudronniere, M. Deckert, E. Teixeiro, P. Mas and A. Altman, A novel functional interaction between Vav and PKCθ is required for TCR-induced T cell activation, Immunity 12, 151–160 (2000). 18. M. Villalba, K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham and A. Altman, Translocation of PKCθ in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C, J Cell Biol 157, 253–263 (2002). 19. M. Villalba, K. Bi, F. Rodriguez, Y. Tanaka, S. Schoenberger and A. Altman, Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells, J Cell Biol 155, 331–338 (2001). 20. K. Y. Lee, F. D'Acquisto, M. S. Hayden, J. H. Shim and S. Ghosh, PDK1 nucleates T cell receptor-induced signaling complex for NF-κB activation, Science 308, 114– 118 (2005). 21. Y. Liu, S. Witte, Y. C. Liu, M. Doyle, C. Elly and A. Altman, Regulation of protein kinase Cθ function during T cell activation by Lck-mediated tyrosine phosphorylation, J Biol Chem 275, 3603–3609 (2000). 22. C. W. Arendt, B. Albrecht, T. J. Soos and D. R. Littman, Protein kinase C-θ: signaling from the center of the T-cell synapse, Curr Opin Immunol 14, 323-330 (2002). 23. N. Isakov and A. Altman, Protein kinase Cθ in T cell activation, Annu Rev Immunol 20, 761–794 (2002). 24. A. Altman and M. Villalba, Protein kinase C-θ (PKCθ): it's all about location, location, location, Immunol Rev 192, 53–63 (2003). 25. C. E. Sedwick and A. Altman, Perspectives on PKCθ in T cell activation, Mol Immunol 41, 675–686 (2004). 26. Q. Li and I. M. Verma, NF-κB regulation in the immune system, Nat Rev Immunol 2, 725–734 (2002). 27. G. Xiao, E. W. Harhaj and S. C. Sun, NF-κB-inducing kinase regulates the processing of NF-κB2 p100, Mol Cell 7, 401–409 (2001). 28. G. Xiao, A. Fong and S. C. Sun, Induction of p100 processing by NF-κB-inducing kinase involves docking IκB kinase α (IKα) to p100 and IKKα-mediated phosphorylation, J Biol Chem 279, 30099–30105 (2004). 29. L. P. Kane, J. Lin and A. Weiss, It's all Rel-ative: NF-κB and CD28 costimulation of T-cell activation, Trends Immunol 23, 413–420 (2002). 30. J. C. Sun and M. J. Bevan, Defective CD8 T cell memory following acute infection without CD4 T cell help, Science 300, 339–342 (2003). 31. Y. Li, C. E. Sedwick, J. Hu and A. Altman, Role for protein kinase Cθ (PKCθ) in TCR/CD28-mediated signaling through the canonical but not the non-canonical pathway for NF-κB activation, J Biol Chem 280, 1217–1223 (2005). 32. M. Thome and J. Tschopp, TCR-induced NF-κB activation: a crucial role for Carma1, Bcl10 and MALT1, Trends Immunol 24, 419–424 (2003). 33. J. Ruland, G. S. Duncan, A. Wakeham and T. W. Mak, Differential requirement for Malt1 in T and B cell antigen receptor signaling, Immunity 19, 749–758 (2003). 34. M. Thome, CARMA1, BCL-10 and MALT1 in lymphocyte development and activation, Nat Rev Immunol 4, 348–359 (2004).

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35. O. Gaide, B. Favier, D. F. Legler, D. Bonnet, B. Brissoni, S. Valitutti, C. Bron, J. Tschopp and M. Thome, CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-κB activation, Nat Immunol 3, 836–843 (2002). 36. O. Gaide, F. Martinon, O. Micheau, D. Bonnet, M. Thome and J. Tschopp, Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-κB activation, FEBS Lett 496, 121–127 (2001). 37. J. L. Pomerantz, E. M. Denny and D. Baltimore, CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex, EMBO J 21, 5184–5194 (2002). 38. H. Zhou, I. Wertz, K. O'Rourke, M. Ultsch, S. Seshagiri, M. Eby, W. Xiao and V. M. Dixit, Bcl10 activates the NF-κB pathway through ubiquitination of NEMO, Nature 427, 167–171 (2004). 39. D. Wang, Y. You, S. M. Case, L. M. McAllister-Lucas, L. Wang, P. S. DiStefano, G. Nunez, J. Bertin and X. Lin, A requirement for CARMA1 in TCR-induced NFκB activation, Nat Immunol 3, 830–835 (2002). 40. T. Egawa, B. Albrecht, B. Favier, M. J. Sunshine, K. Mirchandani, W. O'Brien, M. Thome and D. R. Littman, Requirement for CARMA1 in antigen receptor-induced NF-κB activation and lymphocyte proliferation, Curr Biol 13, 1252–1258 (2003). 41. H. Hara, C. Bakal, T. Wada, D. Bouchard, R. Rottapel, T. Saito and J. M. Penninger, The molecular adapter Carma1 controls entry of IκB kinase into the central immune synapse, J Exp Med 200, 1167–1177 (2004). 42. E. Shaulian and M. Karin, AP-1 as a regulator of cell life and death, Nat Cell Biol 4, E131–136 (2002). 43. A. Avraham, S. Jung, Y. Samuels, R. Seger and Y. Ben-Neriah, Co-stimulationdependent activation of a JNK-kinase in T lymphocytes, Eur J Immunol 28, 2320– 2330 (1998). 44. G. Werlen, E. Jacinto, Y. Xia and M. Karin, Calcineurin preferentially synergizes with PKC-θ to activate JNK and IL-2 promoter in T lymphocytes, EMBO J 17, 3101–3111 (1998). 45. N. Ghaffari-Tabrizi, B. Bauer, A. Villunger, G. Baier-Bitterlich, A. Altman, G. Utermann, F. Uberall and G. Baier, Protein kinase Cθ, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells, Eur J Immunol 29, 132– 142 (1999). 46. R. Barouch-Bentov, E. E. Lemmens, J. Hu, E. M. Janssen, N. M. Droin, J. Song, S. P. Schoenberger and A. Altman, Protein kinase C-θ is an early survival factor re+ quired for differentiation of effector CD8 T cells, J Immunol 175, 5126–5134 (2005). 47. Y. Li, J. Hu, R. Vita, B. Sun, H. Tabata and A. Altman, SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway, EMBO J 23, 1112–1122 (2004). 48. A. Altman, S. Kaminski, V. Busuttil, N. Droin, J. Hu, Y. Tadevosyan, R. A. Hip2+ skind and M. Villalba, Positive feedback regulation of PLCγ1/Ca signaling by PKCθ in restimulated T cells via a Tec kinase-dependent pathway, Eur J Immunol 34, 2001–2011 (2004). 49. N. N. Berg-Brown, M. A. Gronski, R. G. Jones, A. R. Elford, E. K. Deenick, B. Odermatt, D. R. Littman and P. S. Ohashi, PKCθ signals activation versus tolerance in vivo, J Exp Med 199, 743–752 (2004). 50. P. Wong and E. G. Pamer, CD8 T cell responses to infectious pathogens, Annu Rev Immunol 21, 29–70 (2003).

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51. B. J. Marsland, T. J. Soos, G. Spath, D. R. Littman and M. Kopf, Protein kinase Cθ is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses, J Exp Med 200, 181–189 (2004). 52. S. Salek-Ardakani, T. So, B. S. Halteman, A. Altman and M. Croft, Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase Cθ, J Immunol 173, 6440–6447 (2004). 53. S. Salek-Ardakani, T. So, B. S. Halteman, A. Altman and M. Croft, PKC-θ controls T helper type 1 cells in experimental autoimmune encephalomyelitis, J Immunol 175, 7635–7641 (2005).

2 TRAVEL INFORMATIONS ON THE TEC KINASES DURING LYMPHOCYTE ACTIVATION Fabien Garçon1 and Jacques A. Nunès2

1. INTRODUCTION Antigen receptor signal transduction is a critical step to the development and function of T and B lymphocytes. The non-receptor protein tyrosine kinases (PTKs) of Tec family are emerging as key players of antigen receptor signaling (see reviews [1–4]). The initial interest for working on this family came from the discovery of mutations affecting Btk linked to the human genetic disorder Xlinked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (see review [5]). Btk is a member of the Tec family that includes Tec (tyrosine kinase expressed in hepatocellular carcinoma), Btk (Bruton’s tyrosine kinase), Itk (inducible T-cell kinase, also known as Emt or Tsk), Bmx (Bone Marrow tyrosine kinase gene in chromosome X, also named Etk), and finally Rlk (resting lymphocyte kinase, also named Txk). Tec family members are expressed in cells of the heamatopoietic lineage. Tec family kinases are involved in several aspects of the lymphocyte development, differentiation and activation (see reviews [1– 4]). Upon antigen receptor engagement, these PTKs become activated: in T cells these include Tec, Itk and Rlk, whereas in B cells, Btk and Tec. Initially, these 2+ PTKs have been shown to regulate Ca -dependent pathways by participating to the activation of PLC-γ. More recent reports demonstrated a contribution of Tec family kinases in actin cytoskeletal reorganization and cell adhesion. These PTKs are characterized by an NH2-terminal pleckstrin homology (PH) domain (absent in Rlk), a proline-rich region, Src-homology 3 (SH3) and SH2 domains, and a COOH-terminal PTK domain. These domains can be

1 Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK, 2Centre de Recherche en Cancérologie de Marseille, INSERM UMR599, Institut Paoli-Calmettes, Université de la Méditerranée, 13009 Marseilles, France

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involved in targeting the PTKs at a specific subcellular compartment and in contributing to the formation of signalosomes containing the Tec kinases. Whereas most of the Tec family members reside in the cytoplasm of resting T and B cells, an antigen receptor engagement induces different patterns of subcellular localization. In this chapter, we focus our attention in the regulation of the Tec kinases translocation from cytosol to or near to the plasma membrane, or to the nucleus. To evaluate this dynamic regulation of the Tec family kinases, different methods should be used; we will summarize the techniques available to visualize these intracellular proteins.

2. TEC KINASES LOCALIZATION AT THE PLASMA MEMBRANE Upon antigen receptor engagement, activation of the Tec kinases involves two events: first, membrane localization via interaction of the PH domain with phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] generated by phosphoinositide 3-kinase (PI3-K); and, second, phosphorylation by the Src kinases on a tyrosine in the activation loop, resulting in enhanced Tec kinase activity. Thus, in this general scheme, membrane recruitment corresponds to the first step of Tec kinase activation and the presence of a PH domain on the amino-terminal sequence of Btk, Tec and Itk is essential for this recruitment in B and T cells upon antigen stimulation. The PH domain was first identified in 1993 as a 100–120 residue stretch of amino-acid-sequence similarity that occurs twice in pleckstrin and is found 6,7 in several proteins involved in cellular signaling . The PH domains of the Tec kinases fall in the category of high-affinity phosphoinositide-binding PH domains. The importance of this domain has been highlighted by the mutations in the Btk PH domain known to cause impaired B cell development. The mutations that correlate with XLA in humans are known to decrease the affinity 8 of the Btk PH domain for PI(3,4,5)P3, illustrating the importance of this event in B cell receptor (BcR) signalling. Membrane recruitment of the Btk PH domain is generally weak and occurs rapidly and transiently after BcR crosslinking, and 9 correlates with the kinetics of PI(3,4,5)P3 production . PI3-K activation is critical in the signalling pathway leading to the plasma membrane translocation of the molecules containing a PH domain. A targeted disruption of the p85α regulatory subunit of PI3-K in mice (p85α KO mice) leads to the same phenotype as de10,11 veloped in Xid mice expressing Btk harbouring mutations in its PH domain . Nevertheless, the recruitment of activated Btk to the plasma membrane is not 12 affected by PI3-K inhibitors or in p85α KO B cells . The Xid phenotype in these mice with a loss of the p85α-mediated PI3-K activation could be induced by the impairment of the membrane recruitment of another PH domaincontaining protein: the Akt/PKB serine/threonine protein kinase. These data suggest that other domains of Btk can be involved for its translocation,

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Figure 1. The PH domain-containing Tec family kinases are predominantly cytosolic in resting lymphocytes. The amino-acid sequence of these proteins reveals the presence of several domains, starting at the amino terminus with a PH domain, followed by a domain named TH (for Tec homology) containing one or two proline-rich (PxxP) regions. This TH domain is able to bind the SH3 domain; these interactions are involved in dimerization of the Tec kinases. As in Src family kinases, the SH3 domain is followed by an SH2 domain and the carboxy terminus at the sequence contains the tyrosine kinase domain. Upon receptor engagement, the Tec family kinases are phosphorylated on a tyrosine residue within the activation loop of the kinase domain by an Src kinase, then Tec kinases autophosphorylate a tyrosine residue within its SH3 domain to give a protein with full catalytic activity. Conditioning this activation step, the Tec kinases should be recruited from the cytosol to the plasma membrane. An established model (model A) for Tec localization at the plasma membrane, corresponds to a first step (A-1) where the PH domain interacts with the phospholipids: PI(3,4,5)P3 (PIP3) generated by the PI3-kinase activation, then the SH3 domain can interact with partners at the plasma membrane containing proline-rich regions (PxxP) and/or the SH2 domain will interact with tyrosine-phosphorylated (pY) proteins (A-2). We suggest an alternative model (model B) where the PH domain is not necessary for Tec recruitment and is initially replaced by other domains such as the SH3 and/or SH2 domain (B-1). However, all the data are converging to the point that the PH domain is important for Tec activation, suggesting that also in this second model, PIP3 binding to the PH domain is required for Tec activation (B-2).

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either to maintain Btk at the membrane or to target directly the Tec kinases at the plasma membrane. For two of the Tec kinases present in T cells, Itk and Tec, a general activation model is established where membrane recruitment is mediated by their PH domains, thus the first step is controlled by PI3-K activation, and then an Src family member (Lck or Fyn) will phosphorylate and activate these Tec kinases (see Figure 1A). The Itk and Tec kinase activation is controlled by the PI(3,4,5)P3 levels at the membranes: both SHIP, an SH2-containing inositol phosphatase, and PTEN, a phosphatase and tensin homologue that catalyzes respectively hydrolysis of PI(3,4,5)P3 to PI(3,4)P2 or to PI(4,5)P2, downregulate 13,14 the Itk and Tec kinase activities . Both Itk and Tec co-localize with the activated T cell receptor (TcR) and the deletion of their PH domain abrogates or 15,16 impairs the translocation of these PTKs to the plasma membrane . Moreover, the Tec PH domain alone is able to co-localize partially with the activated TcR in Jurkat T cells (F. Garçon and J.A. Nunès, unpublished data). These studies were performed using antibodies against the TcR/CD3 complex. As a receptor engagement with antibodies or a natural ligand can mediate different intracellu17 lar signals , we and others have performed experiments using antigenpresenting cells (APCs) loaded with either agonist peptide or superantigen to 18, 19 stimulate the TcR expressed on T cells . Upon T cell–APC contacts, the presence of the PH domain is not necessary to induce membrane recruitment of Tec. All these data are showing that the PH domain of Tec is critical for the phosphorylation of PLC-γ1, a substrate of Tec, and activation of gene transcription. However, depending on the strength of T cell activation (antibodies crosslinking versus cellular contacts), the PH domain can be dispensable for the first step of Tec activation such as its targeting at the plasma membrane. In this new activation model (see Figure 1B), the first step of Tec activation could depend of another domain, for instance, the SH2 or SH3 domain. It has been reported that the 16,18 SH2 domain contributes to localization of Tec at the membranes . We also described that the SH3 domain is essential for targeting Tec at the plasma mem19,20 brane . Several partners of the SH2 and SH3 domains of Tec kinases are present at the T cell–APC contact zone, such as the adaptor molecules LAT (linker for activation of T cells) and SLP-76 (SH2-containing leukocyte protein of 76 kDa), Vav a Rac GDP/GTP exchange factor, WASP (Wiskott-Aldrich syndrome protein), or the CD28 co-stimulatory receptor (see review [3]). Several of these interacting proteins are involved in actin cytoskeleton reorganization, and the Tec kinases are good candidates to make the link between the receptors expressed at the cell surface and the assembly of the actin network (see review [21]). Vice-versa, some proteins participating in this network could bring Tec kinases from the cytosol and target them to the membranes. The Tec kinases cannot only translocate to the plasma membrane; it has been reported that Tec, but not Itk or Btk, is able to accumulate in intracellular 18 vesicles under the plasma membrane of T cells . The nature of these vesicles has been recently characterized as a compartment expressing the early en-

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dosomal antigen 1 marker (EEA1), and the PH domain is necessary to locate 22 Tec in these cellular structures . These intracellular compartments can accumu23,24 late several proteins involved in antigen receptor signaling . Interestingly, the translocation of Tec from the cytosol to these vesicles could involve Tec in the regulation of endocytosis and/or regulation of signal transduction at the plasma membrane. The presence of the Tec kinase family member Btk has been described in a 25 subcellular fraction from primary B cells corresponding to the caveolae . The caveolae are invaginations of the plasma membrane that serve as cell-surface 26 microdomains . Smith and colleagues reported some evidence that this localiza25 tion would downregulate Btk activity and that Btk associates with caveolin-1 . The larger isoform of Rlk that lacks the PH domain contains an amino-terminal 27 cysteine-string motif where palmitoylation occurs . This isoform is accumulated also in a detergent-insoluble fraction that is corresponding, in this case, to the 28 lipid rafts .

3. TEC KINASE LOCALIZATION IN THE NUCLEUS In addition to their membrane recruitment, Tec kinases have been shown to travel from the cytoplasm to the nucleus. The first evidence of nuclear localization of Tec kinases appeared in 1999 27 with the identification and characterization of Txk/Rlk . Looking at the intracellular localization of Rlk, the authors showed by immunofluorescence that the shorter form, which lacks the cysteine string motif, is located in the nucleus when expressed without the longer form. In fact, inspection of the amino-acid sequence of Txk/Rlk revealed a nuclear localization site (NLS) — (K(N)10KRKPLPP) — present in both forms of Rlk. Mutation of this NLS induces cytoplasmic localization of Rlk, while mutation of the cysteine string motif induces nuclear localization of the larger form, suggesting that this motif is responsible for maintaining the larger form in the cytoplasm. Thus, the NLS and the cysteine-rich motif play the role of antagonist in the localization of Rlk. Upon TCR activation of Jurkat T cells, a subpopulation of Rlk migrates from the cytoplasm to the membrane, while a second subpopulation translocates in the nucleus. However, after long-term stimulation the membrane fraction disappears and only the nuclear fraction remains. These data suggest that Rlk may have distinct cytosolic and nuclear function. Indeed, the nuclear localization of Rlk has been shown to be important for induction of IFN-γ, as a mutation of the NLS of Rlk abrogates induction of IFN29 γ promoter activity . In fact, Rlk directly binds to the IFN-γ enhancer region and 30 acts as a transcription factor . Remarkably, the region of the IFN-γ enhancer that responds to Rlk is conserved between the human and other mammalian IFN-γ genes, and similar sequences are present in several Th1 cell-associated genes. This suggests an important role of the nuclear translocation of Rlk for

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Th1 development. Interestingly, the longer form seems to be the main isoform that binds to the IFN-γ promoter. However, there is still no evidence that the nuclear function of Rlk is solely dependent of one isoform or the other. Similarly, following a first description of the nuclear translocation of Btk in 31 HeLa cells after SDF-1α stimulation , the nuclear shuttling of Btk in pre-B cell 32 line, B cells, and mast cells has been shown . Like Rlk, Btk presents an NLSlike sequence in the PH domain. However, this sequence is not necessary or required for translocation of Btk. Instead, Btk uses an exportin-1-dependent nuclear export signal to shuttle between nucleus and cytoplasm. Indeed, an NESlike sequence (PLNFKKRLFLL) can be found in the PH domain. It is of interest that this sequence is also found in sequences of the other Tec kinase family members. The PH domain and the Src kinases are critical in the signaling pathway leading to nuclear translocation of Btk. Thus, nucleocytoplasmic shuttling of a PH-deleted form of Btk is dependent on Src activity. Interestingly, deletion of the SH3 domain induces nuclear localization of Btk, suggesting that this domain could also present an NES. However, the precise mechanisms of the shuttling are still not known at the moment. How the PH and the SH3 domain act to regulate the shuttling of Btk is still to be elucidated. Data obtained so far would suggest either that Src-dependent protein, which sequesters Btk in the cytoplasm, may release it when tyrosine is phosphorylated, or that interaction of Btk with other Src substrates may expose masked NLS. 33 Btk can phosphorylate and regulate the transcription factors TFII-I and 34 STAT5 and can associate with the Bright transcription factor complex in a PH35 dependent manner . Interestingly, while Bright is synthesized in activated spleen cells from Xid mice, it did not bind DNA or associate with Btk. Thus, these data suggest a potential mechanism by which Xid mutation could lead to defects in Ig synthesis. Nevertheless, it appears that Btk can still be found in the nucleus of Xid cells. For Itk, an interaction with the nuclear import chaperone karyopherin/ 36 Rch1α has been demonstrated in Jurkat cells . This interaction implicates the SH3 domain of Itk and requires proline 242 of Rch1α. Interestingly, expression of a proline mutant of Rch1α decreased both Itk nuclear localization and CD3mediated IL-2 production in Jurkat T cells. The nuclear localization of Itk has been shown to be enhanced in TcR-activated T cells. If the exact function and the nature of Itk targets in the nucleus still remain to be elucidated, posttranscriptional modifications could be one important step in the nuclear function of Itk. For instance, methylation of proteins on arginine by S-adenosylmethionine-dependent protein arginine methyltransferase (PRMT) may be important during nuclear import/export process. Thus, it has been shown that CD28 engagement induced arginine methylation of several proteins, including Vav1 and 37 Itk . Interestingly, the R-methylated form of Vav1 is localized preferentially in the nucleus. These data suggest that this modification may require appropriate subcellular localization for Vav1. According to the authors, Itk was also found methylated upon CD28 stimulation with kinetics similar to Vav1. The role of R

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methylation is just emerging, and the impact of this modification on protein function remains to be clarified. Interestingly, even if the NLS sequence can be found in the sequence of all the Tec kinases, only translocation of Rlk has been shown to be NLS-dependent so far. Both Btk and Itk use different ways to shuttle between nucleus and cytoplasm. Finally, Tec is also able to interact with Rch1α but the mutation of its SH3 domain does not abolish this interaction (F. Garçon and J.A. Nunès, unpublished data). However, Tec can be found in the nucleus, and this localization seems to 19 be dependent on its SH3 domain . The exact function of this localization is still not known.

4. VISUALIZATION METHODS Over the past decade, genetically encoded fluorescent proteins have become widely used as markers in living cells. The development of these fluorescent proteins, coupled with advances in digital imaging has allowed addressing questions of the recruitment, colocalization and interactions of specific proteins within particular subcellular compartments. However, analyses of the localization of the Tec kinases poorly use these new technologies, whereas these techniques are now widely used in the field of immunology and more specifically in signal transduction. In the following section, we will briefly review some recent developments in imaging that could be used to monitor subcellular distribution and colocalization of Tec kinases. 4.1. TIRF In 2004 Tec was shown to have a unique pattern of subcellular localization as it 18 was found in small vesicles at the plasma membrane . Recently, the authors used TIRF (total internal reflection fluorescence) microscopy in order to exam22 ine these Tec-containing structures at the plasma membrane in live cells . TIRF imaging relies on an evanescent wave generated when light reflects off a surface at an incident angle equivalent to or greater than the critical angle. This allows better resolution due to a smaller optical section depth. The major advantage of this method is a higher signal-to-noise ratio than the other fluorescence imaging method, e.g., less background and more sensitivity to small changes in fluorescence. Furthermore, as just a small number of molecules are excited, photobleaching and phototoxicity are reduced. Using TIRF, it has been possible to demonstrate that Tec segregates in specific vesicles containing several important 22 signaling molecules like Lck and PLCγ . Recently, such clusters of proteins 38 have been described in Jurkat cells . Once again, TIRF allowed the authors to follow and characterize the behavior of single signaling molecules like Lck,

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LAT, or CD2 at the surface of a live cell. These clusters, or “microdomains,” are supposed to be formed by diffusional trapping through protein–protein interaction, and concentrate or exclude cell surface proteins to facilitate T cell signaling. One can wonder if these “microdomains” overlap with the Tec-containing vesicles, and where the others Tec kinases are. TIRF microscopy is certainly the best approach to answer this kind of question. 4.2. FRET The molecular dynamics of immunoreceptors and signaling proteins are thought to be important for cell activation but have not been extensively investigated in the environment of the immunological synapse. Classical biochemical approaches to the study of localized interactions cannot give a complete and dynamic picture of the events. One of the few techniques capable of giving such views of protein–protein interactions relies on Fluorescence Resonance Energy Transfer (FRET). FRET is a technique used to measure the interaction between two molecules labeled with different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. This transfer is only possible when the distance between the donor and the acceptor is very small (about the nanometer range). In biological applications, this technique has become popular to map protein–protein interactions (for some example of applications in T cells see review [39]). So this technique can give dynamic information about the real interaction between molecules, as opposed to simply the subcellular colocalization provided by fluorescence microscopy. Based on the idea that mutation in the PH domain of Btk leads to changes in its affinity for PI(3,4,5)P3, a miniaturized FRET-based assay for drug screening has been developed by monitoring interaction between the PH domain of Btk and 40 PI(3,4,5)P3 . This system could be easily adapted in a cellular system and allow researchers to monitor the effects of the compounds of interest on Tec localization. Recently, a new FRET method using three chromophores instead of two 41 has been developed . This technique allows multiple-interaction screening in living cells. Thus, the authors were able to detect and analyze interactions between EGF receptor and two intracellular partners: the adaptor molecule Grb2 and the E3 ubiquitin ligase Cbl. This 3-FRET technique could facilitate an understanding of the formation of protein networks containing Tec kinases during T cell activation. By its sensibility, FRET will be a great help in visualizing the dynamic of the interactions of Tec kinases with their partners during T cell activation. 4.3. FRAP The current knowledge of the changes in intracellular localization and dynamic movements of signaling molecules during lymphocyte activation is limited. Fluorescence recovery after photobleaching (FRAP) uses a short pulse of intense

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laser light to irreversibly destroy fluorescence in a very small area. The recovery of fluorescence into that area occurs as a result of signaling molecule mobility. This technique is then very useful to study the dynamics of signaling molecules during lymphocyte activation. For example, LAT has been shown to have lower mobility when present at the site of aggregated rafts after TCR stimulation than 42 that that of LAT found in other areas of plasma membrane . Then, FRAP can provide useful information on the mobility of proteins in the membrane or between several subcellular compartments. It could then help us determine if Tec kinases are trapped into a specific location at the synapse or if they are able to 43 diffuse in the plasma membrane like PI(3,4,5)P3 .

5. CONCLUDING REMARKS A well-established mechanism of activation of Tec family kinases suggests that the first step of activation is driven by plasma membrane recruitment of these Tec kinases via their PH domains. In this chapter we provide some arguments that this first step can be driven by other domains. Thus, the notion that Tec kinases are effectors of PI3-K should be discussed. The PH domain of Tec kinases interacts with some 3-phosphoinositides generated by the PI3-K activation such as PI(3,4,5)P3. The Akt/PKB serine/threonine kinase is a downstream target of PI3-K, and contains a PH domain that is necessary and sufficient to 44 recruit Akt/PKB at the plasma membrane . Several animal models demonstrated 12,45 that Akt/PKB, but not Tec kinases, is directly involved in PI3-K signaling . A major difference between the PH domain of Akt/PKB and those found on the Tec kinases corresponds to the specificity of theses domains: the Akt/PKB PH domain recognizes both PI(3,4,5)P3 and PI(3,4)P2, in contrast to the Tec PH domain, which binds only PI(3,4,5)P3 with high affinity (see review [46]). Upon antigen receptor engagement in lymphocytes, PI(3,4,5)P3 production appears to 9 be very transient compared to PI(3,4)P2 production . Thus, the Tec kinases might develop other strategies to be located at the plasma membrane using their SH2 or SH3 domains. Localization of the different Tec family kinases in the nucleus has been reported, but the issue of the nuclear functions of these kinases remains to be addressed. Many of the studies on the organization of cellular networks containing Tec kinases will profit from the now available methodologies (TIRF, FRAP, FRET, etc.) that allow visualization of two or more partners. As the Tec kinases are differently expressed during lymphocyte maturation (see review [4]), these studies should be addressed in the different stages of lymphocyte differentiation and activation.

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6. ACKNOWLEDGMENTS The authors thank Daniel Olive and members of his laboratory for helpful discussions. J.A.N acknowledges support from the INSERM and the Ligue Nationale contre le Cancer (équipe labellisée). F.G. is supported by a grant form the BBSRC.

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3 INDUCIBLE T CELL TYROSINE KINASE (ITK): STRUCTURAL REQUIREMENTS AND ACTIN POLYMERIZATION Constantine D. Tsoukas1, Juris A. Grasis1, Cecille D. Browne2 and Keith A. Ching3 1. INTRODUCTION Protein tyrosine kinases play critical roles in regulating signals initiated by the 1 engagement of the T cell receptor for antigen (TCR) . These kinases belong to 2-4 various families, such as Src, Syk, and Tec among others . The Tec family of protein kinases is one of the more recently discovered families that includes 4 members such as the prototypical member Tec, as well as Btk, Rlk, Etk, and Itk . 4 All of these proteins are specifically expressed in leukocytes . Itk, also known as 4 Tsk or Emt, is expressed in T lymphocytes, Natural Killer cells, and Mast cells . The biological significance of Itk in T cell biology has been demonstrated 4 using both in vitro as well as in vivo models . Using mice with disrupted Itk genes (KO mice), investigators have demonstrated the important role that this 5–7 kinase plays in both T cell development and activation . Itk KO mice display + 8 profound defects in T cell development, more specifically that of CD4 T cells . These defects have consequences in the production of cytokines, particularly 8 those produced by Th2 type helper cells . During the past several years, our laboratory has been interested in studying the involvement of Itk in T cell signaling through the TCR. Our research has focused on the structural requirements for Itk activation. Recently, we discovered a novel role for Itk, which is regulation of the TCR-induced actin polymeri9 zation . This chapter summarizes pertinent data in the field, focusing on our contributions that were presented during the 3rd Lymphocyte Signal Transduction Workshop (May 27–June 1, 2005, Crete, Greece).

1

Department of Biology and the Center for Microbial Sciences, San Diego State University, San Diego, CA 92181, USA, 2The Burnham Institute, La Jolla, CA 92037, USA, and 3Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.

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2. STRUCTURE OF ITK Itk, similar to Src family kinases, is modular in its organization as it includes SH1, SH2, and SH3 domains (Figure 1). However, unlike Src kinases, Itk also contains Pleckstrin Homology (PH) and Tec Homology (TH) domains at its N terminus (Figure 1).

Figure 1. Schematic representation of the Itk domains.

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Itk co-localizes inducibly with activated TCR–CD3 complexes . Upon transfection of GFP-tagged Itk into Jurkat T cells and examination by confocal 10 microscopy, we found Itk to be distributed primarily in the cytoplasm (see also Figure 2A). However, upon engagement of the TCR the cellular distribution and 10 localization of Itk changed and became similar to that of TCR complexes (also compare panels A and B to E and F in Figure 2). Interestingly, Itk also co10 localized with CD28 complexes under the same activation conditions .

3. PH DOMAIN The PH domain plays an important role in the recruitment of Itk to the cell 10,11 membrane and in mediating its interaction with membrane phospholipids . Recruitment of Itk to the membrane has consequences for its ability to associate with activated TCR complexes and in the subsequent transphosphorylation and 10 activation of the kinase . This is evidenced by genetic analysis where PH deletion mutants of Itk fail to associate with activated TCR complexes and remain 10 cytoplasmic upon T cell activation . Similar results were obtained with a variant of Itk that bears a point mutation (R29C) at the lipid-binding site of the PH domain (Figure 2K,L). Furthermore, overexpression of a gene product coding for the PH–TH domains of Itk in tandem interfered with the ability of endogenous Itk to become transphosphorylated and activated (Figure 3). Presumably the PH–TH protein acts as a competitive inhibitor for membrane phospholipid binding sites, thereby preventing endogenous Itk from being recruited to the cell membrane in order to become transphosphorylated. Interestingly, targeting PH domain deletion mutants of Itk to the cell membrane via addition of myristoylation/acylation target sequences restores their 10 ability to associate with activated TCR complexes . Deletion of the PH domain 10 appears to result in hyperactivation of Itk enzymatic activity , suggesting that the PH domain is not only important for membrane targeting but also for regulation of the enzymatic activity of Itk.

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Figure 2. Colocalization of WT and mutant Itk with activated TCR complexes. Jurkat cells were transfected with chimeric GFP constructs of WT Itk (panels A, B, E, and F), Itk SH3 mutant W208K (panels C and D), Itk kinase dead mutant K390R (panels G and H), Itk SH2 mutant R265K (panels I and J), or Itk PH mutant R29C (panels K and L). Cells were left unstimulated (panels A and B) or they were stimulated by treatment with a mouse anti-TCR monoclonal antibody reactived with the epsilon chain of the TCR complex (panels C–L). Cells were fixed and analyzed by confocal microscopy for the intracellular distribution of the transfected GFP-Itk (panels A, C, E, G, I, and K). The surface distribution of TCR was assessed by staining with an anti-mouse Texas Red-conjugated secondary antibody (panels B, D, F, H, J, and L). Upon stimulation, the distribution and polarity of WT–, W208K–, and K390R–Itk is similar to that of TCR (compare panels E, C, and G to F, D, and H). In contrast, R265K– and R29C–Itk do not distribute the same way as the TCR (compare panels I and K to J and L).

4. TH AND SH3 DOMAINS The TH domain of Itk contains a proline-rich motif that is hypothesized to engage intramolecularly with the SH3 domain and stabilize a closed inactive con12 formation . Thus, it appears that both PH and TH domains function as internal regulators of Itk enzymatic activity. Evidence supporting such a regulatory role is also obtained by the observation that a point mutation (W208K) in the SH3 domain that disrupts its ability to interact with proline-rich regions results in 16 elevated enzymatic activity of Itk . In contrast to the intracellular distribution of the PH domain mutants, the SH3 mutants of Itk display a distribution and localization similar to that of activated TCR complexes (Figure 2C and D). This suggests that the SH3 domain, although critical in the regulation of the enzymatic activity of Itk, is not essential for interaction of the kinase with the mem-

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Figure 3. Expression of PH–TH domains of Itk inhibits endogenous Itk phosphorylation. Jurkat cells were transfected with an expression vector (pCMV4) coding for the PH–TH domains of Itk in tandem along with a selection marker (neo). Transfectants were cloned by limiting dilution and individual clones were selected with G418 for expression of the transgene. The figure displays representative data with two of the clones where cells were stimulated (+) with anti-TCR receptor antibody (anti-CD3 epsilon OKT3 antibody) or left unstimulated (–). Control non-transfected Jurkat cells were treated the same way. Cells were lysed and lysates were subjected to immunoprecipitation (IP) with anti-Itk antibody. Immune complexes were resolved by SDS-gel electrophoresis and then immunoblotted (IB) with anti-phosphotyrosine (pY) antibodies followed by anti-Itk antibodies (loading controls) as indicated. Control non-transfected cells display increased tyrosine phosphorylation upon TCR stimulation. This is in contrast to the two PH–TH expressing clones where phosphorylation of endogenous Itk does not increase over non-stimulated levels. The bottom panel is an immunoblot of lysates from the three cell cultures using an antibody (H902) specific to the tag in the PH–TH construct. It can be seen that lysates from the two clones contain a band of approximately 25 kDa (arrow) that represents the PH–TH protein.

brane or its intracellular distribution and localization. In addition to its interaction with the TH domain, the SH3 domain of Itk has been shown to interact with 13 proline-rich regions of other signaling proteins . In this context, an interesting observation that prompted us to uncover the unique role of Itk in regulating the actin cytoskeleton is the in vitro interaction between WASP and Itk-SH3 GST 14 15 fusion proteins, first described by Cory et al and by Bunnel and colleagues .

5. SH2 DOMAIN The SH2 domain of Itk plays a critical role in both localization and activation of 16 Itk . Similar to WT-Itk, SH2 mutants carrying a mutation (R265K) that disrupts the interaction of Itk with phosphotyrosine residues on other signaling partners,

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associate with the cell membrane (Figure 2E, I). However, in contrast to the WT protein, SH2-mutant Itk has a different intracellular distribution and localization than activated TCR complexes (Figure 2, compare panels E and F to I and J). Interestingly, the clustering of TCR complexes (caps) that is induced by treatment with anti-TCR antibodies is disrupted in cells that express SH2-mutant Itk (Figure 2J). This result suggests that the SH2 mutant Itk interferes with the pathway that leads to TCR clustering in a dominant-negative fashion. It is interesting that a similar phenomenon was seen with cells expressing the PH domain mutant of Itk (Figure 2L) suggesting that this mutant may also interfere with the TCR clustering machinery. Mutations in the SH2 domain of Itk have consequences on the enzymatic 16 activity of the kinase . SH2 mutants of Itk do not become transphosphorylated 16 16 and lack enzymatic activity . This is in sharp contrast to the SH3 mutants . The lack of enzymatic activity is shared with kinase-dead mutants, which, however, 16 do become transphosphorylated . Thus, it appears the SH2 domain plays a critical role in the targeting of Itk to the relevant cellular sites and/or its interaction with relevant signaling partners that are critical for transphosphorylation. Adapter proteins are important for the recruitment of signaling molecules to the appropriate cellular sites and for guiding the interaction of relevant signaling 17 partners . The Linker for Activation of T cells (LAT) is an adapter that is critical for T cell activation, as it is involved in the formation of signaling complexes 18 essential for TCR-mediated T cell activation . We have shown Itk to inducibly associate with LAT in an interaction bearing consequences on the function of 16,19 Itk . Thus, TCR-induced activation of Itk is found to be defective in LATdeficient T cells and can be restored by transgenic expression of LAT in these 16,19 cells . The SH2 domain of Itk plays a critical role in this interaction as both deletion and point (R265K) mutants fail to associate with LAT and remain en16 zymatically inactive upon TCR engagement . Another important adapter protein that Itk interacts with is the SH220 containing Leukocyte-Specific Protein of 76 kDa (SLP-76) . In vitro experiments using Itk–SH2, –SH3 GST-fusion proteins have shown that Itk associates with SLP-76 in an interaction dependent on the cooperative binding of both of 15 these Itk domains to SLP-76 . However, in contrast to the studies with LATdeficient T cells described above, Itk retains the ability to become inducibly 21 phosphorylated in the absence of SLP-76 . Furthermore, the absence of SLP-76 does not affect the ability of Itk to associate with LAT (Tsoukas lab, in progress). Collectively, the above data suggest that the association of Itk and LAT is not dependent on SLP-76, and thus Itk may participate in distinct signaling complexes with these two adapter proteins. The association with LAT appears to be important for the phosphorylation and activation of Itk, whereas association with SLP-76 is not.

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6. REGULATION OF THE ACTIN CYTOSKELETON Expression of either Itk-SH2 deletion or point (R265K) mutants in Jurkat T cells reproducibly caused disruption of “cap” formation upon TCR crosslinking (Figure 2J). The formation of caps upon such clustering of TCR molecules has been 22 proposed to represent a structure similar to the immunological synapse . The formation of caps, as well as the immunological synapse, has been shown to 23,24 require actin reorganization and polymerization . Therefore, we suspected that expression of the Itk SH2 mutants might have an effect on the actin cytoskeleton. To test this possibility we adopted an assay in which Jurkat cells, incubated with latex beads coated with anti-TCR antibodies, extend cytoplasmic protru25 sions around the beads as to endocytose them . This event is dependent on actin 25 polymerization . Using laser scanning confocal microscopy, we visualized endocytosis of anti-TCR antibody coated beads by Jurkat cells that were transfected either with GFP–WT or GFP–SH2 mutant Itk. In contrast to WT–Itk 9 transfectants, those expressing SH2 mutants failed to endocytose beads . This observation suggested that the SH2 mutants exert a dominant-negative effect on the actin polymerization process. In order to analyze this effect more directly, we utilized the same assay and quantified the polymerization of actin at the cell– bead contact site by measuring pixel intensity after intracellular staining of cells with TRITC-conjugated phalloidin. Phalloidin is known to bind only to filamen26 tous actin . Similar to the bead endocytosis assay, we observed impaired actin polymerization in cells expressing Itk–SH2 mutants, as compared to WT–Itk9 expressing cells (see also Figure 4B,H). Since Itk–SH2 mutants fail to become phosphorylated and are enzymatically inactive, we hypothesized that the dominant-negative inhibitory effect mediated by these mutants may be related to the fact that they are catalytically inactive. Surprisingly, however, transfection with kinase-dead (K390R) mutants of Itk does not interfere with actin polymerization in our assay, suggesting that the kinase activity of Itk may not be involved in 9 actin polymerization . These observations have been confirmed and further extended by Dombroski et al., who inhibited Itk gene expression in Jurkat and pe27 ripheral blood T cells by using small interfering (si) RNA technology . In these cells actin polarization was impaired and could be rescued not only by transfec27 tion of WT–Itk, but also by kinase-dead Itk . Furthermore, transfection with an SH2 mutant of Itk failed to reconstitute actin polarization in siRNA-inhibited 27 cells . Thus, the SH2 domain, but not the kinase domain, of Itk appears to play an important role in TCR-mediated actin reorganization and polymerization. Interestingly, Dombroski and his colleagues found that a PH mutant of Itk that disrupts lipid binding failed to reconstitute actin polarization in siRNA inhibited cells thus, indicating that Itk has to interact with membrane phospholip27 ids in order to regulate actin polymerization . This observation is consistent with our data in Figure 2L, where a similar Itk mutant appears to diminish the ability of T cells to form TCR clusters. The importance of this interaction

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Figure 4. SH2 mutant Itk interferes with TCR-induced actin polymerization. Jurkat cells transfected with chimeric GFP constructs of WT– or SH2 mutant (R265K)–Itk were incubated with latex beads coated with either anti-TCR (anti-CD3 epsilon OKT3) or control antibody as indicated. Actin polymerization was visualized by intracellular staining with FITC-phalloidin followed by confocal microscopy. The DIC images (panels C, F, and I) are shown to facilitate orientation of cells and beads. WT–Itk is localized at the cell–bead interface similar to polymerized actin (panels A and B). SH2 mutant Itk does not localize at the cell–bead interface, and cells transfected with SH2 mutants fail to display actin polymerization (panels G and H). In this respect the SH2 mutant Itk-expressing cells behave like non-stimulated WT–Itk transfected cells (panels D and E).

is further supported by the experiment shown in Figure 5, where clones of Jurkat transfectants expressing the PH–TH domains of Itk in tandem fail to display actin polymerization as assessed by the bead endocytosis assay described above. This result suggests that the expression of the PH–TH transgene product competes with endogenous Itk for lipid binding sites at the membrane. The biological significance of Itk as a regulator of the actin cytoskeleton in T cell activation is also confirmed by experiments with primary lymphocytes. Using the anti-TCR coated bead assay described above, we observed that in contrast to splenic T cells of normal mice those from Itk-deficient (KO) animals do 9 not display significant levels of actin polymerization . Labno et al. have confirmed this by stimulating T cells from TCR transgenic mice deficient in Itk 28 with specific antigen-pulsed APC . In addition, these investigators showed that Itk is required for localized activation of Cdc42 and recruitment of Vav, both of which are critical in the TCR-mediated reorganization and polymerization of

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Figure 5. Expression of PH–TH domains of Itk inhibits TCR-induced actin polymerization. Clone 8 of Jurkat cell transfectants described in Figure 3 above were assessed for TCR-mediated actin polymerization using the bead endocytosis assay described above. The expression of Itk PH–TH domains in tandem interferes with bead endocytosis.

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actin . Interestingly, Dombroski and his colleagues have shown that Itk associates constitutively with Vav and that this interaction is important for proper Vav 27 localization . This finding provides some insight into the possible mechanism through which Itk may regulate the actin cytoskeleton during T cell activation.

7. CONCLUSIONS AND FUTURE DIRECTIONS Early studies indicated that Itk plays a critical role in signal transduction through 4 the TCR . Initially, studies conducted on Itk focused on the role of its enzymatic activity on T cell activation. For its enzymatic activation Itk requires a two-step 29,30 phosphorylation process . Upon T cell engagement, the tyrosine at position 29 511 of Itk becomes transphosphorylated by Lck . This event is followed by a 30 second step where Tyr 180 is autophosphorylated . Interestingly, LAT is re16,19 21 quired for the phosphorylation of Itk , but SLP-76 is not . Studies with T cells from Itk-deficient mice have shown the enzymatic activity of Itk to be important for the phosphorylation and activation of PLC6,7 2+ gamma1 in T cells . This is further supported by the deficient intracellular Ca 6,7 mobilization seen in T cells from these animals . Itk specifically regulates the + function of CD4 –Th2 lymphocytes, as evidenced by the profound effect that the 8 absence of Itk has on IL-4 and other Th2-type cytokines . Recent data by Miller and his colleagues, as well as Hwang et al., suggest that Itk may regulate Th2

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cytokine production indirectly by controlling the transcriptional activity of T-bet 31,32 through tyrosine phosphorylation . It was back in early 2001 when some of our preliminary experiments led us to predict that Itk may play an important role in TCR-induced cytoskeletal reor33 ganization . At that time, however, we suspected that the enzymatic activity of Itk would be critical in this process. Two years later, both our studies and those of Dombroski et al. confirmed our prediction and, surprisingly, established that the kinase activity of Itk is not essential. Thus, Itk may function as an adaptor 9,27 module to regulate actin remodeling . It is important that the mechanism through which Itk regulates TCR-induced actin remodeling be elucidated. One possibility could be that Itk interacts with and regulates the function of proteins involved in the actin polymerization machinery. One such protein might be WASP. This possibility is supported by the data of Bunnel et al., who have dem15 onstrated that the SH3 domain of Itk interacts with WASP . Furthermore, Labno and her colleagues have shown that in activated T cells from Itk-deficient mice WASP is recruited to the immunological synapse, but does not become acti28 vated . The interaction of the Itk–SH3 domain and WASP has been established by 15 in vitro analysis using Itk–SH3 GST-fusion proteins . Although these observations are important, as they provide preliminary clues, they must be reproduced with the intact proteins and confirmed in a cellular context. An experiment addressing this issue is shown in Figure 6. Using Jurkat T cells, it is shown that Itk associates with WASP and that this association increases one minute following TCR-induced stimulation and then declines. The association of Itk with WASP may not be direct and most likely involves several other proteins in a signaling complex. Ongoing studies in our laboratory focus on the structural requirements

Figure 6. Itk associates with WASP. Jurkat T cells transfected with Itk were stimulated for various periods of time with an antibody to the TCR (anti-CD3 epsilon OKT3). Cell lysates were immunoprecipitated (IP) with anti-WASP antibodies, and after resolution the immune complexes were blotted (IB) with the indicated antibodies. Itk associates with WASP and this association increases upon stimulation for 1 minute and declines thereafter. Tyrosine phosphorylation of WASP correlates with peak association with Itk.

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of this association and on the role of other signaling molecules. Interestingly, the peak of Itk–WASP association coincides with tyrosine phosphorylation of WASP (Figure 6). Data from other laboratories have suggested that several events need to occur for WASP to become activated and mediate actin polymerization. WASP has to interact with membrane phospholipids and with GTPbound cdc42, both of which facilitate the conformational change of WASP into 34,35 an active state that promotes actin polymerization . In addition, studies by Cory et al. have suggested that phosphorylation of WASP on tyrosine 291, in the 36 GTPase binding domain, enhances actin polymerization . Even though the seeming coincidence between peak WASP phosphorylation and its association with Itk seen in Figure 6 may suggest that Itk phosphorylates WASP, the data of Badour et al. indicate that Itk does not mediate the phosphorylation of WASP. These investigators demonstrated that TCR-mediated tyrosine phosphorylation 37 of WASP is not affected in T cells from Itk-deficient mice . Instead, it was 37 shown that Fyn could phosphorylate tyrosine 291 of WASP . Labno et al. have reported that in T cells from Itk-deficient mice WASP is 28 recruited to the T cell–APC contact site but does not become activated . This 28 observation was established using an antibody-recognizing active WASP . This antibody has been described to react with a site on WASP that is secluded in inactive WASP (closed conformation) but becomes exposed when WASP is 28 activated (open conformation) . Unfortunately, however, this reagent has not been described in any detail and its characterization is lacking. Therefore, the possible effects of Itk on WASP must be assessed using alternative approaches. In currently ongoing studies in our lab, we are collaborating with Mike Lorenz (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany) in studying the effects of Itk on WASP using Fluorescence Resonance Energy Transfer (FRET) technology. In conclusion, the study of Itk has led investigators to an exciting path of findings that underscore the importance of this protein on T cell development and activation. Itk appears to be one of the first paradigms of tyrosine kinases that participate in signaling pathways not by virtue of its catalytic properties alone, but also as a module that serves an adapter function exclusive of its enzymatic activity.

8. ACKNOWLEDGMENTS The studies in our laboratory were supported by grants (to CDT) from the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIH) and from the California State University Program for Education and Research in Biotechnology (CSUPERB). The authors would like to thank past and present members of the Tsoukas lab for their contributions and insightful discussions.

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4 DIFFERENTIAL REQUIREMENTS OF PI3K SUBUNITS FOR BCR OR BCR/CD19-INDUCED ERK ACTIVATION Elena Vigorito and Martin Turner

1. INTRODUCTION Phosphoinositide 3-kinase (PI3K) comprises a family of enzymes that generates D3-phosphorylated phosphoinositides. The phospholipids generated by PI3K mediate a variety of biological functions in different cell types by recruiting specific proteins to cellular membranes. They are classified in three groups: class I, II, or III, depending on their subunit structure, regulation, and substrate 1,2 selectivity . Class I PI3Ks are the only enzymes able to generate the second messenger phophatidylinositol-(3,4,5) triphosphate (PIP3) at the inner leaflet of the plasma membrane. In resting cells the levels of PIP3 are very low, but they can sharply increase upon stimulation, where they appear to be distributed in 2 particular subcellular compartments . This leads to membrane recruitment of certain proteins containing pleckstrin homology domains such as PKB or Btk. Class I PI3K are heterodimers composed of a p110 catalytic subunit and a regulatory subunit and can be further subdivided into class IA and B. Subclass Ia includes three catalytic subunits — α, β or δ — encoded by three distinct genes. Three genes also encode for the regulatory subunits, giving rise to five proteins: p85α, p55α, p50α, p85β, and p55γ. The p85α, p55α, and p50α subunits are generated as alternative transcripts of the same gene. It is believed each of the catalytic subunits can associate with any of the regulatory subunits. Some differences in their biological activities could be attributed to differences in their ex3 pression pattern. Whereas p110δ is mainly expressed in haematopoietic cells , 4,5 the other catalytic subunits are ubiquitously expressed Moreover, among the different regulatory subunits, p85α is the predominant form in B cells, p55α is primarily detected in brain and muscle, whereas p50α is

Laboratory of Lymphocyte Signaling and Development, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.

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highly detected in brain, muscle, liver, and kidney . In contrast to class Ia proteins, only one catalytic subunit, p110γ, and one regulatory subunit, p101, comprise class IB PI3Ks. Binding to the regulatory subunit increases the thermal stability of the catalytic subunit and allows its translocation to membraneassociated signalling complexes. The best understood mechanism of activation of class IA PI3K is downstream of tyrosine-kinase associated receptors, such as antigen receptors and its co-receptors, whereas for class IB is downstream of Gprotein couple receptors. B cell receptor (BCR) signals are essential for B cell development and function. Antigen engagement of the BCR on mature B cells drives their proliferation and differentiation. The surface expression and signalling capacity of the BCR is mediated by the associated non-polymorphic proteins CD79a and CD79b (Igα and Igβ). CD79a and CD79b contain immunoreceptor tyrosinebased activation motifs (ITAMs) within their cytoplasmic domains that, when phosphorylated, recruit tyrosine kinases, including Syk and Lyn. CD79a may also bind the adapter protein B cell linker protein (BLNK, also named SLP-65/ 10 BASH) through a non-ITAM tyrosine in its cytoplasmic domain . The recruitment of further signalling proteins is facilitated by BLNK, which may serve as a scaffold protein for the assembly of a multi-protein complex that includes phospholipase-C-γ2 (PLCγ2), Bruton’s tyrosine kinase (Btk), Vav, and phospho11-13 inositide 3-kinase (PI3K) and has been termed the signalosome . The signalosome controls downstream events, including the accumulation of phosphatidylinositol 3,4,5 trisphosphate, the initial peak of intracellular calcium and the activation serine–threonine mitogen-activated protein kinases (MAPKs). The MAPK cascade is one of the most ancient and evolutionary conserved signaling pathways. Of the mammalian MAPKs, the extracellular signal regulated protein 14 kinases (ERK) are the best characterized . The activation of ERK in many cell types, including B and T cells, is dependent on the Ras–Raf–MEK signaling 14 cascade . Phosphorylation at two positions in their activation loop result in a 1000-fold increase in activity. In addition, ERK phosphorylation promotes dimer formation — a step required for nuclear translocation and subsequent 15 phosphorylation of its targets . There are two isoforms of ERK, designated ERK1 and ERK2, which share 84% amino-acid identity and are thought to be 16,17 functionally redundant . Genetic disruption of ERK1 does not affect B cell development or basal levels of serum Ig, suggesting that ERK2 may compensate 18 19,20 for a lack of ERK1 . ERK2-deficient mice are embryonic lethal .

2. CONTRIBUTION OF P85α AND P110δ PI3K SUBUNITS TO ERK ACTIVATION TRIGGERED BY THE BCR Gene targeting strategies constitute a valuable experimental approach for dissecting the contribution of particular catalytic and regulatory subunits of PI3K to

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B cell development and function. The p85α locus has been disrupted in two different ways. In one strategy, all isoforms (p85α, p55α, and p50α) were de21 leted ; a distinct strategy eliminated only the long form p85α (hereafter referred 22 to as p85α mice) . In both studies reduced numbers of B1 cells, mature periph22,23 eral B2 cells, as well as defective proliferative responses were reported . Further analysis of p85α mice showed decreased numbers of marginal zone B 24 cells . Although transgenic expression of Bcl-2 restored numbers of mature follicular cells, proliferative responses to BCR crosslinking remained severely im24 paired and marginal zone B cell numbers remained low . By contrast, deletion 25 of p85β had no apparent effect on B cell development and function . The function of the catalytic subunits has been more difficult to address as deletion of 26,27 either p110α or β produces embryonic lethal phenotypes . Deletion of p110δ leads to decreased numbers of B1, marginal zone B cells, and defective pro28-30 liferative responses . Use of the pharmacological inhibitors wortmannin and LY294002 has implicated PI3K in BCR-induced proliferation and proximal sig2+ 31 nalling events, including PKB phosphorylation or Ca mobilisation . We were interested in studying the effect of PI3K and, in particular, the contribution of the p85α and p110δ subunits to ERK activation. We and other investigators have previously demonstrated that p85α and p110δ are required for 2+ optimal PKB activation and Ca release from intracellular stores in response to 28,29,32,33 BCR engagement , suggesting that both subunits contribute to overall PI3K activity. We monitored ERK activation by measuring phosphorylation 14 at positions Th202/Tyr204, which correlates with its activity . In agreement 34 with previous reports , we observed that wortmannin almost completely inhibited Erk phosphorylation elicited by BCR crosslinking (Figure 1A). We next tested the requirement for p110δ and p85α in ERK activation in response to BCR crosslinking. To this end, purified B cells from wild-type p110δ- or p85αdeficient mice were treated with anti-IgM antibodies for the indicated time points (Figure 1B). To our surprise, we observed almost normal levels of ERK phosphorylation both in p110δ- as well as p85α-deficient B cells (Figure 1B). These results suggest that other catalytic and regulatory subunits may be involved in ERK activation triggered by the BCR. We previously found that 28 p110δ-deficient B cells expressed normal levels of p110α and p110β . In the case of p85α-deficient B cells we observed upregulation of the p55α and p50α isoforms, suggesting that those molecules may contribute to ERK activation (Figure 1C). The contribution of PI3K to signalling pathways elicited by the BCR that result in ERK activation has not been fully clarified. A key step for ERK activation is the membrane targeting of guanine exchange factors for Ras. Studies based on cell lines have shown that BCR signalling triggers the association of 34 Sos to the adapter proteins Shc and Grb2, facilitating its membrane targeting .

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Figure 1. Effect of PI3K on ERK phosphorylation in response to α-IgM. (A) Splenic B cells from wild-type mice were pretreated with 100-nM wortmannin or vehicle control for 15 minutes followed by stimulation with 10 µg/ml of F(ab)′2 α-IgM fragments for the indicated time points. ERK phosphorylation was analyzed by immunoblot using anti-phospho ERK1/2 (T202/Y204) antibodies. After stripping, blots were re-probed with anti-ERK1/2 antibodies as loading control. (B) Splenic B cells from the indicated genotypes were stimulated and analyzed for ERK phosphorylation as described in part (A). (C) Lysates from wild-type or p85α-deficient B cells were immuno-blotted with anti-p85α antibodies. The antibody also recognises the alternative spliced forms p55α and p50α.

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Inhibition of PI3K, however, does not interfere with this complex formation . Instead, it has been shown that PI3K may affect ERK activation by regulation of 34 35 PLCγ . More recently, Kurosaki et al. proposed a model, based on studies on DT40 cells, in which DAG generated upon BCR stimulation recruits the guanine exchange factor RasGRP3 to the plasma membrane, where it interacts with Ras. An additional level of regulation of RasGRP3 comes from PKC phosphorylation, which enhances RasGRP3 enzymatic activity. Therefore, it is possible to speculate that PI3K induction of PLCγ2 activity could regulate levels of DAG, which, in turn, regulate RasGRP membrane localisation and enzymatic activity. This would lead to Ras and, subsequently, ERK activation.

3. P85α AND P110δ ARE REQUIRED FOR ERK ACTIVATION ELICITED BY BCR:CD19 COLIGATION B cell responses to foreign antigens typically result from the integration of signals from the BCR and its co-receptors. The CD19–CD21 complex links innate immune responses to the acquired immune system by binding antigens that have activated complement, resulting in coligation with the BCR. In this context, B cell responses such as germinal center formation and antibody secretion are en-

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hanced by several orders of magnitude . CD19 is rapidly phosphorylated as a consequence of BCR engagement, and the cytoplasmic domain of CD19 contains nine tyrosine residues with the potential to recruit SH2 domain-containing proteins. PI3K is recruited to CD19 through binding of the SH2 domain of the 37 regulatory subunit to tyrosines 482 and 513 . Mutated forms of CD19 at residues 482 and 513 expressed in CD19-deficient mice have illustrated the importance of these tyrosines in vivo. Remarkably, tyrosines 482 and 513 contribute to all aspects of CD19 function, such as T-dependent responses and germinal 38 center reaction . Moreover, the association of CD19 to PI3K is abolished in 38 Y482/Y513 CD19-mutant mice , highlighting the importance of PI3K activation in CD19 signalling. It is well established that PI3K is synergistically acti39 vated by coligation of the BCR with CD19 . We became interested in addressing the contribution of p110δ and p85α to the overall PI3K activity elicited by BCR and CD19. As phosphorylation of PKB is dependent upon PIP3 generated by PI3K, we monitored phosphorylation 40 of PKB at serine 473 as readout of PI3K activation . For the coligation of suboptimal numbers of BCR with CD19, biotinylated Fab’ fragments of monoclonal antibodies against κ-light chain and CD19 were co-crosslinked by addi41 tion of avidin, a system previously described . Our results showed a significant drop in the levels of PKB phosphorylation upon BCR and CD19 coligation in p110δ- or p85α-deficient B cells. This allowed us to conclude that both subunits are important contributors to the overall PI3K activity elicited under synergistic conditions. The synergistic coligation of BCR and CD19 also leads to MAPK 42 activation, in particular ERK activation . This is illustrated in Figure 2A, which shows ERK phosphorylation in response to ligation of the BCR alone, CD19 alone, or BCR in combination with CD19. Stimulation with α-IgM was included as a control for comparative purposes. Crosslinking of the BCR alone or CD19 alone promoted a dose-dependent phosphorylation of ERK (Figure 2A). Moreover, phosphorylation of ERK was synergistically amplified by coligation of BCR with CD19 at levels that were by themselves suboptimal (Figure 2A). Coligation of BCR with CD19 at 0.1 and 0.5 µg/ml resulted in similar levels of ERK phosphorylation, as we observed with optimal doses of anti-IgM (Figure 2A). We next analyzed the phosphorylation status of ERK following optimal ligation of BCR alone, CD19 alone, or the synergistic response of BCR and CD19 in p110δ- or p85α-deficient B cells (Figure 2B). Following BCR crosslinking using maximal levels of anti-κ B cells lacking p110δ showed similar levels of ERK phosphorylation to that observed in wild-type cells (Figure 2B). This result resembles what we observed using anti-IgM antibodies (Figure 1B), suggesting that stimulating the BCR either by IgM or IgM/IgD crosslinking leads to similar levels of ERK phosphorylation. In terms of the synergistic response of BCR and CD19, ERK phosphorylation in p110δ-deficient B cells was

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Figure 2. Effect of p85α and p110δ in the synergistic phosphorylation of ERK following coligation of Igκ and CD19. Purified splenic B cells were treated. (A) Samples were incubated for 15 min with 0.1, 0.5, 1, 5, or 10 µg/ml of biotinylated Fab fragments of kappa (a-kappa) or CD19 (a-CD19) respectively or synergistic (S) combinations of anti-kappa and anti-CD19. S* correspond to 0.1 µg/ml of a-kappa plus 0.1 µg/ml α-CD19. S** correspond to 0.1 µg/ml of a-kappa plus 0.5 µg/ml of antiCD19. After incubation, cells were washed and stimulated for 2 min at 37oC by the addition of 20 µg/ml avidin. Alternatively, cells were stimulated with F(ab)2 α-IgM (10 µg/ml) for 2 minutes. Samples were analyzed by Western blot as described in Figure 1. Cell lysates were blotted with the indicated antibodies. The blots were subsequently stripped and re-probed with indicated antibodies as controls. (B) ERK phosphorylation activated by coligation of Igκ and CD19. Cells of the indicated genotypes were stimulated with optimal concentrations of a-κ or CD19 Fab′ fragments (10 µg/ml) or under synergistic conditions of a-κ and CD19 (0.1 and 0.5 µg/ml, respectively) as described in part (A).

severely reduced. We found that p85α-deficient B cells behaved similarly, although the extent of reduction of BCR-stimulated ERK phosphorylation was less severe than that seen in p110δ-deficient B cells following optimal BCR crosslinking (Figure 2B). Taken together these data show that p110δ and p85α contribute significantly more to ERK activation by BCR:CD19 coligation than to BCR ligation alone. Moreover, as there is still some residual level of ERK phosphorylation in p85α- or p110δ-deficient B cells, our results implicate additional catalytic and adapter subunits to the BCR:CD19 synergy response. Regulation of ERK activation in the BCR:CD19 synergy response appears 44 to be different to that induced by anti-IgM alone . This could explain the differ-

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ential requirements of p85α and p110δ in both kinds of responses. In contrast to IgM crosslinking, coligation of BCR:CD19 does not lead to synergistic activa43 tion of Ras . Under synergistic conditions signals from the BCR and CD19 ap43 pear to converge at the level MEK activation . CD19 signals for ERK activation are not well characterised, although it is known they are not sensitive to changes +2 43 in intracellular Ca levels or PKC inhibition . Our results with p110δ−deficient cells also show that CD19-dependent ERK activation is highly dependent on PI3K (Figure 2B).

4. CONCLUSIONS In this paper we have explored the requirements of the PI3K catalytic and regulatory subunits p110δ and p85α for ERK activation in the signalling pathways emanating from BCR or BCR:CD19 coligation. While neither subunit is required for ERK phosphorylation in response to IgM crosslinking, both subunits substantially contribute to ERK phosphorylation in the BCR:CD19 synergistic response.

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5 PROTEIN TYROSINE PHOSPHATASES IN HUMAN DISEASE Tomas Mustelin

1. INTRODUCTION Protein tyrosine phosphatases (PTPs) play key roles in numerous cellular processes in immune cells and are implicated in numerous human diseases, from cancer to neurological, cardiovascular, metabolic, immunological, and infectious diseases. In this chapter I review the ways that PTPs can contribute to pathophysiology and I discuss the potential of PTPs as drug targets. 1.1. The Protein Tyrosine Phosphatases It has been estimated that more than a third of all cellular proteins contain covalently bound phosphate. Among the hydroxyl-containing phospho-acceptor amino acids, phosphorylation on serine is the most common, constituting some 95% of all protein-bound phosphate, followed by phosphate on threonine in approximately 5% of phosphoproteins. By comparison, phosphorylation of pro1 teins on tyrosine is very rare, only 0.01–0.1% of phosphoproteins, but nevertheless plays a crucial role in many processes that are characteristic of multicellular organisms, such as cell-to-cell communication and functions that coordinate the 2-5 behavior of cell populations . Nevertheless, tyrosine phosphorylation also oc6-8 curs in bacteria and Archea , the genomes of which typically contain two to four genes for protein tyrosine phosphatases (PTPs). Thus, tyrosine phosphorylation may have been an important tool for the emergence of multicellular organisms, but it already existed from much earlier times. Tyrosine phosphorylation is a rapidly reversible posttranslational modification, which is catalyzed by protein tyrosine kinases (PTKs) and reversed by PTPs. At any given moment in time, the state of phosphorylation of a protein is the net result of the opposing activities of the relevant PTK(s) and PTP(s). A

Inflammatory and Infectious Disease Center, and Program of Signal Transduction, Cancer Center, The Burnham Institute, La Jolla, CA 92037, USA.

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change in phosphorylation state can be brought about by altering the activity (or access) of either enzyme. With very few exceptions (e.g., Lck Y505), the balance is skewed very far toward the dephosphorylated state. In fact, most tyrosine phosphorylated proteins are not phosphorylated at all under normal conditions and become phosphorylated to a stoichiometry of maximally a few percent even under the strongest inducing conditions. Thus, one could even argue that PTPs are more important than kinases in setting the levels of tyrosine phosphorylation and that they should be much better drug targets. PTPs often play highly spe9-17 cific, non-redundant, and strictly regulated roles in cellular processes . Many 18-20 PTPs play “positive” roles , rather than negative, and more than half of all 21-29 reported PTP knockout mice have unique and complex phenotypes . Finally, 3,30 the human genome contains more PTP genes than PTK genes : 107 versus 90. We refer to the human genome complement of PTPs as the “human PTPome.” We defined “PTPs” as all known enzymes with PTP activity, as well as all 3 proteins with structural homology to their catalytic domains . This structural definition includes catalytically inactive or undefined enzymes and enzymes that have evolved to have other than phosphotyrosine-specific activity, e.g., PTEN, a phosphoinositide 3-phosphatase. By this definition, the human genome encodes for at least 107 PTPs, which fall into four evolutionary separate families: the class I, II, and III Cys-based PTPs, and the Asp-based phosphatases, exemplified 14 by the Eya (eyes absent) tyrosine phosphatases . These Asp-based PTPs belong to the very large family of haloacid dehalogenase (HAD)-related enzymes, which is well represented in plants, prokaryotes, yeast, and mammals, and which includes numerous enzymes with other than phosphotyrosine-specificity. The three families of Cys-based PTPs share a common catalytic mechanism, in which a cysteine residue acts as a nucleophile to attack the incoming phosphotyrosine of the substrate and forms a thiophosphate intermediate during catalysis. This is greatly aided by the effect of adjacent amino-acid residues, which lower the pKa of the catalytic cysteine, which also renders these PTPs very sensitive to oxidation. The vast majority of PTPs belong to the class I family, which are structur31 ally related to the first sequenced PTP, PTP1B . There are at least 99 genes for 3 PTPs of this family in the human genome and they can be further subclassified into the classical PTPs (both receptor-like and non-receptor PTPs), and the VH1-like phosphatase group, which contains the eleven MAP kinase phosphatases (MKPs), the 19 atypical dual-specificity phosphatases (aDSPs), the three slingshots, the three PRLs, the four CDC14s, the PTEN/tensin group, and the 16 myotubularins. The two latter groups have evolved to dephosphorylate the 332,33 phosphate of inositol phospholipids . Based on the presence of homologs in all 34 kingdoms of life, including Archea and plants , as well as structural considera34 tions , it seems that the aDSPs are the evolutionary most ancient members of the family. On the other hand, the classical PTPs, particularly the receptor-like group, are only found in multicellular organisms.

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The class II Cys-based PTPs comprise a small group of cell-cycle regulators known as the CDC25 phosphatases. Although their catalytic machinery is very similar to that of the class I enzymes, they are otherwise structurally unrelated 35 and instead bear considerable resemblance to bacterial rhodanese enzymes , from which are thought to have evolved relatively late in eukaryote evolution. Curiously, the eleven MAP kinase phosphatases, which belong to the class I family, have taken up a catalytically inactive rhodanese-like domain to act as a 36 MAP kinase docking module . Finally, the human genome contains a single gene for a class III PTP, the low Mr PTP (LMPTP), which undergoes alternative splicing to yield two active and one inactive isoforms. Although a polymorphism in this gene correlates with 4 numerous common human diseases , the function of LMPTP has remained obscure. 1.2. Why So Many PTPs? One may wonder why there are so many PTPs in the human genome. Likely, part of the answer is that PTPs have a high degree of specificity, important and non-redundant functions, and, consequently, relatively few substrates each. However, these premises remain largely untested, and there are some indications also for the opposite: while many of the >30 reported PTP knockout mice support the notion of important and unique functions (e.g., the embryonic lethal 21 22 deletions of PTP–PEST and SHP2 ), some of these mice have a milder pheno37 38 type than expected, e.g., MKP1 or PEP . Thus, it seems reasonable to assume that closely related PTPs have at least partly overlapping sets of substrates. This view has important consequences for the consideration of PTPs as drug targets: strictly monospecific PTP inhibitors may have a much smaller impact than inhibitors of a small group of closely related PTPs. Another possible explanation for the abundance of PTP genes in the human genome would be that many genes have a restricted expression profile. However, this does not appear to be the case: most PTPs are quite broadly expressed (albeit at different levels in different tissues) and individual cell types express a large portion of the PTPome. We have found that white blood cells express between 65 and 75 different PTPs, with each cell lineage having its own profile of PTPs at certain relative expression levels. Interestingly, this profile undergoes both qualitative and quantitative changes in response to external stimuli, cell activation, differentiation, etc. Remarkably, each response is unique, and identical stimuli elicit different responses in different cells types. Finally, there are individual variations in PTP expression profile between healthy blood donors, suggesting that polymorphisms and genetic heterogeneity affect PTP expression patterns. All these levels of complexity will need to be considered when contemplating the question of redundancy between PTPs and the use of PTPs as drug targets. Clearly, a more systematic analysis of tissue expression profiles,

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relative expression levels, and differential regulation of PTP expression during embryogenesis and development will be needed. 1.3. The Set of PTPs Expressed in Immune Cells Lymphocytes express a remarkably high proportion of the 107 PTP genes in the + human genome. CD4 T cells contain an estimated 58 different PTPs, while + CD8 T cells have 57 PTPs and B cells even more, perhaps as many as 74 (our unpublished data). Some uncertainty in these numbers is introduced by a few PTPs, primarily receptor-PTPs, being detected at levels that may be too low to be physiologically meaningful. There are several PTPs that are restricted to hematopoietic cells, such as CD45, HePTP, SHP1, LYP (PEP in the mouse), and PAC1, and others that are expressed at particularly high levels in lymphocytes, such as PTP-MEG1, PTP-PEST, MKP5, and MTMR1. Interestingly, B lymphocytes express several PTPs involved in cell-to-cell interactions through the cadherin/catenin systems, such as RPTPκ. All lymphocytes are particularly rich in intracellular (non-receptor) classical PTPs, almost all of which are readily detectable by immunoblotting.

2. PTPS AND HUMAN DISEASE Supporting the notion that PTPs have important and non-redundant functions in cell physiology, almost half of all PTP genes in the human genome have already 3,4,39 been implicated in human disease . Disease-related perturbations range from major genetic lesions (e.g., deletion) to point mutations and single amino-acid substitutions. There are also many examples of amplification, overexpression, or ectopic expression of PTPs in human disease, e.g., in cancer. Perhaps the most striking finding is that very subtle alterations in PTP function can precipitate life-long suffering, even fatal disease. It also seems that genetic polymorphisms in PTPs play a significant role in disease predisposition and in the genetic heterogeneity that underlies individual variation in immunity and disease susceptibility, severity, and recovery. Given the importance of tyrosine phosphorylation in so many physiological processes, including many of high relevance for the immune system, and the very limited studies performed so far, it seems likely that many more human health concerns will be found to involve a central role for PTPs. I also predict that the pharmaceutical industry will become increasingly interested in PTPs as drug targets. 2.1. PTPs in Neoplastic Disease As one might expect from the known role of tyrosine phosphorylation in cell growth and the existence of PTK oncogenes, loss of at least 30 different PTPs

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has been reported in numerous experimental and clinical cancers. The genetic basis for these alterations in PTP expression include chromosomal abnormalities, frame-shift mutations, or point mutations, as well as epigenetic mechanisms, such as promoter methylation or changes in transcription There are also a few examples of altered stability of PTPs in cancerous cells. One of the earliest examples of PTP dysregulation in cancer was provided by Brent Zanke and coworkers, who cloned the hematopoietic tyrosine phos40 phatase HePTP and found that its gene is located on chromosome 1q32.1, a site of frequent abnormalities in the preleukemic myelodysplastic syndrome, as well 41 as in leukemias . They also identified a patient with acute myeloblastic leuke41 mia in which the malignant cells overexpressed HePTP several-fold . Similarly, the SH2-containing PTP SHP1, which acts as a negative regulator of many im11 portant signaling pathways in hematopoietic cells , is frequently lost in myelo42 43 dysplastic syndrome and lymphomas . Reduced expression of SHP1 seems to be associated with an early step in carcinogenesis and is indicative of aggressive 42 disease with more rapid progression . By far the most commonly lost PTP in human cancer is PTEN, a class I cysteine-based phosphatase specific for phosphate at the 3-position of inositol phospholipids rather than phosphotyrosine. Loss of PTEN is found in more than half of all glioblastomas and in a high proportion of breast and prostate cancers, 44-53 in lymphomas, and many other common cancers . Because PTEN directly counteracts the many growth, survival, and motility promoting effects of phosphatidylinositol-3-kinase, which involve the Ser/Thr-protein kinase Akt and other pleckstrin homology domain-containing proteins, and signaling pathways downstream of them, the loss of PTEN provides strong growth and survival advantages to the malignant cells. Several transmembrane PTPs have also been reported to be lost in malig54 nant cells, such as DEP1 (PTPRJ) in colon cancer , and GLEPP1 (PTPRO) in 55 56 hepatocellular carcinomas and colon cancer . A more comprehensive analysis 57 of PTPs in human cancer by Wang and colleagues found that RPTPρ (PTPRT), RPTPγ (PTPRG), LAR (PTPRF), PTPH1 (PTPN3), PTP-BAS (PTPN13), and PTPD2 (PTPN14) are frequently mutated in colon cancer. Several of the detected mutations resulted in reduced catalytic activity. The discovery that PTPs sometimes are overexpressed, rather than reduced, in human cancer illustrates the growing notion that PTPs not only act as tumor suppressors, but also as “positive” components of signaling pathways and central cell processes, many of which require a dynamic balance between kinases and phosphatases, rather than either type of enzyme alone. Cell motility is a great example of this concept, which may explain why a high level of overexpression of the small farnesylated phosphatase PRL3 is found in metastatic co58 lon cancer , but not in the primary tumor. Similarly, overexpression of the MAP 59 kinase phosphatase MKP1 in prostate cancer may serve to prevent excessive activation of MAP kinases, which can result in cell cycle arrest and cell senes60 cence. Finally, the increased levels of PTPα in breast cancer is clearly related

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to the ability of this PTP to activate Src family PTKs by dephosphorylating their negative regulatory site. Finally, there are many PTPs that regulate cell cycle progression, such as the three Cdc25A, Cdc25B, Cdc25C, Cdc14A, and Cdc14B phosphatases, which dephosphorylate and activate or inactivate cyclin-dependent kinases at key transition points in the cell cycle. These phosphatases have long been considered as drug targets for cancer therapy. 2.2. PTPs in Monogenic Inherited Syndromes Several PTPs were originally discovered as the loci of inherited genetic dis3 eases . The responsible lesions include frame-shift mutations that cause loss of protein due to premature termination of translation, and point mutations that result in amino-acid substitutions. Examples of the former mechanism include Lafora’s epilepsy, which is caused by loss of a small PTP, called Laforin, which 61,62 has a glycogen-binding domain , as well as X-linked muscular dystrophy, which is caused by loss of a phosphoinositide-specific PTP called myotubu63 larin . A related PTP, myotubularin-related protein 2 (MTMR2), was found to be 64 mutated in Charcot-Marie-Tooth syndrome type 4B , an inherited nerve myelination disease. Surprisingly, a subset of patients were found to have a normal MTMR2 gene and instead to have point mutations in the catalytically inactive 65 phosphatase MTMR13 . The disease is the same in both cases, posing the question of how the loss of an inactive phosphatase can lead to the same disease as the loss of an active phosphatase. The answer to this riddle was provided by the discovery that the two proteins form a heterodimer, in which the catalytically inactive MTMR13 is required for proper targeting and function of the active MTMR2. Germline mutations in PTEN are also present in three related monogenic diseases — Cowden disease, Bannayan-Zonana syndrome, and Lhermitte66-68 Duclos disease — all of which are characterized by a propensity to develop benign hamartomas and a greatly increased risk of malignant tumors. Another monogenic disease with elevated risk of leukemia is Noonan syndrome, which is caused by gain-of-function mutations in the SH2 domain-containing phos69 phatase SHP2 . The heart abnormalities, facial dysmorphisms, short stature, cryptorchism, mental retardation, and other developmental problems associated with this disease are compatible with an important role of SHP2 during embryonic development and organogenesis. Knockout of the shp2 gene in mice is early embryonic lethal. 2.3. Involvement of PTPs in Metabolic Syndromes The main reason the pharmaceutical industry recently became very interested in 70 PTPs as drug targets was the phenotype of the PTP1B knockout mouse , which included heightened responses to insulin and resistance to fatty diet-induced

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obesity. This phenotype precipitated the notion that a small-molecule inhibitor of PTP1B would increase insulin signaling and improve glucose uptake and fatty acid metabolism, both highly desirable effects in patients with type 2 diabetes. The biology of PTP1B is also very interesting: PTP1B is mostly located on internal membranes of the endoplasmic reticulum and endosomes, in which it meets the phosphorylated active insulin receptor after it has been internalized by endocytosis. PTP1B can also be released by calpain-mediated cleavage. However, it seems that PTP1B mainly acts rather late in insulin receptor signaling and affects the duration of signaling. In humans, a polymorphism in the gene for 71 PTP1B (PTPN1) appears to correlate with insulin resistance . In addition, it is now clear that PTP1B also regulates other receptor PTKs in a similar manner and that at least some of the functions of PTP1B are shared with the closely related TCPTP (PTPN2). It is also clear that several other PTPs participate in the dephosphorylation of the insulin receptor both before and early after insulin 72 binding . Another PTP with a regulatory role in overall metabolism is the SH2 domain-containing phosphatase SHP2, the brain-specific deletion of which in mice 73 leads to a striking obesity due to decreased catabolism and body temperature . This role of SHP2 in central control of metabolism is presumably related to its function in leptin and STAT3 signaling. 2.4. PTPs in Cardiovascular and Neurological Diseases Endothelial cells express several transmembrane PTPs, which participate in the 13 regulation of adhesion and intercellular junctions between vascular wall cells , 74 and the cadherin–catenin–desmoplakin complexes of tight junctions , including PTPκ (PTPRK), PTPµ (PTPRM), GLEPP1 (PTPRO), and DEP1 (PTPRJ). At least the first two of these are regulated through homophilic interactions during 75-77 cell–cell contact and they dephosphorylate cadherin-associated proteins in endothelial cells that line blood vessels. Several knockout mice have demon-/strated these PTPs have an important function in vascular biology: PTPµ mice 78 -/have defective dilation responses in their mesenteric arteries , while GLEPP1 79 mice had hypertension due to abnormal podocyte development in their kidneys . Since immune and inflammatory responses to local bacterial infections or trauma require transmigration of leukocytes from the blood into the surrounding tissues, it is likely that these and other PTPs that regulate cell–cell junctions between endothelial cells also play important roles in the interactions between endothelial cells and immune cells. 4 80 Two phosphatases, LMPTP and MKP-1 , have been implicated in cardiac hypertrophy, the pathogenesis of which involves signaling from growth factor receptors and MAP kinases. The polymorphism in the gene for LMPTP (ACP1) 4 also correlates with Alzheimer’s disease , perhaps through an (indirect?) influence on tau phosphorylation. Finally, a case report on a severely autistic child found a chromosomal abnormality on 15q24 that included loss of the gene for

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PTP-MEG2 (PTPN9) in addition to one other gene . Our observations concern82 ing the role of PTP-MEG2 in secretory vesicle homeostasis and embryonic development (unpublished data), support the notion that PTP-MEG2 could play a causative role in development of autism. Furthermore, the critical roles of many PTPs in axonal guidance, synaptic morphology and flexibility, glutamate receptor regulation, MAP kinase regulation, learning, memory, motor control, and many other functions of the central nervous system, make it very likely that many of these PTPs are instrumental in neuropathological processes and may be considered as drug targets in the near future. 2.5. PTPs in Immunodeficiency and Autoimmunity An important discovery for our understanding of how important PTPs are for the proper function of the immune system was the finding that the motheaten mouse 83 is deficient in the SH2 domain-containing tyrosine phosphatase SHP1 . These mice have overly active macrophages and their lymphoid cells show enhanced responses to antigen stimulation. As a consequence, the mice develop a hyperin84 flammatory disease of multiple tissues, and die within days to weeks . Interestingly, the human gene for SHP1 (PTPN6) contains several genetic polymor85 phisms , but no associations have so far been reported with autoimmune disease. Nevertheless, loss of SHP1 has been observed in human disease, namely, in hematological malignancies and myelodysplastic syndrome (see above). Another human PTP gene, called PTPN22, which encodes the lymphoid tyrosine phosphatase LYP was recently found to associate with numerous major human autoimmune diseases. Our laboratory discovered that PTPN22 has a single-nucleotide polymorphism (C1858T), which leads to a substitution of argin86 ine for tryptophan at position 620 in the C terminus of LYP . This small change results in complete loss of binding of LYP to the SH3 domain of the inhibitory 86 Csk kinase and correlates with the development of type 1 diabetes , an autoimmune disease characterized by T cell-mediated destruction of the insulinproducing β-cells in the pancreas. Our finding has now been confirmed by nu87-91 92-94 95 merous studies , and extended to rheumatoid arthritis , juvenile arthritis , 94,96 97,98 systemic lupus erythematosus , Graves’ disease , and other autoimmune 99 diseases . Interestingly, multiple sclerosis, celiac disease, and inflammatory 100-102 bowel disease do not seem to associate with the polymorphism in PTPN22 . The molecular mechanisms by which LYP contributes to autoimmune disease seem to be related to our finding that the disease-causing allele of LYP is a gain103 of-function form, i.e. a more active phosphatase . This, in turn, likely compromises negative selection of autoreactive T cells in the thymus, leading to the emergence of such cells into the circulation of carriers of the disease-causing allele. Curiously, a main antigen for autoreactive T cells in the β-islets is also a PTP, namely, the receptor-like enzyme termed phogrin (PTPRN), which is lo104 cated on the insulin-containing secretory vesicles in β-cells .

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Abnormalities in another leukocyte-restricted PTP, the receptor-like CD45, 105 have also been reported in patients with systemic lupus erythematosus and a polymorphism that impairs alternative splicing of CD45 was reported to be as106 107 sociated with multiple sclerosis . This association is somewhat controversial , as might be expected in polygenic diseases with linkage disequilibrium and 108 complex interactions with other genetic and environmental factors . However, autoimmune disease also develops in mice transgenic for a gain-of-function 109 E613R mutant CD45 , while loss of CD45 leads to severe combined immune 110,111 deficiency . Together, these findings are probably explained by the important 19 role that CD45 plays in the regulation of Src family PTKs and lymphocyte ac11 tivation . Accordingly, CD45 is a potential drug target for the treatment of autoimmune diseases, as well as for transplant rejection. Allelic polymorphism in a third PTP, LMPTP (ACP1), correlates with in4,112 creased IgE levels and atopy . In this case, the allelic variation affects the alternative splicing of the biochemically distinct isoforms A and B, as well as the total levels of LMPTP expression. As with LYP and CD45, significant human disease seems to arise from relatively small alterations in PTP function, attesting to the delicate balances that govern the accuracy of our immune system. 2.6. PTPs As Tools for Immune Evasion 11

The key roles that many PTPs play in the immune response has been exploited by several pathogenic bacteria and viruses to subvert or evade the immune system. Perhaps the best example is the highly virulent bacterium that causes bubonic plague, Yersinia pestis, which uses a type III secretion system to directly inject a highly active PTP, called YopH, into the cytoplasm of immune cells, 113,114 including macrophages, T and B cells . Inside these cells YopH efficiently dephosphorylates key signaling proteins and thereby inhibits the initiation of an immune response. As a consequence, the bacterium can multiply in the lymph nodes of the host unopposed by immunity, thereby causing a rapidly fatal disease. We found that YopH very efficiently dephosphorylates the T cell antigen receptor-associated tyrosine kinase Lck at its positive regulatory site, Tyr-394, 115 resulting in a complete paralysis of signaling from this receptor . Virulent Salmonella species also use very similar strategies to inject host cells with the phosphatase SptP, which interferes with activation of MAP 116 kinases . Mycobacteria also secrete a tyrosine-specific PTP involved in viru117 lence . All these PTPs are potential drug targets for pathogen-specific treatments, as well as tools for mechanistic studies of PTP targets in signaling pathways in immune cells.

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3. PTPS AS DRUG TARGETS The growing number of human diseases discovered to be associated with PTP abnormalities (discussed above) has sparked an increasing interest in PTPs as 118-122 drug targets . Some work has already been done both in academic and pharmaceutical laboratories to develop inhibitors against a number of PTPs, notably 122,123 124 PTP1B for type II diabetes and obesity , MKP1 and CDC25 for cancer , and 125,126 YopH to combat Yersinia pestis in the hands of bioterrorists. Other already considered drug targets include PTPα, CD45, SHP2, PRL3, and LMPTP. I predict that the list will grow considerably in the next few years. In the drug development pipeline, PTPs are some five to seven years behind the PTKs, many inhibitors of which already are in advanced clinical trials or even on the market. This is mostly because the first PTP was identified molecu31 larly ten years after the first PTK, and most PTPs were cloned in the mid to late 1990s (several are still unpublished today!). In addition, enthusiasm was initially greatly dampened by the totally unfounded notion that PTPs were less specific than PTKs and that the structure of the active site of PTPs did not allow for generation of potent and selective small-molecule inhibitors. However, it is now becoming clear that PTPs indeed have unique non-redundant and important functions and a great deal of specificity in vivo. The question of how selective small-molecule inhibitors can be is not yet quite resolved, although many promising examples have been published. A stumbling block has been that phosphomimetics tend to be too hydrophilic for penetrating the plasma membrane appears to have been solved and the crystallization of many PTPs has revealed that the surface topology surrounding the catalytic pocket of each PTP has many unique features that can be utilized for rational structure-based design of highly selective inhibitors. The publication that truly kicked off the quest for PTP inhibitors with great 70 market potential was the characterization of the PTP1B knockout mouse , as discussed in §2.3. This mouse showed that PTP1B is a negative regulator of the insulin receptor and that, by extension, inhibition of PTP1B would improve insulin signaling in type II diabetes, having beneficial effects on both glucose balance and fatty acid metabolism. With the trend of increasing obesity in the Western world, the market for type II diabetes and obesity drugs is one of the largest known to the industry. The literature on PTP inhibitors is still relatively small, but a good number of pharmacophores have been published, from nonselective phosphate mimics like vanadate, to phosphotyrosine-like molecules, peptidomimetics, and the newest small-molecule inhibitors that were rationally designed or found through 118 high-throughput screening. Many of these molecules were recently reviewed .

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4. FUTURE PERSPECTIVES In my view, the best times are still ahead for the PTP field. So many PTPs have hardly been touched yet and the biology and function of so few are understood in any detail. However, the entire set of PTP genes in humans and mice is now known, and it is finally possible to venture into global studies of the “PTPome” and to directly address questions of specificity, redundancy, and expression of all PTPs in healthy and diseased tissues. This task is naturally complicated by many factors, including the capacity of many PTP genes to generate alternatively spliced products and the existence of single-nucleotide polymorphisms that subtly alter function or expression. In addition, numerous post-translational modifications and protein–protein interactions impact nearly every PTP, sometimes in unexpected ways. On the other hand, it seems likely that the rate of progress will be much accelerated by the many new emerging technologies, for example, in high-throughput screening, rapid content-based imaging, NMR127 based drug design , and large-scale proteomics. A critical step in the elucidation of PTP function is the identification of their physiological substrates, which likely will be enhanced by the development of mass spectrometry technologies to study the entire phosphoproteome of cells in a comprehensive, detailed, and quantitative manner. Genetic approaches and RNA interference already promote the discovery of physiological functions and will help address important questions of redundancy. All these issues are important for the full utilization of PTPs as drug targets and the treatment of many human diseases in which PTPs play regulatory and sometimes even dominant and causative roles. I predict that discoveries in the coming years will reveal that many more PTPs are involved in human disease and that they will need to be considered as potential drug targets. This will stimulate the efforts to design increasingly potent and selective PTP inhibitors with optimal drug-like properties, which also will drive better discovery research in PTP biology. At this point, I would caution against the notion that PTP inhibitors need to be monospecific. Although the true extent of redundancy between PTPs in vivo remains unclear, I interpret the existing information to mean that groups of related PTPs have at least partly overlapping functions and that even more distantly related PTPs can participate in the same biological process. Thus, the greatest drugs will likely be the “oligospecific” PTP inhibitors, i.e., those that inhibit a subgroup of two to five PTPs rather than a single PTP. Gleevec is a good example of this concept from the world of tyrosine kinase inhibitors. A major challenge in PTP inhibitor development is posed by the hydrophilic and multiply charged nature of the natural substrate for PTPs, which makes it challenging to design substrate-like inhibitors, i.e., phosphotyrosine mimetics, that are sufficiently hydrophobic to penetrate the plasma membrane. Fortunately, many cell-permeable lead compounds have recently been reported, and we believe that these, as well as many new ones, will serve as starting points for

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the development of efficient and selective PTP inhibitors with optimal pharmacological properties. An important lesson from the progress made in the past few years is the realization that although all PTPs have very similar catalytic cores, they differ very much in surface topology and charge distribution in the terrain that sur3 rounds the catalytic cleft . In cells these differences presumably reflect distinct preferences in substrate selection and protein–protein interactions between the PTP and multiple surface features of the substrates surrounding the incoming phosphotyrosine target residue. These same features can be utilized for the development of small-molecule inhibitors with a high degree of specificity. Although this notion has already validated by several published examples, I believe that the true potential of structure-based specificity and selectivity of PTP inhibitors still awaits future realization. Better PTP assays that use their physiological substrates, rather than artificial ones, are likely to be helpful in these efforts. In conclusions, I am convinced that the human PTPome contains numerous potential drug targets and that the next decade will see many successful efforts to develop specific PTP inhibitors for the treatment of human disease. I predict that the first PTP inhibitor on the market will greatly increase the enthusiasm for PTPs and will inspire more research and drug development in this field.

5. ACKNOWLEDGMENTS I wish to apologize to all colleagues whose papers I could not cite here due to space limitations. I thank Nunzio Bottini, Michael David, Jack Dixon, Rob Edwards, Gen-Sheng Feng, Adam Godzik, Andrei Osterman, Robert Rickert, Zhong-Yin Zhang, and many other colleagues and friends for many stimulating discussions about phosphatases. This work was supported by grants AI35603, AI48032, AI53114, AI53585, AI55741, AI55789, and CA96949 from the National Institutes of Health.

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6 THE T CELL-SPECIFIC ADAPTER PROTEIN FUNCTIONS AS A REGULATOR OF PERIPHERAL BUT NOT CENTRAL IMMUNOLOGICAL TOLERANCE Philip E. Lapinski, Jennifer N. MacGregor, Francesc Marti and Philip D. King

1. INTRODUCTION Signal transduction in T cells, as in other cell types, involves an interaction between catalytically active molecules (e.g., protein and lipid kinases and phos1 phatases) and adapter proteins that lack catalytic activity . Typically, adapter proteins participate in signal transduction by juxtaposing catalytically active molecules to their substrates. Physical interaction with catalytically active molecules is mediated either by modular binding domains (e.g., Src-homology-2 (SH2) or SH3 domain) or by defined peptide motifs present in the adapter. Adapter proteins can be broadly classified into two groups depending upon their location within the cell, i.e., intracellular or transmembrane. In T cells, examples of intracellular adapter proteins include growth receptor-bound protein-2 (Grb-2), Grb-2-related adapter protein-2 (GRAP-2), SH2-containing leu2-5 kocyte protein of 76 kD (SLP-76) and Fyn-binding protein (FYB) . Transmembrane adapters in T cells include the linker for activated T cells (LAT) and 6-8 Lck-interacting transmembrane protein (LIME) . Testimony to their important role in T cell signal transduction, knockout, or knock-in mutations of adapter proteins in mice frequently result in abnormalities of T cell development and/or peripheral T cell function. Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA

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T cell-specific adapter protein (TSAd) is a relatively recently described 9 adapter protein that has received less attention than most other T cell adapters . However, accumulating evidence indicates that TSAd is critical for normal T cell function and that deficient expression of TSAd in T cells results in a breakdown of immunological tolerance resulting in the development of autoimmunity. In this report we provide and discuss recent data that clarifies the cellular mechanism responsible for the development of autoimmune disease in TSAddeficient mice. In addition, we discuss recent data relating to the precise role of TSAd in T cell signal transduction that allows an understanding of the function of this molecule as a regulator of immunological tolerance in a molecular context.

2. TSAd TSAd was first identified in humans as part of a subtractive cDNA library 10 screen of activated peripheral blood CD8+ T cells . Subsequently, TSAd was identified in mouse as an interacting partner of different bait proteins in yeast11-13 hybrid screens (see below) . The structure of TSAd is depicted in Figure 1. TSAd resembles a typical intracellular adapter protein that lacks any recognizable catalytic domain but instead contains a modular binding domain, in this case an SH2 domain, positioned at the center of the linear sequence. The SH2 domain of TSAd permits binding to phosphorylated tyrosine residues present in protein ligands (see below). Aside from the SH2 domain, no other protein or lipid interaction domains have been identified from the primary sequence. However, the carboxyl region of TSAd contains a conserved proline-rich stretch and four conserved tyrosine residues that could potentially be bound by the SH3 14 and SH2 domains respectively of other signaling proteins .

Y292

Mouse

Y317 Y302

Y275 SH2

Y290

Y260

Human

Y280

Y305

Figure 1. T cell-specific adapter protein (TSAd). Shown is the location of the SH2 domain, conserved proline-rich stretch (represented as black bar) and conserved tyrosine residues.

Northern blot analyses show that TSAd is expressed predominantly in T 10-12 lineage and NK cells . In addition, TSAd appears to be expressed at low levels 13,15 in epithelial cell lines and in primary endothelial cells . Within the T cell line-

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age, TSAd is not expressed in CD4–CD8– double-negative (DN) thymocytes. It is first detected at the CD4+CD8+ (DP) stage of thymocyte development where 11 it is strongly and constitutively expressed . Thereafter, TSAd is expressed at lower levels in CD4+ and CD8+ single-positive (SP) thymocytes and peripheral T cells. In human peripheral T cells, expression of TSAd is substantially upregulated in response to stimulation with T cell antigen receptor (TCR) and 16,17 CD28 costimulatory receptor antibodies . Likewise, expression of TSAd is increased in murine peripheral T cells in response to activation although to a 11 lesser degree compared to human peripheral T cells .

3. TSAd REGULATION OF T CELL ACTIVATION Initial investigations aimed at determining if TSAd functions as a positive- or a negative-regulator of T cell activation yielded conflicting results. In one study, transfection of TSAd into the Jurkat human T leukemia cell line inhibited TCRinduced activation of the promoter for the T cell cytokine, IL-2, suggesting a 16 negative-signaling role for this adapter . However, in another study, TCRinduced IL-2 promoter activation in Jurkat was blocked as a consequence of anti-sense knockdown of endogenous TSAd expression, indicating a role for 12 TSAd as a positive-regulator . It seems likely that inhibition of TCR signaling resulting from transfection of TSAd into Jurkat is an artifact of strong overexpression. In subsequent studies, it was shown that when TSAd was only mildly overexpressed in Jurkat, TCR-mediated activation of the IL-2 promoter was 18 augmented, consistent with anti-sense inhibition findings . Moreover, that TSAd acts a positive-regulator rather than a negative-regulator of T cell activa11 tion was confirmed following the production of TSAd-deficient mice . TSAd-deficient mice are viable and for the first few months of life do not show any overt signs of disease. Apart from modest increases in thymocyte numbers and numbers of splenic T cells, ratios of different T cell subsets to one another are unaltered in both organs. However, peripheral T cells from TSAddeficient mice synthesize reduced quantities of several key cytokines, including IL-2, interferon-γ (IFN-γ) and IL-4 following TCR engagement. This has been demonstrated in response to stimulation with TCR and CD28 antibodies in vitro 11,19 and in response to superantigen immunization in vivo . Therefore, at least with regards cytokine synthesis, these findings show unequivocally that TSAd acts as a positive-regulator of TCR signal transduction in peripheral T cells.

4. TSAD AND AUTOIMMUNE DISEASE With increasing age, TSAd-deficient mice show features of systemic lupus-like 19 autoimmune disease . Serum concentrations of all immunoglobulin subclasses become elevated and by 9 months of age autoantibodies against self molecules such as single-stranded (ss-) and double-stranded (ds-) DNA, cardiolipin, and

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self-IgG can be detected. Eventually, immune complexes become deposited in kidneys, leading to glomerulonephritis and kidney dysfunction. Other features of the disease include the accumulation of large numbers of activated T cells in spleen, a breakdown of normal lymphoid architecture, and leukocytic infiltration into non-lymphoid organs, particularly lung. In addition to the spontaneous disease observed in older TSAd-deficient mice, young, 2–3 month old, TSAddeficient mice show increased susceptibility to lupus-like disease induced by immunization with the hydrocarbon, pristane. This is most clearly manifested as an increase in the titers of ss- and ds-DNA antibodies and in the severity of glomerulonephritis in TSAd-deficient compared to wild-type mice. Deficient expression of TSAd has also been linked to the development of autoimmune disease in humans. In this regard, polymorphic variants of the human TSAd gene have been described that differ in the number of GA repeats located 340 bp upstream of the transcription initiation site in the gene pro20 moter . In Norwegian cohorts, alleles that contain a fewer number of GA repeats show a statistically significant association with the autoimmune diseases, 21,22 multiple sclerosis and juvenile rheumatoid arthritis . Importantly, it has been demonstrated that these short alleles drive lower levels of TSAd expression compared to the longer alleles with larger numbers of GA repeats. These findings, therefore, suggest that in humans, as in mice, impaired expression of TSAd may predispose to the development of autoimmune disease.

5. TSAd AND T CELL TOLERANCE The relative restricted expression of TSAd to the T cell lineage and the acquisition of large numbers of activated T cells in older TSAd-deficient mice point to a breakdown of T cell tolerance as the primary cause of autoimmune disease. Theoretically, this could result from a failure of central or peripheral tolerance mechanisms or both. 5.1. Peripheral T Cell Tolerance An increased propensity of T cells to proliferate and/or resist apoptotic death in 23 response to antigen could lead to a failure of peripheral tolerance . As determined in vivo, TSAd-deficient T cells proliferate normally in response to TCR 19 engagement. By contrast, apoptotic death is impaired . This is best illustrated in the in vivo T cell response to superantigen (Figure 2). Following immunization of wild-type C57BL/6 mice with the superantigen, staphylococcal enterotoxin B (SEB), responding TCR-Vβ8 T cells become activated and expand initially but then decline in number as they are deleted from the peripheral T cell pool by 24 apoptosis . In TSAd-deficient C57BL/6 mice, TCR-Vβ8 T cells are initially activated to the same degree as in wild-type mice. However, the subsequent death response is blocked. Exactly which type of apoptotic death leads to

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Figure 2. Defective T cell death in TSAd-deficient mice. (A) Percentages of TCR Vβ6+ or TCR Vβ8+ T cells among total CD4+ T cells in venous blood from wild-type and TSAd-deficient mice (age 2 months) at the indicated times after immunization with SEB were determined by flow cytometry. Data are from eight mice in each group and are represented as means ±1 SE. At days 4 and 11 differences between wild-type and TSAd-deficient mice with respect to percentage of Vβ8+ T cells are statistically significant (both P < 0.005 calculated using the Student’s two sample t test). (B) CD69 activation marker expression on TCR Vβ6+ and Vβ8+ T cells from wild-type and TSAddeficient mice 24 hours after immunization with SEB was determined by flow cytometry. The percentages of Vβ6+ and Vβ8+ T cells that express CD69 are indicated in parentheses. Reproduced from the Journal of Experimental Medicine, 2003, 198, 809-821 by permission of the Rockefeller University Press.

the deletion of TCR-Vβ8 T cells in wild-type mice is controversial. Based upon the finding that the T cell death response is intact in mice that are deficient in expression of both Fas and TNF receptors (which constitute the main death receptors of T cells), the conclusion has been drawn that this type of death is independent of death receptors and is instead mediated by mitochondrial dysfunc25,26 tion . Balanced against this, others have reported that superantigen-induced T 27,28 cell death is impaired in Fas-deficient mice . Regardless of whether superanti-

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gen-induced death is death receptor- or mitochondrial-mediated, both forms of death are thought to guard against the development of autoimmune disease at least in part by promoting the demise of potentially autoreactive peripheral T cells that have escaped thymic censorship programs. Therefore, the finding of impaired superantigen-induced T cell death in TSAd-deficient T cells points to defective peripheral T cell death as at least a contributing factor to the development of autoimmune disease in these animals. 5.2. Central T Cell Tolerance Alterations in the efficiency of thymic positive and negative selection processes could both lead to a loss of central T tolerance that could promote autoimmune 23 disease . Impaired positive selection might result in the survival of developing T cells that express only the highest affinities for self-major histocompatability complex (MHC)-peptide. Without any alteration in the efficiency of negative selection, this could skew the peripheral T cell repertoire toward self reactivity. Impaired negative selection, on the other hand, would allow the emergence into the periphery of T cells with overtly high affinity for self MHC-peptide, which would also result in autoreactivity. To examine if TSAd plays a role in positive or negative selection of developing T cells, we used the well characterized H-Y TCR transgenic model that 29 has been used extensively to dissect thymic selection processes . The H-Y TCR b expresses an affinity for the MHC class I molecule, H-2K . In female C57BL/6 H-Y TCR transgenic mice, DP H-Y TCR-expressing thymocytes are positively b selected on H-2K and consequently develop into CD8 SP T cells. In contrast, in male C57BL/6 H-Y TCR transgenic mice, developing DP H-Y TCR-expressing b thymocytes are negatively selected on a complex of H-2K and the male-specific H-Y peptide and are thus removed from the repertoire. Therefore, by examining the ratio and number of different thymocyte subpopulations within female and male C57BL/6 H-Y TCR transgenic mice that express or do not express TSAd, information on the role of TSAd in either selection process can be obtained. To assess positive selection efficiency, the ratio of CD8 SP to DP thymocytes among H-Y TCR positive thymocytes was compared in female TSAd wild-type, heterozygote and TSAd-deficient mice (Table I, Figure 3). Should positive selection be impaired or augmented in the absence of TSAd, then this ratio should be reduced or increased, respectively. However, as shown in three independent experiments, the CD8 SP:DP ratio within the H-Y TCR-expressing populations was largely unaltered. Therefore, TSAd does not appear to play a significant role in positive selection. To examine negative selection, absolute numbers of H-Y TCR-expressing DP and SP thymocytes in male H-Y TCR transgenic thymi (and their ratio to DN cells) were compared with those in female H-Y TCR transgenic thymi (Table 1, Figure 3). In TSAd wild-type H-Y TCR transgenic male thymi, massive

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Table 1. Thymocyte development in TSAd-deficient H-Y TCR transgenic mice Thymocyte number X 10 6 T3.70hi Genotype (TSAd) Expt. 1 (4 wk) Female

Male

Expt. 2 (8 wk) Female

Male

Expt. 3 (6 wk) Female

Total

CD4-CD8DN

CD4+C8+ DP

CD8+ SP

CD4+ SP

+/-

55

11.7(61.9)

4.9(25.9)

1.9(10.1)

0.4(2.1)

-/-

104.5

16.3(46.2)

13.3(37.7)

5.0(14.2)

0.7(2.0)

+/+

11.0

5.6(88.9)

0.1(1.6)

0.3(4.8)

0.3(4.8)

+/-

21.5

10(90.2)

0.09(0.8)

0.3(2.7)

0.7(6.3)

-/-

12.5

7.3(8.6)

0.1(1.2)

0.4(4.7)

0.7(8.2)

+/-

142.5

24.8(39.9)

18.1(29.1)

18.3(29.4)

1.0(1.6)

-/-

128.5

22.7(43.1)

17.1(32.4)

11.8(22.4)

1.1(2.1)

+/+

18.5

15.1(93.5)

0.05(0.3)

0.4(2.5)

0.6(3.7)

+/-

17.0

13.0(94.0)

0.03(0.2)

0.7(5.1)

0.1(0.7)

-/-

20.5

15.7(92.6)

0.06(0.4)

0.9(5.3)

0.3(1.8)

+/+

116

21.6(43.0)

13.8(27.4)

13.6(27.0)

1.3(2.6)

+/-

143

23.9(36.7)

21.3(32.7)

18.9(29.0)

1.0(1.6)

-/-

149

26.5(41.3)

20.3(31.7)

16.1(25.2)

1.2(1.9)

Thymocytes from littermate H-Y TCR transgenic TSAd wild-type, heterozygote and TSAddeficient mice on H-2Kb positively-selecting (female) and negatively-selecting (male) backgrounds were stained with labeled-CD4, CD8 and H-Y TCR clonotypic (T3.70) antibodies and analyzed by flow cytometry. Shown are the total numbers of thymocytes within each of the indicated populations. The percentages of DN, DP and SP cells within the T3.70 population are shown in parentheses. In female mice, the ratio of T3.70hi DP to CD8+ SP cells is similar in the presence and absence of TSAd showing that positive selection is largely unaffected by TSAddeficiency in this model. Similarly, in male mice, the massive deletion of T3.70hi DP and CD8+ SP cells (compared to female mice) is observed to the same degree in the presence and absence of TSAd indicating that TSAd is not required for thymic negative selection.

deletion (negative selection) of H-Y TCR transgenic DP and consequently SP thymocytes was noted. Similarly, massive deletion of H-Y TCR transgenic DP and SP thymocytes was noted in the thymi of TSAd-heterozygote and TSAd-

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deficient mice. These results, therefore, show clearly that TSAd is not required for thymic negative selection. H-Y TCR Females TSAd (-/-)

100 101

10 4

10 2

25.9

10 0

10.1 101

102

10 3

10 4

10 0 10 1 10 2 10 3

103

104

TSAd (+/-)

10 0

37.7

14.2 10 1

102

103

10 4

10 0 10 1 10 2 10 3 10 4

10 0

TSAd (+/-) 0.8

2.7 101

102

10 3

10 4

10 0 10 1 10 2 10 3 10 4

H-Y TCR Males

CD4

10 0

TSAd (-/-)

1.2

4.7 10 1

102

103

10 4

CD8

Figure 3. Thymocyte development in TSAd-deficient H-Y TCR transgenic mice. Shown are CD4 versus CD8 plots of T3.70hi cells from Expt. 1 of Table 1. Percentages of DP and CD8+ SP cells are indicated.

Another important component of central tolerance is generation of 30,31 CD4+CD25+ T regulatory cells (Tregs) in the thymus . Tregs have the potential to block the expansion of autoreactive T cells in the periphery. Therefore, defective generation or activity of Tregs in TSAd-deficient mice could contribute to the development of autoimmune disease. However, preliminary analyses indicate that Tregs are present in normal numbers in TSAd-deficient mice and are fully functional as suppressors of T cell proliferation (unpublished observations). This finding is consistent with the idea that Tregs are generated as a byproduct of negative selection, which is unperturbed in TSAd-deficient mice (see above).

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IL-2 c-myc CLIC4 IFN-gamma BAP29 Hsp60

0

1

2

3

4

5

WT/KO

Figure 4. Pro-apoptotic genes regulated by TSAd in T cells. CD4+ peripheral T cells from wild-type (WT) and TSAd-deficient (KO) mice were stimulated for 20 hours with TCR and CD28 antibodies. Shown is the relative ratio of expression of six identified pro-apoptotic genes that are positively regulated by TSAd.

6. MOLECULAR BASIS FOR IMPAIRED DEATH OF PERIPHERAL T CELLS IN TSAd-DEFICIENT MICE As an inducer of peripheral T cell death, TSAd could participate in signaling pathways that either positively regulate the transcription of pro-apoptotic proteins or negatively regulate the transcription of anti-apoptotic proteins. To identify such genes, gene expression profiles of resting and TCR/CD28-stimulated CD4+ peripheral T cells from wild-type and TSAd-deficient mice were com19 pared . With the use of Affymetrix gene chips containing approximately 12,000 probe sets, representing all described murine genes, approximately 100 genes were identified to be positively regulated by TSAd (no negatively regulated genes were identified). Of these, six have previously been shown to function as pro-apoptotic genes (Figure 4). These include the cytokine, IFN-γ; the transcription factor, c-myc; the chloride channel, CLIC-4; the death receptor associated protein, BAP29; and the molecular chaperone, HSP60. Also contained within this group is IL-2. Although classically thought to function as a T cell growth 32 33-35 factor , IL-2 is known to promote T cell death in vivo . Indeed, amongst this group of genes, it is transcription of IL-2 that is most affected by TSAd deficiency, indicating that impaired synthesis of IL-2 may be the predominant factor accounting for the development of autoimmune disease in TSAd-deficient mice.

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7. ROLE OF TSAD IN T CELL SIGNAL TRANSDUCTION Available evidence indicates that TSAd regulates IL-2 synthesis in multiple different ways, acting in both the nucleus and cytoplasm. Each of these functions is reviewed briefly below. 7.1. Nuclear Function of TSAd 18

In Jurkat T cells a substantial fraction of TSAd is found in the nucleus . Entry of TSAd into the nucleus is not a result of passive diffusion but is an active process that depends upon the integrity of the TSAd SH2 domain. No obvious nuclear localization sequence is discernible within the TSAd primary amino-acid sequence. Instead, the SH2 domain is thought to mediate nuclear entry through recognition of a tyrosine-phosphorylated nuclear-importing protein ligand. This active nuclear import is strongly suggestive of a specific function for TSAd within this compartment. The exact role of TSAd within the nucleus remains to be determined. However, it is of note that when fused to a heterologous DNAbinding domain, TSAd shows potent transcription-activating activity both in 18 yeast and in human and murine T cell lines . This suggests that TSAd may participate in the process of gene transcription in T cells, although transcription activation does not appear to be an intrinsic property of TSAd. Similar to nuclear import, transcription activation requires SH2 domain recognition of a tyrosine-phosphorylated protein ligand. In this sense, TSAd may function as a transcription-related adapter protein acting to position molecule(s) with intrinsic transcription-activating ability to gene promoters though dual recognition of those molecules(s) and DNA-binding protein(s). Recently, the ligand of the TSAd SH2 domain that is involved in TSAd nuclear import has been identified as the highly conserved Valosin-Containing 36 Protein (VCP), also known as CDC48 in yeast . Interestingly, VCP/CDC48 has been implicated in the nuclear import of transcription factors beforehand, 37-39 namely, SPT23 and Mga2 in yeast and NFκB in mammalian cells . SH2 domain-mediated binding of TSAd to VCP is dependent upon tyrosine residue 805 of VCP, which has long been recognized to be inducibly phosphorylated in T 40,41 cells in response to TCR ligation . This suggests that TSAd entry into the nucleus may not be constitutive but may be regulated (induced) by TCR engagement through phosphorylation of VCP tyrosine 805. However, from a structural standpoint it is unlikely that TSAd binds directly to tyrosine 805, which is the penultimate residue of the VCP protein. Instead, phosphorylation of tyrosine 805 may allow some other covalent modification of VCP that promotes interaction 36 with TSAd .

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7.2. Cytoplasmic Functions of TSAd Within the cytoplasm of peripheral T cells, TSAd functions in signaling pathways that result in the activation of AP-1 and NFAT transcription factors that 42,43 induce the transcription of IL-2 gene and other genes (unpublished observations). TSAd participates in AP-1 activation in part through its ability to promote conversion of the small G-protein, Ras, from its inactivated GDP-bound form to its activated GTP-bound form. Activated Ras triggers a MAP-kinase signal cascade, resulting in phosphorylation of AP-1 components in the nu42,44,45 . Ras is known to be directly regulated by Ras guanine nucleotide excleus change factors (RasGEFs), such as SOS and RasGRP1, and by such Ras GTPase-activating proteins as RasGAP1. RasGEFs activate Ras by ejecting GDP from the Ras guanine nucleotide-binding pocket, allowing Ras to bind additional GTP molecules. By contrast, RasGAPs inactivate Ras by greatly augmenting the ability of Ras to hydrolyze bound GTP to GDP. The role of TSAd in Ras activation in peripheral T cells appears to be twofold. First, TSAd is able to act as an inhibitor of RasGAP. In response to TCR engagement, TSAd becomes phosphorylated on carboxyl-region tyrosine residues, allowing physical interaction with RasGAP SH2 domains. This physical interaction then inhibits RasGAP activity probably through the induction of conformational changes. Second, TSAd promotes the recruitment of RasGEFs to the T cell plasma membrane, which consequently become juxtaposed to Ras, allowing effective GDP– GTP exchange. The mechanism by which TSAd controls RasGEF membrane recruitment has recently been determined. In this regard, TSAd appears to have a major role in TCR-induced activation of the LCK Src-family protein tyrosine kinase at the 46 outset of TCR signaling (unpublished observations). In TSAd-deficient peripheral T cells, LCK is only poorly activated by the TCR, resulting in weak activation of the ZAP-70 Syk-family PTK and reduced tyrosine phosphorylation of the 7 LAT adapter protein . Phosphorylation of LAT normally leads to interaction with phospholipase C-γ1 (PLC-γ1), which subsequently becomes activated, leading to phosphoinositide hydrolysis and membrane recruitment of RasGRP1. In addition, the Grb-2 adapter protein interacts with tyrosine-phosphorylated LAT and in so doing recruits SOS to the membrane. Therefore, impaired activation of LCK in TSAd-deficient T cells ultimately accounts for impaired recruitment of both RasGEFs. Furthermore, since PLC-γ1 triggers intracellular calcium fluxes, which results in NFAT nuclear mobilization, then impaired LCK activa43 tion in TSAd-deficient T cells also explains blocked activation of this pathway . During TCR signaling, we have found that TSAd activates LCK by a process that involves physical interaction between the two proteins. Indeed, murine TSAd was originally cloned as an LCK-binding protein in a yeast hybrid system, although the functional significance of this interaction was not appreciated 12 at the time . In overexpression studies performed in cell lines, TSAd was shown 16 to inhibit LCK kinase activity . However, as discussed above, overexpression of

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TSAd blocks TCR-induced IL-2 secretion, indicating that the finding of LCK inhibition may also be an overexpression artifact. As revealed through the study of TSAd-deficient mice, at physiological levels of TSAd expression TSAd is very much required for LCK activation. Aside from LCK, murine TSAd has also been cloned as an interacting partner of the Tec-family kinase, Itk, and the Mekk2 kinase in yeast-hybrid sys11,13,47 . Hitherto, physical interaction between endogenously expressed protems teins in primary T cells has not been demonstrated and the functional significance of these interactions remains obscure. In an epithelial cell line, physical interaction of TSAd with Mekk2 has been shown to be necessary for 47 activation of Mekk2 and the downstream ERK5 kinase . However, T cells from Mekk2-deficient mice synthesize increased amounts of IL-2 in response to TCR 48 stimulation . Therefore, if TSAd were to play a similar role in the activation of Mekk2 in T cells, then this is counter to the finding that TSAd is necessary for proper induction of IL-2.

8. CONCLUSIONS In this chapter we have discussed both new and published data that show that the TSAd adapter protein regulates peripheral but not central T cell tolerance. As a regulator of peripheral tolerance, TSAd appears to function in both the cytoplasm and nucleus of T cells to regulate synthesis of IL-2 and, hence, apoptotic death. TSAd is not required for thymic selection processes despite the fact that TSAd is well expressed in DP thymocytes where such processes take place. Recent data show that several of the signaling pathways regulated by TSAd in peripheral T cells are not similarly regulated by TSAd in thymocytes, consistent with its lack of influence upon thymic selection. The basis for this uncoupling of TSAd in thymocytes remains to be determined.

9. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant AI050699 awarded to PDK.

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36. F. Marti, and P. D. King, The p95-100 kDa ligand of the T cell-specific adaptor (TSAd) protein Src-homology-2 (SH2) domain implicated in TSAd nuclear import is p97 Valosin-containing protein (VCP), Immunol Lett 97, 235–243 (2005). 37. R. M. Dai, E. Chen, D. L. Longo, C. M. Gorbea, and C. C. Li, Involvement of valosin-containing protein, an ATPase Co-purified with IκBα and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IκBα, J Biol Chem 273, 3562– 3573 (1998). 38. M. Rape, T. Hoppe, I. Gorr, M. Kalocay, H. Richly, and S. Jentsch, Mobilization of UFD1/NPL4 , a ubiqprocessed, membrane-tethered SPT23 transcription factor by CDC48 uitin-selective chaperone, Cell 107, 667–677 (2001). 39. N. Shcherbik, T. Zoladek, J. T. Nickels, and D. S. Haines, Rsp5p is required for ER bound Mga2p120 polyubiquitination and release of the processed/tethered transactivator Mga2p90, Curr Biol 13, 1227–1233 (2003). 40. M. Egerton, O. R. Ashe, D. Chen, B. J. Druker, W. H. Burgess, and L. E. Samelson, VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation, EMBO J 11, 3533–3540 (1992). 41. M. Egerton, and L. E. Samelson, Biochemical characterization of valosin-containing protein, a protein tyrosine kinase substrate in hematopoietic cells, J Biol Chem 269, 11435–11441 (1994). 42. C. Dong, R. J. Davis, and R. A. Flavell, MAP kinases in the immune response, Annu Rev Immunol 20, 55–72 (2002). 43. P. G. Hogan, L. Chen, J. Nardone, and A. Rao, Transcriptional regulation by calcium, calcineurin, and NFAT, Genes Dev 17, 2205–2232 (2003). 44. E. Genot, and D. A. Cantrell, Ras regulation and function in lymphocytes, Curr Opin Immunol 12, 289–294 (2000). 45. D. A. Cantrell, GTPases and T cell activation, Immunol Rev 192, 122–130 (2003). 46. E. H. Palacios, and A. Weiss, Function of the Src-family kinases, Lck and Fyn, in Tcell development and activation, Oncogene 23, 7990–8000 (2004). 47. W. Sun, X. Wei, K. Kesavan, T. P. Garrington, R. Fan, J. Mei, S. M. Anderson, E. W. Gelfand, and G. L. Johnson, MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src, Mol Cell Biol 23, 2298–2308 (2003). 48. Z. Guo, G. Clydesdale, J. Cheng, K. Kim, L. Gan, D. J. McConkey, S. E. Ullrich, Y. Zhuang, and B. Su, Disruption of Mekk2 in mice reveals an unexpected role for MEKK2 in modulating T-cell receptor signal transduction, Mol Cell Biol 22, 5761– 5768 (2002).

7 PROXIMAL SIGNALS CONTROLLING B-CELL ANTIGEN RECEPTOR (BCR) MEDIATED NF-κB ACTIVATION Miguel E. Moreno-García1, Karen M. Sommer2, Ashok D. Bandaranayake3, and David J. Rawlings1,2,,3

1. INTRODUCTION The development and function of the immune system is dependent upon constitutive, or antigen-dependent, triggering of surface immunoreceptors, including the T cell antigen receptor (TCR) and B cell antigen receptor (BCR) on T or B lymphocytes, respectively. Activation of these receptors triggers a variety of intracellular signaling cascades that control and propagate immunological processes. BCR-dependent activation of Nuclear Factor-κB (NF-κB) represents one of the most important signaling events induced in B lymphocytes. In these cells, NF-κB is involved in a variety of immune cell functions — including prolifera1 tion, survival, differentiation, and immunoglobulin class switching . The NF-κB family of transcription factors consists of five members — NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and c-Rel — that homo- and heterodimerize. In unstimulated lymphocytes, IκBs (a family that includes IκBα, IκBβ, and IκBε) bind NF-κB dimers, preventing their translocation into the nucleus (IκBβ), or altering their dynamic partitioning between the nucleus and cytosol to favor cytosolic localization (IκBα and ε). Importantly, NF-κB activation by surface receptors is tightly regulated by the IKK complex, comprised of two catalytic kinase subunits, IKKα and IKKβ, and one regulatory subunit, 2 IKKγ (NEMO) .

1

Departments of Pediatrics and 3Immunology, University of Washington School of Medicine, Seattle, WA 98195; 2Section of Immunology, Children’s Hospital and Regional Medical Center, 307 Westlake Ave N. Suite 300, Seattle, WA 98109, USA.

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There are two known pathways by which cells stimulate the release of NFκB from its IκB tether in the cytosol: the canonical and non-canonical path3 ways . The non-canonical pathway is not stimulated by BCR engagement, and is not discussed in this review. In the canonical pathway, induced by BCR stimulation, downstream NF-κB activation requires phosphorylation of IκBs bound to NF-κB dimers via the IKK complex. IKK-dependent phosphorylation promotes IκB ubiquitination and subsequent degradation by the 26S proteasome. This 4 releases NF-κB dimers (predominantly p50/c-Rel and p50/p65) to the nucleus, 2 where they activate transcription of specific gene programs . Despite the considerable knowledge of these highly conserved events required for NF-κB activation as well as downstream gene programs, knowledge of specific upstream signaling events that initiate the conserved IKK/IκB/NF-κB pathway is incomplete. In this review, we describe the signaling pathways that lead to NF-κB activation in B lymphocytes, starting with the initial signals triggered by the BCR, and followed by the sequential events that conclude with NF-κB activation. We will focus predominantly on new evidence identifying the mechanism(s) that link(s) specific protein kinase C (PKC) isoforms and the adaptor protein CARMA1 in initiation of NF-κB activation.

2. BCR-INDUCED PROXIMAL SIGNALS The BCR is a complex of a transmembrane immunoglobulin molecule (commonly of the IgM or IgD isotype) that is non-covalently associated with a single disulfide-bound CD79a/CD79b (Igα/Igβ) heterodimer. While surface immunoglobulin interacts with antigens, the co-associated Igα/Igβ heterodimer is responsible for transducing this extracellular interaction into intracellular signaling 5 pathways . Each Igα or Igβ molecule contains a conserved domain known as an ITAM (immunoreceptor tyrosine-based activation motif) in the intracellular portion of these proteins. The ITAM is comprised of six conserved amino acids and includes two tyrosine residues that are phosphorylated after BCR crosslink6 ing . Upon BCR interaction with its specific antigen, or crosslinking with agonist antibodies, one of the earliest signaling events is the phosphorylation of the tyrosine residues of the Igα/Igβ ITAMs (Figure 1A). Initial ITAM phosphorylation is considered to be dependent on the function of the Src family of protein 7 tyrosine kinases (Src-PTK), including Lyn, Fyn, Lck and Blk . This process is facilitated by the translocation of the BCR into semi-rigid lipid structures called

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Figure 1. Schematic model illustrating the main signaling pathways leading to PKCβ activation in B lymphocytes stimulated via the BCR. (A) Antigen recognition induces BCR raft recruitment and CD45-dependent dephosphorylation of the inhibitory Y508 residue in Lyn (and other Src-PTKs), which induces Lyn activation. (B) Lyn phosphorylates the Igα/Igβ ITAMs and tyrosine residues within the intracellular domain of CD19. SH2 domains of Syk kinase and PI3K are required for their recruitment to the phosphorylated BCR ITAMs or CD19, respectively. (C) PI3K phosphorylates PtdIns-4,5-P2 to generate PtdIns-3, 4,5-P3 that recruits Btk via its PH domain. Full Btk activation requires Lyn-dependent Y551 phosphorylation and Y223 autophosphorylation. Btk also associates with and recruits PIP5K to the plasma membrane, promoting further PtdIns-4, 5-P2 generation (not shown). (D) Syk phosphorylates the adaptor BLNK and promotes BLNK recruitment to the plasma membrane. (E) BLNK recruits PLCγ2, which is then phosphorylated by Btk at residues Y753 and Y759. PLCγ2 hydrolyzes PtdIns-4,5-P2 and generates DAG and IP3. IP3 induces Ca2+ release from ER. DAG and Ca2+ are required for classical PKC activation.

lipid rafts. The Src-PTKs undergo posttranslational acylation, which promotes their constitutive association with lipid rafts. Movement of the antigen-engaged BCR into rafts places the BCR within close proximity to Src-PTK proteins, fa8 voring Igα/Igβ phosphorylation . The phosphatase CD45 (B220) and the tyrosine kinase Csk are important co-regulators of Src-PTK activation, and play an opposing role in modulating the phosphorylation and dephosphorylation balance of a regulatory C-terminal tyrosine residue (Y508 in Lyn) in the catalytic region 9, 10 of the Src-PTKs . Phosphorylation of this regulatory site maintains the SrcPTKs in their catalytically inactive state. Upon BCR crosslinking, CD45 pro-

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motes Src-PTK activation by dephosphorylating this inhibitory tyrosine, thereby 10 enhancing Src-PTK-dependent ITAM phosphorylation . Phosphorylated ITAMs serve as scaffolding domains that recruit specific SH2-domain-containing proteins to the BCR signalosome, most notably includ11 ing the PTK Syk . Syk belongs to the Syk/ZAP-70 kinase family, characterized by the presence of two tandem SH2 domains in the N terminus that allow Syk recruitment to the BCR. Activation of Syk is induced upon recruitment of Syk to the phosphorylated Igα/Igβ ITAMs, and is further enhanced by transphos11 phorylation and autophosphorylation events (Figure 1B).

3. SIGNALING PATHWAYS LEADING TO PKC ACTIVATION Following Src-PTK and Syk recruitment and activation, transmission of the BCR signal to distal signaling pathways relies on a cascade of additional tyrosine phosphorylation events triggered by these PTKs. Activation of Lyn and Syk in B cells precedes the activation of another important kinase, Bruton’s tyrosine 12 kinase (Btk) . Btk belongs to the Tec family of tyrosine kinases that also includes the Tec, Itk, Txk, and Bmx kinases. Structurally, Btk contains SH1 (kinase), SH2, and SH3 domains, and family-specific Tec homology (TH) and Pleckstrin homology (PH) domains. Activation of Btk involves both its recruit13 ment to the plasma membrane and its transphosphorylation (Figure 1C) . Recruitment of Btk to the plasma membrane is mediated by the interaction of its PH domain with the phospholipid phosphatidylinositol-3,4,5-trisphosphate 14 (PtdIns-3,4,5-P3) . The importance of this interaction in B cell activation was revealed in XID mice, which have a point mutation in the PH domain of Btk that eliminates binding to PtdIns-3,4,5-P3. In XID mice, Btk translocation is ablated, 15, 16 resulting in severe B cell activation defects . PtdIns-3,4,5-P3 is generated by phosphatidylinositol-3 kinase (PI3K) phosphorylation of PtdIns-4,5-P2 in position 3 of the inositol ring. Activation of PI3K occurs primarily as a consequence of its inducible association with the phosphorylated intra-cytoplasmic region of 17 the B cell surface co-receptor CD19 . Src-PTKs, including Lyn, phosphorylate 18 CD19 , promoting the recruitment of PI3K and its lipid-kinase activity; and placing PI3K in proximity with its phospholipid substrate. Membrane recruitment of Btk promotes its transphosphorylation by Src-PTKs at tyrosine Y551, followed by Btk autophosphorylation at the Y223 residue, leading to enhanced 19, 20 kinase activity (Figure 1C). Propagation of Src-PTK, Syk, PI3K, and Btk signals is also critically dependent upon phosphorylation and plasma membrane recruitment of the adaptor 21, 22 protein BLNK (also known as SLP-65 or BASH) (Figure 1D) . Structurally, BLNK contains an SH2 and a polyproline domain, several N-terminal tyrosine phosphorylation sites that may interact with SH2 domains of other proteins, and an N-terminal leucine zipper motif. The recruitment of BLNK to the plasma membrane has been attributed to both the BLNK SH2 domain (through interac-

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tion with a non-ITAM phospho-tyrosine of Igα subunit) , and its leucine zipper 24 motif . While initial studies suggested that BLNK plasma membrane transloca23 tion is dependent on Igα expression , more recent data strongly support the model that BLNK is predominantly recruited through a coiled–coil interaction 24 with an undetermined interacting protein . BLNK acts as a “master” adaptor for 25 multiple intracellular molecules including Grb2, Vav, and Nck . Most notably, tyrosine phosphorylation of BLNK, likely by Syk, also plays a crucial, non26,27 redundant role in recruitment of PLCγ2 to the plasma membrane . Through this mechanism, PLCγ2 is brought in close proximity to its activators. In vitro studies suggested that Src-PTKs, Syk, and Btk each might be activating kinases for PLCγ2. However, recent data clearly demonstrate a non-redundant role for Btk in phosphorylation of residues Y753 and Y759 (within the SH2-SH3 linker of PLCγ2), the critical regulatory phosphorylation sites mediating activation of 28 PLCγ2 (Figure 1E). Activated PLCγ2 hydrolyzes PtdIns-4,5-P2, present at the cytoplasmic face of the plasma membrane, to generate two important second messengers: diacylglycerol (DAG) and inositol-1,4,5-P3 (IP3). IP3 binds to the 2+ IP3 receptors on the endoplasmic reticulum (ER), releasing ER Ca stores. Besides catalyzing its activation via direct phosphorylation, Btk plays a second role in PLCγ2 regulation. Btk membrane recruitment also leads to co29 recruitment of associated PIP5K . Here, PIP5K encounters and phosphorylates its substrate, PtdIns-4-P, a limiting precursor of PtdIns-4,5-P2. This adaptor function of Btk facilitates continued availability of PtdIns-4,5-P2, the substrate for both PLCγ2 and PI3K. Together, these combined activities maintain produc2+ 30 tion of PtdIns-3,4,5-P3 and IP3 and, in turn, sustained Ca signaling in B cells (Figure 1C). Of note, BCR engagement also promotes additional important signals, including the Shc/Grb2/Sos/Ras signaling cascades leading to ERK1/2 activation, the Rac/Cdc42/Vav interactions that facilitate JNK1/2 and P38 activation and cytoskeleton rearrangement, and the PI3K-dependent Akt pathway and its crucial role in cell survival and other signals (reviewed in [7] and [31]). Due to the specific focus of this review, these signaling cascades are not discussed in detail here.

4. PKC ISOFORMS INVOLVED IN B CELL SIGNALING AND NF-κB ACTIVATION 2+

Together, generation of DAG and release of intracellular Ca triggers activation of classical PKC isoforms, including PKCβ. As detailed below, activation of PKCβ directly and specifically links BCR signaling to the IKK/NF-κB path32 way (Figure 1E). The PKC protein family is comprised of 11 known isoforms that are classi2+ fied by their differential requirement of Ca and/or DAG for their full activation. The family of classical PKCs, including the α, βΙ−βΙΙ, and γ isoforms, re-

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quires both DAG and Ca to be activated; whereas the novel PKCs, including δ, ε, θ, and η, require only DAG; and atypical PKCs, including ζ and τ/λ iso33 forms, require neither messenger . Most of these PKC isoforms are expressed in B lymphocytes, and the generation of mice deficient in individual PKC isoforms has revealed specific functions for these molecules in B cell signaling. For example, generation of PKCδ-deficient mice revealed that this isoform promotes B 34,35 cell peripheral tolerance and anergy . In contrast, PKCζ-deficient mice revealed a positive role for this isoform in B cell function, with a partial requirement for BCR-mediated proliferation and survival, and the humoral response of 36 B cells . PKCζ can directly phosphorylate p65 (RelA) to control NF-κB activation in embryonic fibroblasts and lung cells, where this isoform is highly ex37 pressed . However, neither PKCδ nor PKCζ induce B cell-specific effects through activation of proximal NF-κB pathways. Indeed, reduced ERK activation in PKCζ-deficient B cells may be sufficient to explain its partial phenotype 37 in B cells . Analysis of PKCβ knockout mice has demonstrated a critical role for this isoform in B cell development and function. PKCβ-/- mice have reduced numbers of splenic B cells, peritoneal B1 B cells, and bone marrow recirculating B 38,39 cells; and exhibit reduced T-independent humoral responses . Splenocytes from PKCβ-/- mice do not proliferate in response to BCR engagement, and are 38-40 highly sensitive to induction of apoptosis . Consistent with these findings, PKCβ-/- B cells exhibit a lack of BCR-dependent IκBα phosphorylation and degradation, IKK activation, and reduced NF-κB nuclear translocation. Interestingly, PKCβ deficiency leads to reduced BCR-dependent induction of the antiapoptotic Bcl-xL and A1 genes, but not the cell cycle progression gene cyclin39,40 D2 . These results suggest that the main role of PKCβ in B lymphocyte function is induction of NF-κB-dependent cell survival rather than proliferation.

5. A PKCβ-DEPENDENT MECHANISM PROMOTING NF-κB ACTIVATION Our laboratory has previously shown that loss of PKCβ activity, either through genetic knockout or pharmacological inhibition, disrupts BCR signalosome for39 mation and IKK translocation into the lipid raft fraction . These data indicate that PKCβ controls NF-κB activation through raft recruitment and activation of the IKK complex. The mechanism connecting PKCβ to activation of the IKK/NF–κB signaling complex is not completely understood. However, it is unlikely that the IKK complex is a direct substrate of PKCβ serine/threonine kinase activity in B cells. As the kinase activity of PKCβ is required for IKK recruitment, it is also unlikely that PKCβ merely acts as a scaffold for IKK. Therefore, it appears that another molecule(s) downstream of PKCβ recruits and activates IKK in the BCR–NF-κB pathway.

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Recently, three adaptor molecules have been shown to be crucial for NF-κB activation in both B and T cells. These molecules are Bcl-10 (also known as CIPER, c-E10, mE10, c-CARMEN, CLAP), MALT1 (paracaspase), and 41 CARMA1 (CARD11, Bimp3) . It was previously demonstrated that Bcl-10 and MALT1 proteins are involved in development of mucosa-associated lymphoid tissue (MALT) lymphomas. Several chromosomal abnormalities have been observed in these lymphomas, the best characterized of which is the translocation 42 t(1; 14)(p22; p32) . This translocation juxtaposes the Bcl-10 coding region with the strong Ig transcriptional enhancers on chromosome 14, promoting Bcl-10 overexpression. Another important translocation, t(11:18)(q21; p21), creates a fusion protein between the N-terminal portion of API2 and the C terminus of 43 MALT1 . The main signaling phenotype of both of these molecular abnormalities is promotion of NF-κB activation. These events are enhanced by the physi44 cal association of Bcl-10 with MALT1 , indicating that these molecules collaborate in this signaling pathway. A third member of this complex, CARMA1, was identified using database searches and screens for Bcl-10 interacting pro45,46 teins . Functional analysis of CARMA1 (or the related family members, CARMA2 and CARMA3) in cell lines revealed that CARMA proteins associate with and promote phosphorylation of Bcl-10. Additionally, CARMA1-induced NF-κB activation is synergized by co-expression of Bcl-10, suggesting that CARMA1 and Bcl-10 form a complex necessary for efficient NF-κB activa45,46 tion . Formal genetic demonstration that the CARMA1/Bcl-10/MALT1 complex is specifically required for NF-κB induction in lymphocytes has been provided by the generation of murine knockout models of each of these proteins. Defi47-50 51,52 53,54 ciency in CARMA1 , Bcl-10 , or MALT1 each led to failure to upregulate the activation markers CD69, CD44, CD25 (in T cells), and CD86 (in B cells); and loss of BCR-, TCR- or PMA/Ionomycin- (pharmacological activators of PKCs) induced B and T cell proliferation, respectively. These defects are reflected in low basal immunoglobulin levels, impaired T-cell dependent and Tindependent humoral responses, and deficient IL-2 production, CD28 costimulation, and CTL responses in T cells. In contrast, other signaling events 2+ such as total tyrosine phosphorylation, Ca mobilization, and ERK activation are unaffected. Most notably, IKK activation, IκBα degradation, and NF-κB nuclear translocation are abrogated in lymphocytes from each of these knockout strains. Taken together with observations made in cell lines, these data demonstrate that CARMA1, Bcl10, and MALT1 are each required for immunoreceptor-induced activation of the NF-κB signaling pathway. Several lines of evidence suggest that the hierarchy of signaling components downstream of BCR leading to NF-κB activation proceeds first with activation of PKC, followed by CARMA1, Bcl-10, MALT1, and then IKK: 1. PKC activation can be placed upstream of CARMA1 by a number of findings. First, PMA/ionomycin treatment of CARMA1-deficient lymphocytes (or cell lines 47-49,55,56 expressing dominant negative CARMA1) fails to activate NF-κB . In addi-

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tion, CARMA1-deficient T cells exhibit normal recruitment of PKCθ into the 57 cSMAC upon antigen stimulation . Second, we have observed that CARMA1, PKCβ, IKK, and Bcl-10 rapidly translocate into lipid rafts of human B cell lines after BCR crosslinking. However, CARMA1, IKK, and Bcl-10 (but not PKCβ) raft recruitment is blocked by specific PKCβ inhibitors. This indicates that the enzymatic activity of PKCβ is required for CARMA1 signalosome formation in 58 B cells . Finally, constitutive NF-κB activation mediated by the over-expression of dominant active CARMA1 (CARMA1-∆PRD, see below) is unaffected by 58 pharmacological inhibition of the PKC enzymes . 2. While CARMA1 is consti56 tutively associated with lipid rafts in T cells, Bcl-10 is predominantly cytosolic . CARMA1 deficiency, or overexpression of dominant-negative CARMA1 (with mutations in the SH3 domain, or with a deleted CARD motif that removes the Bcl-10 binding site), inhibits TCR-induced raft recruitment of Bcl-10 and NFκB activation, implying that CARMA1 is required upstream of Bcl-10 func56,57 59,60 tion . 3. Finally, the suggestion that MALT1 functions downstream of activated Bcl-10 is supported by the observation that overexpressed Bcl-10 is unable to rescue NF-κB activity in MALT1-deficient murine embryonic fibroblasts 53 (MEFs) . Similarly, expression of a constitutively active MALT1 in Bcl-1054 deficient MEFs promotes constitutive NF-κB activation . Together, these data provide compelling evidence to support a model of signal propagation in B lymphocytes from PKCβ:CARMA1:Bcl-10:MALT1:IKK. Evidence that PKCβ regulates NF-κB activation by direct serine phosphory58 lation of CARMA1 has recently been described by our laboratory (Figure 2). The protein structure of CARMA1 is comprised of five domains that can mediate protein–protein interactions including an N-terminal CARD; a coiled-coil (CC); a PDZ (sequence motifs present in the PSD-95, Dlg, and ZO-1 proteins); 41 and C-terminal SH3 and GUK domains . The PDZ–SH3–GUK domain architecture at the C terminus is analogous to that of the MAGUK (membraneassociated guanylate kinase homologue) family proteins that act as signaling 61 scaffolds in neuronal synapses . CARMA1 also has two undefined linker sequences that connect: the CC with the PDZ domain (233 amino acids), and the SH3 with the GUK domain (121 amino acids), respectively. In the MAGUK family, the SH3–GUK linker has been shown to be important for oligomeriza41 tion . However, there were no clues based on homology or structural motifs to suggest a role of the CC–PDZ linker region. In this chapter, we will initially refer to this region as the “linker”. To determine whether PKCβ could bind to a specific domain of CARMA1, we performed in vitro GST pull down assays using a panel of CARMA1 domains fused to GST with the addition of recombinant PKCβ. These experiments demonstrated that PKCβ specifically interacted with CARMA1 linker, while no significant interaction was observed with other CARMA1 domains or with Bcl58 10 . This observation is supported by another report showing that PKCθ could

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Figure 2. Model of PKC-CARMA1-dependent activation of the NF-κB cascade. (A) In resting cells, CARMA1 is present within the cytosol, as well as at the cell membrane [via interaction with its SH3 domain and an unidentified membrane protein (X?)]. This resting pool of CARMA1 is maintained in a closed conformation, mediated by intramolecular binding of CARD and the (PRD) linker. Upon BCR stimulation, activated PKCβ phosphorylates the (PRD) linker region, releasing the CARD– (PRD) linker interaction and triggering an open CARMA1 conformation; promoting CARMA1 oligomerization and markedly enhanced membrane translocation. (B) The Bcl-10/MALT1 complex is recruited to the CARD domain of activated CARMA1. (C) Next, both the ubiquitination (TRAF6/Ubc13/TAK1) and IKK complexes are recruited to the assembled CARMA1/Bcl-10/MALT complex (via protein interactions that remain to be identified). These events promote IKKγ ubiquitination, and phosphorylation of IKKβ (most likely via TAK1), leading to full IKK and NF-κB activation.

be co-immunoprecipitated with the CARMA1 linker in HEK293 fibroblasts, 59 suggesting that a parallel process may occur in T lymphocytes . In vitro kinase (IVK) assays, using purified GST-CARMA1 domain fusion proteins incubated with recombinant active PKCβ (or PKCθ) and radiolabeled ATP, conclusively demonstrated that the CARMA1 linker was directly and specifically phosphorylated by PKCs. Significant phosphorylation was not observed in other CARMA1 58 domains or Bcl-10 GST fusion proteins . We additionally verified that CARMA1 and PKCβ co-associate in vivo in B cells, and that CARMA1 is inducibly serine phosphorylated downstream of PKCβ in response to BCR engagement. We found that PKCβ co-immunoprecipitated with Myc-tagged CARMA1 from stably transfected Ramos B cells, and that this association was further increased after BCR crosslinking. Further, employing immunoblotting using antibodies specific for serine residues phosphorylated by conventional PKCs, we demonstrated that CARMA1 immunoprecipitated from BCR-stimulated Ramos B cells was phosphorylated by a classical

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PKC isoform. Chemical inhibitors (that specifically blocked the kinase activity of PKCβ) blocked this phosphorylation, indicating that inducible CARMA1 58 serine phosphorylation is controlled by PKCβ in B cells . Computational analysis of the linker identified six serine residues that may act as PKC substrates. Generation of point mutations in specific serine residues of the linker demonstrated in vitro and in vivo that three serine residues — S564, S649, and S657 — were the major PKC phosphorylation sites. Most importantly, NF-κB reporter gene assays in Jurkat T cells verified that serines 564 and 657 (but not 649) are essential for NF-κB activation in response to antigen receptor engagement. Because of the compelling evidence that the linker functions as the site for PKC binding and its activating phosphorylation, we have 58 named this region the “PKC regulated domain” (PRD) . One of the most striking features of the PRD is the dearth of significant secondary structural motifs as judged by computational analyses. Indeed, the Nterminal portion of the PRD scores as a No-Regular Structure (NORS) region. NORS regions are evolutionarily conserved — flexible loops that link and regu62 late the conformation of their associated protein domains . Interestingly, deletion of the PRD from CARMA1 (CARMA1-∆PRD) results in extremely highlevel constitutive NF-κB activation in reporter gene assays in both B and T cell lines. The observation that PRD phosphorylation by PKC leads to the same effect on CARMA1 activation, as does PRD deletion, strongly hints that the PRD regulates CARMA1 activity by modulating its structural conformation. Several lines of evidence support the idea. Immunofluorescent imaging of B and T cells shows that WT CARMA1 localizes to the cytosol, and cannot constitutively colocalize to downstream binding partners Bcl-10 and phosphorylated IKK. In striking contrast, deletion of the PRD promotes its constitutive colocalization with Bcl-10 and pIKK at the cell membrane. Not only is CARMA1 constitutively activated by deletion of the entire PRD region (233 amino acids), deletion of two regions with predicted secondary structure proximal to either of the key phosphorylated residues, S564 (beta-strand-helix; 16 amino acids) and S657 residues (helix, 17 amino acids), or of a 19-amino-acid segment from the NORS region, also resulted in high levels of constitutive NF-κB activation. Conversely, deletion of two other predicted helices, not located near these phosphorylation 58 sites, had no effect on downstream NF-κB activation . Together, these data suggested the hypothesis that PRD deletion or PKCβ phosphorylation relieves an inhibitory intramolecular interaction that requires both flexibility at the N terminus of the PRD, and certain structural motifs at the PRD C terminus. Consistent with this, GST pull-down analysis demonstrated that the CARMA1 N-terminal CARD domain and PRD were able to physically associate, and that phosphorylation of the PRD abrogated this interaction. Taken as a whole, we hypothesize that, under resting conditions, CARMA1 may be folded in a “closed” conformation, where the CARD domain and the PRD are physically associated, blocking Bcl-10/MALT1 association, and hence NF-κB

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activation (Figure 2A). Upon BCR crosslinking, PKCβ−dependent PRD phosphorylation relaxes the CARD–PRD interaction, inducing an open CARMA1 conformer that allows recruitment of the Bcl–10/MALT1 complex and leads to NF-κB activation (Figure 2B).

6. EVENTS DOWNSTREAM OF CARMA1 LEADING TO IKK ACTIVATION According to our model, a conformational shift in CARMA1, induced by PKCβ phosphorylation, improves the accessibility of protein-binding domains to downstream effector molecules. One essential downstream binding interaction of CARMA1 is with Bcl-10 via the CARD domain. This recruitment promotes Bcl-10 activation by an unknown mechanism, possibly through phosphory45,46,56 . Bcl-10 recruitment and activation induces that of MALT1. It is likely lation that Bcl-10 recruits MALT1 by direct binding interactions between the C termi44 nus of Bcl-10 and the Ig domains of MALT1 , although it has also been re63 ported that MALT1 can bind the CC domain of CARMA1 (Figure 2B). In addition, CARMA1 and CARMA3 were found to physically associate with IKKγ 64 (NEMO) through a region spanning the PDZ–SH3 domains . It has been shown that oligomerized MALT1 directly binds TRAF6, thereby oligomerizing TRAF6 65 and greatly enhancing poly-ubiquitination of both TRAF6 and NEMO . The requirement for TRAF6 rather than MALT1 in vitro using complementation 66 assays of NEMO polyubiquitination make it less likely that MALT1 acts directly as a ubiquitin ligase at lysine 399 of NEMO, as was initially suggested. Ubc13 and Uev1A are the main E2 conjugating enzymes for NEMO ubiquitina65,66 tion . Together, these findings suggest that activated CARMA1 recruits and allows oligomerization of the Bcl–10/MALT1 complex, which in turn recruits and activates TRAF6 (Figure 2C). Activated TRAF6 then induces NEMO polyubiquitination. Besides NEMO ubiquitination, a second step that is required for 65 IKK activation is likely to be the phosphorylation of IKKβ by TAK1 . Current evidence supports the possibility that TRAF6-ubiquitinated NEMO promotes IKK interaction with the TRAF6/TAK1 complex, allowing TAK1 to activate IKKβ by serine phosphorylation. Genetic proof of the specific requirement for TAK1 in these events, however, remains to be demonstrated; and it remains equally plausible that poly-ubiquitination and oligomerization of the IKK complex alone may be sufficient to trigger its transphosphorylation and activation. As these studies have been carried out primarily in T cells or cell-free systems, it will also be important to determine if the same mechanisms control IKK com65 plex activation in B cells. Finally, Sun et al. describe molecular activation events downstream of Bcl-10 as an “oligomerization cascade.” Our data suggest that this cascade may actually begin even earlier via the coiled–coil domain mediated oligomerization of CARMA1 (see Figure 2B). Consistent with this idea,

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mutation within the CARMA1 coiled–coil domain, as in the “unmodulated” 47 mouse strain, abolishes antigen receptor induction of NF-κB activation . There is currently conflicting data concerning the requirement for Caspase8 in BCR activation of NF-κB. Using several models of Caspase-8-deficient T cells, including human T cells genetically deficient in Caspase-8, Caspase-8deficient Jurkat T cells, Caspase-8 conditional knockout mice, siRNA, and chemical inhibition, Su et al. describe a requirement of Caspase-8 for antigen 67 receptor induction of NF-κB nuclear translocation and reporter gene activation . They observe a parallel requirement for Caspase-8 in B cells from genetically deficient humans, and from studies using Caspase-8-specific siRNA in the Ramos B cell line. Additional data were also presented that suggest that Caspase-8 can physically associate with the CARMA1–Bcl-10–MALT1 complex as 68 well as with IKK. Recently, Beisner et al. , using a B-cell specific conditional Caspase-8 knockout murine model, observed no effect of Caspase-8 deficiency on B cell proliferation, or on proximal markers of NF-κB activation in response to BCR activation. Instead, this group observed a proliferation defect only with TLR3 and TLR4 agonists. In our own laboratory, we have observed no effect of z-IETD-FMK or z-VAD-FMK (Caspase8-specific and pan-Caspase inhibitors, respectively) on NF-κB reporter gene assays performed using Jurkat T cells transfected with either wild-type or constitutively active (∆PRD-CARMA1) CARMA1. The time course and concentrations of the chemical caspase inhibitors we utilized were designed to specifically inhibit Caspase-8 activity (unpublished data), but were considerably less than the concentrations (10 and 20 µM vs. 40 µM) and length of time (6, 12, 24, and 48 hours vs. 4 to 6 days) used by Su et al. to show an effect on NF-κB activation. The explanation for these divergent findings remains to be clarified.

7. CONCLUSION AND PERSPECTIVES In this review we have presented the recent advances in understanding of the signaling events that control BCR-mediated NF-κB activation. These observations clarify the molecular events that regulate activity of the scaffolding molecule CARMA1 and the events directly linking PKCβ with IKK/NF-κB complex activation. The primary event controlling CARMA1 activation is the specific PKC-dependent phosphorylation of the CARMA1 linker, which we term the PRD region (PKC-regulated domain). Our data suggest that this phosphorylation sequence triggers a conformational change in CARMA1 facilitating its membrane association, and its subsequent recruitment of a protein complex formed by Bcl-10, MALT1, NEMO, TRAF6, TAK1, and Ubc13/Uev1A. To complete our understanding of CARMA1 function in the NF-κB activation pathway, it will be important to formally identify the CARMA1 domains, as well as the interacting molecules, required for its raft recruitment and oligomerization. Additionally, it will be important to determine how the assembled, active

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CARMA1 signalosome is downregulated; including whether this requires dephosphorylation of CARMA1 and/or whether CARMA1 enters a specific ly69 sosome degradation pathway, as suggested for Bcl-10 . Finally, it remains of great interest to determine whether additional signaling molecules associate with, and are regulated by, CARMA1. Notably, CARMA1 is clearly required for activation of the JNK signaling cascade, which is completely abrogated in 47,48,56 CARMA1-deficient animals or cell lines . Additionally, as MAGUK domains often facilitate association with plasma membrane and cytoskeleton proteins, it will be important to determine the role for CARMA1 in cytoskeletal 70 dynamics. Finally, we and others have observed via microscopy that Bcl-10 appears to decorate cytoskeletal structures in resting lymphocytes. It will therefore be important to determine whether the recruitment of Bcl-10 to activated CARMA1 requires an active role via the cytoskeleton, rather than a model of passive diffusion. We believe that improving our understanding of the role and regulation of CARMA1 in B cells will significantly advance our knowledge of the mechanisms that control NF-κB both in normal as well as immune-pathological conditions. We also predict that such information may be used successfully for development of new and more specific therapies for the modulation of these pathologies.

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8 THE ADAPTER 3BP2: HOW IT PLUGS INTO LEUKOCYTE SIGNALING Marcel Deckert1 and Robert Rottapel2

1. INTRODUCTION 3BP2/SH3BP2 is an adapter protein composed of an amino-terminal PH domain, a central proline-rich (PR) region, and a carboxyl-terminal SH2 domain that was originally identified as a c-Abl SH3 binding protein in 1993. Functional studies have implicated a role for 3BP2 in immunoreceptor signaling through its interaction with a number of signaling molecules, including Src and Syk families of protein tyrosine kinases, LAT, Vav, PLCγ, and 14-3-3. Recently, the 3bp2/sh3bp2 locus was shown to be mutated in a rare human disease involved in cranial-facial development called cherubism, suggesting a role for 3BP2 in regulating osteoclast function. This review will discuss the current understanding of how the adapter 3BP2 plugs into leukocyte cell signaling.

2. STRUCTURE, EXPRESSION AND THE 3BP2 SIGNALING COMPLEX 3BP2 (or SH3BP2 for Abl-SH3 Binding Protein-2) was initially cloned — together with 3BP1 — in a screen to identify binding partners of the Src1 homology 3 (SH3) domain of the kinase c-Abl . It was subsequently identified 2 as a binding partner of Syk in a yeast two-hybrid screen . In humans, the gene encoding for 3BP2 is located on chromosomic region 4p16.3, which is fre3 4 quently deleted in bladder cancer and Wolf-Hirschorn syndrome . The 3bp2 2,3 gene is composed of 13 exons, encoding a major message of 2.4 kb . The product of this gene is a protein of 561 and 559 amino acids in human and mouse,

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INSERM Unit 576 and Hopital de l’Archet, Nice, France, 2Princess Margaret Hospital and Ontario Cancer Institute, Toronto, Ontario, Canada

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Figure 1. Domain structure of 3BP2 adapter protein. The proteins known to interact with specific domain, and residues subjected to tyrosine and serine phosphorylation or mutated in cherubism are shown.

respectively, with a remarkable conservation suggesting similar functions both in human and rodents. The 3BP2 transcript is ubiquitously expressed in human 2,3,5-7 tissues, with high expression in cells of hematopoietic and lymphoid origin . Analysis of 3bp2 mRNA expression in a wide variety of normal mouse tissues by Affimetrix chip hybridization has shown that the 3BP2 message is restricted to bone osteoclasts, oocytes, and lymph nodes and is most highly expressed in B lymphocytes (Mouse GeneAtlas, GNF, San Diego, CA) (R. Rottapel, meeting communication). The 3BP2 protein has a modular organization of an N-terminal pleckstrin homology (PH) domain, a central proline-rich (PR) domain, and a C-terminal Src-homology 2 (SH2) domain (Figure 1). The domain composition of 3BP2 is reminiscent of the Grb7 family of adapters, which includes Grb 10 and Grb 14, as well as the closely related proteins APS, Lnk, and SH2-B, each of which has been demonstrated to participate in signaling events downstream of various cell 8 surface receptors . The PH domain suggests that 3BP2 binds membrane phospholipids. However, this has not yet been addressed experimentally. Nevertheless, 3BP2 PH domain was shown to be critical for 3BP2’s function as measured by NFAT luciferase assay as its deletion suppresses the ability of signal trans2,5 duction in T and B cells .

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The proline-rich (PR) domain that lies between the PH and SH2 domains is more than 200 aa long and contains multiple canonical PxxP motifs that bind SH3 domains (reviewed in [9]). The ten-amino-acid minimal PR motif for the Abl SH3 domain encompassed by amino acids 201 to 210 was originally identi1 fied as the first SH3 binding motif . This motif also partially overlaps with the 5 binding site of Vav SH3 domains (M. Deckert, meeting communication). The 2 10 SH3 domain of Fyn and Lyn have also been described to bind to 3BP2 within the proline-rich domain. In addition to harboring SH3 binding motifs, the 3BP2 PR domain contains additional uncharacterized motifs predicted to function as 11 binding sites for WW and EVH1 domain interaction sites . The optimal binding sequence for the 3BP2 SH2 domain was determined to be Tyr–Glu–Asn (YEN) using a degenerate peptide library screening method developed by Cantley and colleagues. This motif is found in diverse signaling molecules, including the receptor tyrosine kinase Flt3/Flk2 and the growth fac12 tor receptors EpoR and G-CSFR . The bacterially expressed recombinant 3BP2 SH2 domain can interact with G-CSFR at a phosphorylated tyrosine found 13 within a membrane-distal YEN motif, also known to bind to Grb2 and Shc . The 3BP2 SH2 domain can also bind to the YEN motif found in the transmem2,7 brane adapter LAT protein expressed in T and NK cells . The YEN motif is also present in the B cell expressed co-stimulatory molecule CD19 and in NTAL/LAB, an adapter related to LAT expressed in B cells, suggesting that 3BP2 may also couple to these proteins. The 3BP2 SH2 domain can interact with several tyrosine phosphorylated proteins including the Syk-kinases Syk and 2,5 5 2,5-7 ZAP-70 , Vav2 , PLCγ, and Cbl , though they do not display a YEN motif in their sequences. These interactions may be mediated by an uncharacterized YEN-containing linker molecule or may suggest some degree of flexibility in the binding specificity of the 3BP2 SH2 domain. 3BP2 is modified by both tyrosine and serine phosphorylation. 3BP2 is inducibly tyrosine phosphorylated following ligation of the T cell, B cell antigen receptors and Fc receptors, and can be phosphorylated in vitro on Tyr 174, Tyr 10,14 183, and Tyr 446 by Syk-family kinases . Tyr183 mediates interaction with 6 the Vav1 SH2 domain while Tyr446 specifies Lyn and Lck SH2 domain bind10,14 ing . Mutation of either Tyr 183 or Tyr 446 resulted in decreased activity of 14 3BP2 (M. Deckert, unpublished results), indicating that phosphorylation of these tyrosine residues is required for optimal 3BP2 function. Although 3BP2 has been shown to bind to the Abl SH3 domain, it remains to be demonstrated whether 3BP2 is a substrate for Abl and/or whether 3BP2 can regulate Abl kinase activity. In resting cells 3BP2 is constitutively phosphorylated on serine residues and 15 forms a complex with 14-3-3 through Ser 225 and Ser 277 . In vitro kinase assays have shown that PKC can directly phosphorylate 3BP2 on serine, but these experiments do not exclude the possibility that other AGC family kinases may 15 be physiologic 3BP2 kinases . Disruption of 14-3-3 binding sites increased the

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ability of 3BP2 to activate NFAT as measured by luciferase assays ing that serine phosphorylation negatively regulates 3BP2 activity.

, suggest-

3. ROLES IN LEUKOCYTE CELL SIGNALING What might be the function of 3BP2 in leukocyte signaling? Most data to date that have relied on overexpression approaches have implicated 3BP2 as a positive regulatory protein for antigen and immunoglobulin receptors. What remains unclear is precisely where 3BP2 exerts its function along the signaling pathway(s) lying downstream of the antigen receptors. A growing body of evidence supports the notion that 3BP2 is a component of an Src/Syk kinases-dependent signaling complex that may include LAT, Vav, and PLCγ proteins. In T cells and mast cells, 3BP2 associates with LAT in lipid 2,7,17 raft fractions and potentiated PLCγ activation . In overexpression studies 3BP2 potently induces NFAT- and AP-1-dependent transcription in Jurkat T 2,14 cells and is dependent on calcineurin . Short interfering RNA-mediated suppression of 3BP2 expression substantiated the need for 3BP2-dependent NFAT 5,14 activation in both T and B cell lines . Several observations point to a role of 3BP2 in a PLCγ-dependent pathway involved in BCR-mediated gene activation. 5 Following BCR aggregation 3BP2 interacts with Vav1/2 (M. Deckert, meeting communication), which play pivotal roles in PLCγ, calcium, and NFAT regula18 tion following immunoreceptor stimulation . In basophils, overexpression of a dominant inhibitory form of 3BP2 decreased FcεRI-mediated tyrosine phos7 phorylation of PLCγ1/2, calcium mobilization, and degranulation . Together, 2+ these findings suggested that 3BP2 regulates Ca -mediated signals from the immunoreceptors and NFAT-dependent gene transcription. The precise mechanism by which 3BP2 regulates calcium flux and AP-1 activation still remains obscure. One likely candidate is the Vav proteins, as 3BP2 interacts with Vav in three distinct ways: first, a basal interaction mediated by the proline-rich domain of 3BP2 and the SH3 domain(s) of Vav proteins; second, an activation-induced interaction between the SH2 domain of 3BP2 and phosphorylated Vav1/2; and third, an interaction between phosphorylated Tyr 183 on 3BP2 and the SH2 domain of Vav1. Mutation of 3BP2 Tyr 183 indicated 6 14 that this residue was required for optimal adapter function of 3BP2 in NK , T , and B cells (Deckert M., unpublished observations). Vav proteins are required for 3BP2-induced NFAT activation in B cells. Moreover, the dominant negative form of Rac1, a small GTPase regulated by Vav1, inhibited 3BP2-mediated 5,19,20 NFAT/AP-1 activation . Evidence has recently been reported that 3BP2 can induce the loading of GTP on Rac1 presumably through Vav protein activa5,16 5,6,21 tion . Vav proteins can associate with both 3BP2 and the adapter protein 21-24 SLP-76 through their SH2 domain. Thus, one important question to be addressed is whether 3BP2/Vav and SLP-76/Vav constitute two distinct signaling complexes involved in distinct afferent signaling limbs that regulate Rho

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GTPases. Vav proteins may have distinct functions in T cells, as Vav3 is preferentially required for coupling the TCR to serum response element (SRE)mediated gene transcription, whereas Vav1 preferentially affected TCR21 mediated IL-2 promoter activity , suggesting that 3BP2 may be involved in regulation of several Vav-dependent pathways in lymphocytes. Recently, an additional role of the 3BP2–Vav complex has been described in NK-mediated cytotoxicity downstream CD244 (2B4), a member of the CD150/SLAM family 25 of CD2-related receptors .

4. 3BP2 AND PATHOLOGY 26

Genetic evidence has linked 3BP2 to a rare human disease called cherubism . Cherubism is an autosomal dominant disorder characterized by erosion of maxillar and mandibular bone with resultant dental and facial deformity due to exces27 sive osteoclast activity and giant cell granuloma formation . All mutations identified in cherubism patients are located in exon 9, leading to single-amino-acid substitution in 3BP2 mapping to a six-amino-acid strech — RSPPDG — lying 26 between the end of the Pro-rich and SH2 domains . The most frequent mutation is a substitution of Pro 418 to leucine, argine, or histidine, but mutation of Gly 420 to glutamic acid or arginine, and Arg 415 to proline or glutamine have also 26 been identified . Subsequent studies by other groups have also identified point 28,29 mutations within this region occuring in cherubism individuals . The signaling alterations of the mutant forms of 3BP2 are currently unknown. One characteristic of cherubism is the presence of multiple cysts in the jaw bones that are filled with multinucleated osteoclasts. Consequently, Reinchenberger and colleagues hypothesized that 3BP2 mutations may lead to a gain of function or act in a dominant-negative manner in the signaling pathways of cells involved in jaw26 bone development, including osteoblasts and osteoclast progenitors . A recent study by Sada and colleagues has shown that overexpression of cherubism mutants of 3BP2 in the rat basophilic leukemia RBL-2H3 cells resulted in the loss of function and acted in a dominant-negative manner on Lyn and Vav1-Rac1 signaling activation, antigen-induced degranulation, and cytokine gene tran16 scription . Interestingly, various binding partners of 3BP2 have also been involved in 30 31 bone development, including kinases c-Abl and c-Src , the ubiquitin ligase c32 33 Cbl , and the Rho GTPase activator Vav3 . Moreover, the expression of the ITAM-containing adapter DAP12 and Syk was shown to be conditional to func34 tional osteoclast development . In addition, AP-1 and NFAT, two transcription factors whose activities are regulated by 3BP2 in leukocytes, play pivotal roles 35 in bone homeostasis . Together, this raises the possibility that endogenous 3BP2 regulates Src/Syk and Vav signaling pathways involved in the early development of osteoclasts from hematopoietic stem cells, an idea, however, that requires additional studies in patient cells or animal models. Finally, the 3bp2

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gene is located on chromosomic region 4p16.3, which is frequently deleted in 3 4 bladder cancer and Wolf-Hirschorn syndrome . With the critical involvement of BCR-Abl gene fusion in leukemogenesis, these observations might suggest a role for 3BP2 as a tumor suppressor gene.

5. CONCLUSION AND PERSPECTIVES Genetic and biochemical analysis has revealed that cytoplasmic adapter proteins lacking intrinsic enzymatic activities have crucial roles in leukocyte biology. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of its biological function during immune and allergic responses has yet to emerge. Clearly, its exact function in the mechanisms regulating leukocyte activation, proliferation, and survival need to be clarified. This would require identification of specific cellular events and gene patterns regulated by 3BP2 in primary leukocytes. The interaction of 3BP2 with Abl kinases and receptor tyrosine kinases (RTKs) involved in hemapoietic cell activation and development should also be evaluated in the future. 3BP2 may couple to multiple cell surface receptors since the YENX motif that comprises the optimal binding sequence for the 3BP2 SH2 domain is present on diverse receptors and costimulatory molecules. Finally, the involvement of 3BP2 in the rare genetic bone disease cherubism raises the exciting possibility that 3BP2 acts as a ‘’tumor suppressor’’ downstream from PTKscoupled receptors in hematopoietic cells.

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9 CTLA-4 REGULATION OF T CELL FUNCTION VIA RAP-1-MEDIATED ADHESION Helga Schneider, Elke Valk, Silvy da Rocha Dias, Bin Wei, and Christopher E. Rudd

1. INTRODUCTION T cell activation is mediated by a combination of signals delivered from various cell surface receptors. The conventional view is that an initial signal is sent by the antigen–receptor complex (TcRζ/CD3) in response to specific peptide agonists that are presented by major histocompatibility complex (MHC) molecules on the surface on antigen-presenting cells (APCs). This is then followed by a second signal provided by co-receptors such as CD28, an inducible costimulatory molecule (ICOS), cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD1), T cell immunoglobulin mucin-1 (TIM-1), and other costimulators. These receptors can be broadly expressed on T cells as in the case of CD28, or restricted to subsets of T cells as with TIM-1. The actual sequence of signals is not fully understood and may involve early additional signals from adhesion molecules such as lymphocyte-function-associated antigen-1 (LFA-1), and from chemokines (i.e., stromal cell-derived factor-1 (SDF1)). The combination of these different receptors and signals determines the ultimate nature of the T cell response. Of the co-receptors, members of the CD28 family (i.e., CD28, ICOS, and CTLA-4) are the best characterized. CD28 and CTLA-4 bind to the same 1-3 ligands (CD80 and CD86) on the APC surface . CD28 enhances and sustains T cell responses, while CTLA-4 negatively modulates cytokine production and proliferation. CD28-deficient mice show a marked reduction in the ability to

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respond to most short-lived antigens , while CTLA-4 deficient (CTLA-4-/-) mice show a post-thymic autoimmune phenotype with massive tissue infiltration and 5,6 organ destruction . Despite this, the mechanism by which CD28, ICOS, and CTLA-4 generate intracelllular signals is unclear and the subject of much investigation. One common property involves their binding to the lipid kinase phosphatidylinositol 37 kinase (PI3K) via a classic YxxM motif in the cytoplasmic domain . Although the TcR can also engage the PI3K pathway, the main feature of TcRζ/CD3 sig8,9 naling is the engagement of CD4- and CD8-associated p56lck and associated 10 ZAP-70 to generate a tyrosine phosphorylation cascade . However, little if any tyrosine phosphorylation is induced via ligation of CD28 co-receptor family members. While the intensity of the TcR/CD3-induced signal varies with the affinity of the MHC–peptide complex (i.e., low- vs. high-affinity ligand), the binding of CD80/86 to CD28 and CTLA-4 is constant. This difference may be important in the case of low-affinity ligands, where signaling is more dependent on CD28 ligation. The role of the co-receptors in amplifying or skewing the response is of particular importance in the case of low-affinity ligands (i.e., lowaffinity auto-antigen). Despite the similarity between CD28 and CTLA-4, the difference in functional outcomes of ligating one co-receptor vs. another is remarkable. Reported mechanisms to explain the inhibitory effect of CTLA-4 have included ectodo11 main competition for CD28 binding to CD80/86 , disruption of CD28 localiza12 tion at the immunological synapse , modulation of phosphatases PP2A and 13-17 18-20 SHP-2 , and interference with lipid raft expression . While CD28 enhances 18,21,22 surface raft expression induced by TcR ligation , CTLA-4 coligation with either TcR or the combination of TcR and CD28 blocks surface expression of 18 lipid rafts, which parallels inhibition of cell proliferation . CTLA-4 engagement of CD80/86 on dendritic cells can also induce the release of indoleamine 2,323,24 dioxygenase (IDO) . However, the degree to which this pathway plays a significant role in CTLA-4 function is uncertain due to the fact that the IDO-/- mice 25 show a normal immune response .

2. INTEGRINS AND ADHESION Integrins are heterodimeric transmembrane receptors that consist of an α subunit non-covalently associated with a β subunit. They mediate adhesion to cells and to the extracellular matrix (ECM). At present, 18 α-subunits and 8 β-subunits, which form 24 αβ dimers, have been identified in mammals (Figure 1). Members of the β2 integrin family are the main integrins expressed on leukocytes, and they play major role in leukocyte cell-cell and cell-matrix adhesions

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Figure 1. T cell integrins. Diagrammatic representation of integrins on mammalian cells with the leukocyte-specific integrins of the β2 family.

during inflammation and other immune responses. β2 integrins are signaling receptors, but they are also targets of and are functionally affected by intracellular signals. The β2 integrins are designated CD11/CD18, because they are composed of a common β chain (CD18) and one of four unique α chains: CD11a, CD11b, CD11c, and CD11d. These integrins play a prominent role in T cell function such as migration of T cells to peripheral lymph nodes, in antigen presentation, and cytotoxic killing. Under normal conditions, the integrins are inactive, but exposure to cytokines or chemokines and engagement of other cellsurface receptors results in rapid integrin activation and ligand binding. Integrins can be activated by increasing the affinity of individual molecules for their ligands or by increasing the avidity of adhesion. Increased avidity may result from an increase in number of clustering of integrins at the plasma membrane, therefore increasing adhesion due to a high local concentration of the molecules (Figure 2). Inside-out signaling molecules include ITK (interleukin-2 inducible kinase), VAV-1, ADAP (adhesion and degranulation-promoting adap26,27 28,29 tor protein; also known as FYB or SLAP 130) , SKAP-55 , Rap-1, and Rap30-32 L . After a T cell encounters an APC displaying a peptide–MHC complex that is recognized by its T cell receptor, the avidity of the αLβ2 integrin (LFA-1) for intercellular adhesion molecule-1 (ICAM-1) is rapidly increased, leading to the formation of an adhesion complex between the T cell and the APC. This

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Figure 2. Two modes of LFA-1 activation. Conformational changes for increased affinity, and avidity maturation (i.e., clustering).

complex is known as an immunological synapse (IS) or a supramolecular activation cluster (SMAC), and it facilitates antigen recognition. The IS is a characteristic structure in which an external LFA-1 ring (peripheral SMAC, pSMAc) sur33,34 rounds central TcR clusters (central SMAC, cSMAC) .

3. CTLA-4 MEDIATED UPREGULATION OF LFA-1 ADHESION AND CLUSTERING One of the hallmarks of the CTLA-4-/- mouse is an extensive lymphocytic infil5,6 tration of tissues . Intriguingly, this event is dependent on integrins for migra-

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Figure 3. CTLA-4 up-regulates LFA-1 function in primary T cells, while anti-CD3 upregulation of LFA-1 clustering requires CTLA-4 expression. Panel A, left panel: Pre-activated human peripheral T cells were stimulated with anti-CD3, anti-CD3/CTLA-4, anti-CTLA-4, anti-CD3/CD28, and antiCD28 antibodies and assessed for LFA-1 binding to plates coated with ICAM-1. Right panel: FACS profiles showing CTLA-4 expression on peripheral T cells using phycoerythrin (PE)-labelled CTLA4 antibody. Panel B, left panel: Pre-activated primary CD4+ T cells from CTLA-4 WT and CTLA-4 KO mice were stimulated with anti-CD3 and anti-CD3/CTLA-4 antibodies and assessed for LFA-1 binding to plates coated with ICAM-1. Right panel: Proliferation of unstimulated CTLA-4+/+ and CTLA-4-/- T cells at 24 hr is shown.

ation of T cells to different organs. Given this, we reexamined CTLA-4 function in the context of LFA-1 integrin adhesion. Human and mouse primary T cells were activated for 48 hours to induce CTLA-4 expression followed by coligation with anti-CD3 or anti-CD3/CTLA-4 (Figure 3). With human peripheral T cells, anti-CD3 increased adhesion from 4 to 9 percent of cells, while CTLA-4 coligation increased this further to 14 percent (Figure 3A, left panel). AntiCTLA-4 alone increased adhesion to ICAM-1 to the same level as observed for anti-CD3 alone. This occurred with CTLA-4 expression on 17 percent of cells

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(right panel). No enhancing effect was observed with anti-CD3/CD28 coligation. Similar results were obtained in a comparison of activated age-matched CTLA-4+/+ versus CTLA-4-/- primary mouse T cells (Figure 3B). While antiCD3 increased ICAM-1 binding from 5 to 11 percent of CTLA-4+/+ T cells, CTLA-4 coligation increased this further to 14–15 percent (Figure 3B). This occurred with CTLA-4 expression on 20 percent of cells (data not shown). No increase in binding was evident with CTLA-4-/- T cells, although they were hy5,6 perproliferative, as reported (right panel). A similar difference between CTLA-4+/+ and CTLA-4-/- T cells was observed for LFA-1 clustering (Figure 4). Anti-CD3 increased clustering from 2 to 13 percent of cells, while CTLA-4 coligation augmented this further to 28 percent. Anti-CTLA-4 alone increased clustering to levels seen with anti-CD3 alone. As expected, no effect was evident with CTLA-4-/- T cells. The right panel shows immunofluorescence images of LFA-1 distribution. As a control, neither anti-CD28, CD2, nor CD8 coligation was able to increase adhesion under the short-term incubation conditions of the study (Figure 4B). Our findings confirm that CTLA-4 acts as a selective integrin activator of LFA-1 on primary human and mouse T cells by increasing receptor clustering. In addition to antiCTLA-4 effects on LFA-1 adhesion, our findings uncovered another surprising observation, namely, that CTLA-4 expression is required for the optimal ability of antigen receptor to increase LFA-1 adhesion (Figures 3B, 4A). This was observed in a comparison of CTLA-4+/+ and CTLA-4-/- primary T cells. AntiCD3 induced only a marginal increase in ICAM-1 binding with CTLA-4-/- T cells when compared with CTLA-4+/+ T cells (i.e., from 5 percent on unstimulated cells to 11 percent on CTLA-4+/+ T cells versus 7 percent of CTLA-4-/- T cells). Similarly, anti-CD3-induced LFA-1 clustering was impaired in CTLA-4-/T cells (i.e., from 2 percent of unstimulated cells to 13 percent on CTLA-4+/+ T cells relative to 6 percent for anti-CD3 ligated CTLA-4-/- T cells (Figure 4A). These findings underscore the importance of CTLA-4 in adhesion whereby the loss of CTLA-4 partially decouples the TcR/CD3 complex from the upregulation of LFA-1 clustering/adhesion. Given the ability of anti-CTLA-4 to induce “inside-out” signaling, a question concerned the nature of the pathway. 30-32,35-39 The GTPase Rap-1 has been reported to increase LFA-1 adhesion . This suggested a possible link between our observed effects of CTLA-4 and Rap-1. To assess this, DC27.10-CTLA-4 T cells were ligated with anti-CD3, antiCD3/CTLA-4, or anti-CTLA-4 and followed by precipitation with GST30,35 RalGDS and blotting with anti-Rap-1 . RalGDS binds only to the active GTP40 bound form of Rap-1 . Anti-CD3 induced a 2-fold increase in precipitation of 41 GTP-bound Rap-1 (Figure 5A, lane 2 vs. 1) as described . Significantly, CTLA4 coligation increased this activation by an additional 2.5-fold (lane 3 vs. 2). Further, anti-CTLA-4 ligation alone also induced Rap-1 activation (lanes 4,5).

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Figure 4: CTLA-4 upregulates LFA-1 clustering on primary T cells. Panel A: Pre-activated primary CD4+ T cells from CTLA-4 WT and CTLA-4 KO mice stimulated with anti-CD3, anti-CTLA-4 and anti-CD3/CTLA-4 were stained with FITC-labelled anti-CD18 and analyzed by fluorescent microscopy for LFA-1 capping. Left panel: Histogram showing the percentage of cells with LFA-1 clusters. Right panel: Immunofluorescent images showing examples of cells with LFA-1 clusters. Panel B: Pre-activated primary CD4+ T cells stimulated with anti-CD3, anti-CD3/CTLA-4, anti-CD3/CD28, anti-CD3/CD2, and anti-CD3/CD8 antibodies were stained with FITC-labelled anti-CD18 and analyzed by fluorescent microscopy for LFA-1 capping.

The presence of Rap-1 was confirmed by anti-Rap-1 immunoblotting (lower panel). A time-course of Rap-1 activation by anti-CD3/CTLA-4 showed peak activation by 5–10 min followed by a decrease at 15 min (Figure 5B). During 42 preparation of this manuscript, Stork and coworkers reported a similar result . Our combined findings indicate that CTLA-4 ligation activates Rap-1 beyond that observed with TcR/CD3 ligation alone. To test for a link between CTLA-4

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Figure 5: CTLA-4 ligation activates Rap-1. Panel A: DC27.10-CTLA-4 cells were activated with combinations of anti-CD3, anti-CD3/CTLA-4 and/or anti-CTLA-4 antibodies for 7 min followed by precipitation with GST-RalGDS and blotting with anti-Rap-1. Upper panel: Medium (lane 1), antiCD3 (1 µg/ml; lane 2), anti-CD3/CTLA-4 (1 µg/ml/10 mg/ml; lane 2), anti-CTLA-4 (10 µg/ml; lane 4) or anti-CTLA-4 (20 µg/ml; lane 5). Lower panel: immunoblotting of cell lysates with anti-Rap-1. Panel B: Time course of anti-CD3/CTLA-4 activation of Rap-1. DC27.10-CTLA-4 cells were activated with anti-CD3/CTLA-4 for various times followed by precipitation with GST-RalGDS and blotting with anti-Rap-1. Lane 1: 0 min; lane 2: 2 min; lane 3: 5 min; lane 4: 10 min and lane 5: 15 min.; lane 6: control. Panel C: Rap-1-N17 reverses the upregulation of LFA-1-ICAM-1 adhesion by CTLA-4. Mock and Ha-Rap-1-N17 transfected DC27.10-CTLA-4 cells (left panel) and pre-activated primary human T cells (right panel) were stimulated with anti-CD3, anti-CD3/CTLA-4, and antiCTLA-4 antibodies followed by an assessment of cells bound to ICAM-1. Lower panel: Active Rap1-V12 can substitute for CTLA-4 in the up-regulation of LFA-1-ICAM-1 adhesion. Mock and HaRap-1-V12 transfected DC27.10-CTLA-4 cells were activated with anti-CD3 and anti-CD3/CTLA-4 mAbs for 30 min followed by an assessment of binding to ICAM-1.

activation of Rap-1 and increased LFA-1/ICAM-1 binding, DC27.10-CTLA-4 and activated CTLA-4 positive human T cells were transfected with Rap-1-N17 and assessed for anti-CTLA-4 induced adhesion to ICAM-1 (Figure 5C, left and

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right panels, respectively). Anti-CD3-, CD3/CTLA-4-, and CTLA-4-induced adhesion to ICAM-1 was markedly impaired by Rap-1-N17. Expression of HARap-1-N17 was confirmed by anti-HA immunoblotting (Figure 5C, inset). Significantly, expression of the constitutively active form, Rap-1-V12, substituted for CTLA-4 in promoting adhesion (lower panel). Anti-CD3 stimulation of Rap1-V12-transfected cells increased ICAM-1 binding to levels comparable to antiCD3/CTLA-4 coligation. These combined observations showing that inactive Rap-1-N17 can block CTLA-4 increased adhesion and that Rap-1-V12 can mimic CTLA-4 in its cooperation with anti-CD3 argue strongly for the fact that CTLA-4 increases LFA-1-mediated adhesion via the Rap-1 pathway.

4. CONCLUSIONS 43,44

Integrins play key roles in regulating the migration and localization of T cells . For this reason, the observation that CTLA-4-/- mice show extensive lymphocytic infiltration suggested the possibility that CTLA-4 might regulate integrinmediated adhesion in T cells. In support of this, we have shown that CTLA-4 can generate “inside-out” signals that potently upregulate LFA-1 clustering and adhesion on the surface of T cells. The upregulation of adhesion was observed with TcRζ/CD3–CTLA-4 coligation that led to an inhibition of IL-2 production. Adhesion was also induced by engagement of CTLA-4 alone and not by other coreceptors such as CD28, CD2 and CD8. Further, the importance of the connection between CTLA-4 and LFA-1 adhesion was underscored by the fact that the loss of co-receptor expression in CTLA-4-/- T cells resulted in an impairment of TcR/CD3-induced LFA-1 adhesion and clustering. Overall, our findings show that TcR/CD3–CTLA-4 coligation can have opposing effects that involve inhibition of IL-2 production (i.e., negative signal) and activation of LFA-1 clustering and adhesion (i.e., positive signal). The unexpectedly potent role for CTLA-4 in LFA-1-mediated adhesion provides an alternate potential route by which CTLA-4 modulates T cell immunity. CTLA-4 must now be considered to be an alternate receptor in the regulation of this event. Coligation of CTLA-4 produced an additive increase in adhesion. In the context of antigen presentation, the level of signaling via the TcR/CD3 complex is expected to vary with the avidity of the peptide agonist, and to be lower than that induced with high-avidity anti-CD3 antibody. By contrast, CD80/CD86 binding to CTLA-4 has a constant avidity, and induced a similar level of adhesion as anti-CTLA-4 antibody. The CTLA-4–LFA-1 connection is therefore likely to predominate in the response to many agonists, especially in the case of low-affinity peptide (i.e., self-antigen). In the case of selfantigen, this would be expected to limit the response and the development of autoimmunity. Our findings also show that CTLA-4 depends on Rap-1 for the upregulation of adhesion. Rap-1 is an allosteric regulatory element, switching between inac-

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tive GDP-bound and active GTP-bound conformations . GTP-bound Rap-1 31,36-39 upregulates LFA-1 clustering and T cell/APC conjugate formation . CTLA-4 activated Rap-1 at levels that were higher than anti-CD3. Further, inactive Rap1-N17 abrogated co-receptor-induced adhesion, while active Rap-1-V12 cooperated with anti-CD3 to increase adhesion to levels comparable to antiCD3/CTLA-4. Overall, our observations require a revision of the view that CTLA-4 operates solely as a negative signaling receptor. Other prostimulatory 45 events linked to CTLA-4 include the binding of PI 3K and the differential activation of extracellular signal regulated kinases (ERKs) and c-jun kinases 46 (JNKs) . The ultimate effect of CTLA-4 upregulation of LFA-1 remains to be ascertained. Increased adhesion could favor stable adhesion with limited motility or involve an amplification of both higher adhesion and motility. Preliminary data show that CTLA-4 increases LFA-1-dependent motility, resulting in a reduction of the residency period of T cell/APC interactions (data not shown). This might limit the engagement of TcR receptors and reduce the “threshold” of signaling. Altered adhesion is also likely to affect other integrins, and contribute to the increased lymphocytic infiltration of organs and/or lymphadenopathy observed 5,6 in CTLA-4-deficient mice . Future studies will be needed to assess the full effect of increased adhesion on cell migration and T cell interactions with APCs.

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10 PROTEIN CROSSTALK IN LIPID RAFTS Raquel J. Nunes1,2, Mónica A. A. Castro1, and 1Alexandre M. Carmo1,2 1. INTRODUCTION The view that the T cell receptor (TCR) is part of a multimolecular complex that 1 associates loosely at the surface of T cells and functions as an activation unit , is now widely accepted. More recently, the concept that the assembly of the TCR 2,3 signaling machinery takes place in lipid rafts has been put forward , and whereas the existence of these specialized lipid microdomains seems to be a general assumption, their role in the initiation of T cell signaling has been a matter of intense debate. Much of the controversy results from the experimental approaches used to differentiate these membrane structures and to trail the assemblage of signaling receptors and effectors at the cell surface. In this review we describe the current knowledge and the recent advances toward an understanding of the principles that dictate the organization of the signaling complexes as protein networks in the plasma membrane. 2. LIPID RAFTS DEFINITION: AN OVERVIEW Lipid rafts are membrane microdomains enriched in glycosphingolipids, sphingomyelin, and cholesterol, embedded in the glycerophospholipid-rich fluid plasma membrane. In general, these lipid microdomains can also be designated as GEMs (glycolipid-enriched membranes) or DIGs (detergent-insoluble glycolipid domains), so called due to their property of insolubility in cold 1% Triton X-100. Non-transmembrane proteins with raft affinity undergo lipid modifications that renders them hydrophobic and thus able to anchor to the plasma membrane. These modifications could be either a covalent linkage to glycosylphosphatidylinositol (GPI) in the case of outer leaflet insertion, or the attach-

1

Group of Cell Activation and Gene Expression, Institute for Molecular and Cellular Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. 2Abel Salazar Institute for Biomedical Sciences, University of Porto, Largo do Prof. Abel Salazar 2, 4099-003 Porto, Portugal.

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ment of S-palmitoyl fatty acid, possibly added by myristoylation of amino4,5 terminal glycine residues, for inner leaflet anchoring . A classical example of the latter is the Src-family protein tyrosine kinase Lck, which contains a myris6 toylation site at Gly2 and double palmitoylation sites at Cys3 and Cys5 . The first evidence for the existence of membrane microdomains came from the recognition of different levels or degrees of order in the membrane hydro7 phobic core, detected by fluorescent dyes . Further proof supporting this concept resulted from the observation that cholesterol- and sphingolipid-rich domains were resistant to non-ionic detergent solubilization at low temperatures, and 4,8 could be isolated as low-density fractions on sucrose gradient centrifugation . This is intrinsically related to the organizational levels of the lipids in the membrane; the tightly packed saturated lipid acyl chains and cholesterol, forming a liquid-ordered (Lo) phase will be more resistant to solubilization than the more fluid, liquid-disordered (Ld) phase. The cholesterol dependence of lipid rafts in order to form a liquid-ordered phase in the cell membrane has been demon9 strated in studies using model membranes . However, the principle of heterogeneity based on artificial models as well as the correlation of the lipid rafts concept with detergent insolubility has created much controversy in the field, 10 leading to very critical opinions on the nature and function of lipid rafts .

3. VISUALIZING LIPID MICRODOMAINS: FROM MODELS TO LIVE CELLS One property attributed to Triton X-100 is that it can partially solubilize the Lo phase, which may lead to loss of selected raft components, such as weakly asso11 ciated transmembrane receptors . Results obtained by Simons and coworkers suggest that the composition of detergent-resistant membranes is both dependent on cell type and detergent solubilization properties, raising questions on lipid 12 rafts definition based on a Triton X-100 insolubility criterion . Nevertheless, most detergents do not induce artifactual ordered domains or promote associa13 tion of solubilized components with Lo microdomains ; therefore, detergentresistant lipid microdomains can at most diverge from “physiological” rafts in that some components may be selectively lost during lysis procedures. Given that biochemical approaches produce controversial results, characterization of the lipid rafts and particularly the dynamics in living cells can best be achieved through the usage of advanced microscopy techniques. Contrary to the immunological synapse, which can be readily followed by light microscopy, lipid rafts in resting cells, termed elemental rafts, are submicroscopic complexes 14 (26 ± 13 nm) and cannot be visualized by classical microscopy methods . However, crosslinking raft-resident proteins induces the coalescence of rafts into patches of hundreds of nanometres in diameter that can be viewed by fluores15,16 cence microscopy .

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Several novel technical approaches have been used in an attempt to clarify the raft concept by characterizing the size and structure, and the diversity and dynamics of these microdomains. Fluorescence resonance energy transfer (FRET) measurements have provided evidence that raft markers exhibit an energy transfer pattern that is consistent with cholesterol-dependent clustering in 17-19 small domains . Using homotypic FRET, Sharma and colleagues have shown that a fraction of GPI-anchored proteins at the plasma membrane can be detected 20 in small (4-nm) and dense clusters of as few as 4 GPI protein molecules . Techniques such as fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) were brought into play to address questions concerning the diffusion and dynamics of individual raft proteins or lipids. The FRAP technology showed that the diffusion rate of the non-raft mutant of influenza hemagglutinin (HA) was superior to the rate of lateral diffusion of the 21 wild-type molecule . However, a different study using a similar approach showed that the dynamics of a set of raft and non-raft proteins were comparable, 22 and all molecules diffused freely over large areas . One limitation of FRAP measurements is that the temporal resolution is below the residence time of a protein in the rafts. With the SPT technique and the development of single fluorophore imag23 ing it is now possible to follow an individual molecule and measure its diffusion coefficient and confinement times within certain boundaries, allowing spa24-26 tial and temporal information on the molecule’s behaviour . Using total internal reflection fluorescence microscopy (TIRF) to track the trajectory of single raft-associated Lck and LAT, as well as the non-raft molecule CD2, Douglass and Vale surprisingly observed, in non-activated cells, a much larger 27 diffusion coefficient for raft molecules than for CD2 , although it can be argued that the relative immobility of CD2 may be due to its association with the cyto28 skeleton . The fact that any of these proteins can in some circumstances move in or out of lipid rafts still hinders any definitive conclusion on the mobility of lipid rafts based on protein markers, though.

4. T CELL RECEPTOR SIGNALING ASSOCIATION WITH LIPID RAFTS Upon productive engagement of the T cell receptor to peptide/MHC complexes presented at the surface of antigen-presenting cells, co-receptor (CD4 or CD8) binding to non-polymorphic regions of MHC molecules results in approximation of CD4/CD8-associated Lck to the TCR/CD3 complex. Lck is thus able to phosphorylate tyrosine residues within ITAM sequences present in the different CD3 chains, which become docking sites for the cytoplasmic kinase ZAP-70. Once ZAP-70 is targeted to the membrane, the TCR/CD3 complex is armed with a cocktail of protein tyrosine kinases, also including Fyn, that extensively phosphorylate tyrosine residues in a number of receptors and adaptors.

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The fact that several signaling molecules were found in association with lipid raft microdomains led to an assumption that rafts were more than just a structural peculiarity. In unstimulated cells, important signaling proteins such as 29 30 31 the adaptors LAT or PAG , or the Src-like kinase Fyn , are resident in rafts, whereas most transmembrane receptors are found outside these platforms. However, the protein content of the rafts as well as the activity of raft-associated proteins changes with cell stimulation. Signaling effectors like Lck that are in equilibrium between the two phases can change their behaviour or activity upon TCR triggering; while in the resting cell the fraction of Lck found in the rafts is in a close and inactive conformation, upon cell activation the open/active form 32 31 of Lck is most abundant in the rafts , being able to activate resident Fyn . The lipid modifications and consequent localization in lipid rafts seem crucial for the effective function of Lck, as a non-acylated form of the kinase failed to phos6 phorylate the TCR/CD3 complex . A recent study tackling the functional significance of membrane localization on the initiation of signal transduction, using mutant molecules engineered to not address rafts, produced intriguing results on the function of the lipid moiety of the molecules. A LAT mutant (C26/29A) that cannot be palmitoylated and a fusion protein consisting of the extracellular domain of LAX fused to intracellular LAT (LAX-LAT) were both detected outside rafts only. Just the LAX-LAT fusion could rescue T cell function both in vitro, as well as in vivo, 33 in transgenic mice expressing LAT mutants in a null background . So, whereas raft residency does not seem to be essential for the signaling activity, lipid modifications of raft-based molecules possibly tether membrane proteins stably to the membrane. As many signaling effectors do concentrate in lipid rafts, these membrane microdomains are thus considered as signaling platforms where optimal conditions for cell activation are met. A further function suggested for lipid rafts is to link the signaling machinery with the cytoskeleton. The requirement of cytoskeleton reorganization for sustained signaling has been previously demon34 strated , and the observation that filamentous actin and ξ chains are enriched in 35 lipid microdomains following CD48/TCR coligation strengthens that view . All these findings show or imply that the TCR itself associates with lipid rafts at some stage after selective stimulation of T cells, although there is some controversy on the kinetics of such an association: the TCR is either induced to 2,36 associate with lipid rafts or it is already resident, at least a small fraction, in a 37,38 subset of lipid rafts , these disputes again falling in the general discussion on the correct methodology. However, given that TCR engagement to MHC/peptide should precede all changes in cell behavior due to antigen recognition, including membrane and cytoskeleton reorganization, it is more appropriate to envision TCR association with lipid rafts from the receptor-centered point of view: T cell receptors are constrained to the immunological

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Figure 1. A model for membrane microdomain organization in T cell receptor signaling. For simplicity, only illustrative molecules are represented. In resting cells, lipid rafts may exist as regions of heterogeneous composition and size. Some proteins already partition into these domains either by GPI anchoring or by acylation, as is the case with the Src kinases Lck and Fyn as well as several adaptor proteins (e.g., LAT). Most transmembrane proteins are found outside these platforms. Upon antigen stimulation, rafts coalesce, bringing together the TCR and the signaling machinery. The current view sustains the idea that lipid rafts are highly dynamic structures, which is probably based on the high diffusion rate of resident lipid raft proteins. This mobility requires the ability of these proteins to interact with other proteins. When the proper protein–protein interaction is established this results in the creation of a signaling complex.

synapse, and lipid rafts/raft-resident molecules move to these cellular inter39 faces . Protein–protein interactions can thus be foreseen as the driving forces for redistribution of raft-associated proteins, as well as for the lipid phases (Figure 1).

5. DYNAMICS OF PROTEIN INTERACTIONS IN LIPID RAFTS Results mainly from raft patching experiments suggest that the association of the TCR with lipid rafts increases when the TCR is crosslinked together with acces40-42 sory proteins such as CD2, CD5, CD9, and CD28 . This has been suggested as a mechanism of costimulation, in the sense that the induced clustering of lipid 41,42 rafts resulted in increased tyrosine phosphorylation events . Additionally, CD28 costimulation induces the movement of actin cytoskeleton to the TCR 43 engagement site .

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Raft-controlled interactions, by their dynamic nature, should facilitate transient encounters between raft-resident proteins, whereas the interactions of raft with non-raft proteins could be limited because of the physical segregation in different membrane phases. This constraint is overcome if protein–protein interactions are prevalent over the raft/non-raft separation, and indeed no obstruction on the recruitment of non-raft molecules to the ordered lipid phase is evident from current experimental research. The co-receptor CD8 exists as a CD8αα homodimer or a CD8αβ heterodimer. Whereas the β subunit facilitates raft association, the α unit provides an extracellular binding site for MHC class I and an intracellular motif that binds to Lck. Since CD8αβ, but not CD8αα, partitions into rafts, it promotes the association with lipid rafts of a significant frac44 tion of TCR/CD3 that binds to CD8α/Lck . Therefore, protein–protein interactions can dictate localization at a certain moment for proteins within lipid raft microdomains. Recently, Douglass and Vale were able to show that while non-raft CD2 single molecules are mainly immobile, Lck and LAT showed abrupt changes from highly mobile to a highly immobile state, the latter occurring when they 27 spatially overlapped a signaling cluster . Using a series of mutants that interfere either with lipid raft targeting or with protein associations, they concluded that protein–protein interactions are determinant in selecting which non-raft molecules are retained or excluded from lipid rafts, rather than any type of raftaddressing label. As rafts were not found to be the primary factor in the clustering and diffusional immobility of CD2/Lck/LAT clusters, the authors propose that highly mobile signaling proteins diffusing in the membrane randomly encounter activated signaling complexes, and are captured by the emerging signaling complex provided the correct bonds can be established. CD2 and Lck have been shown to interact directly, and the recruitment of CD2 to lipid rafts relies 45-47 on such interactions . However, these specific protein interactions were not addressed in the study, so complementary work is required, with these and other molecules, before a solid model can be built.

6. CONCLUSIONS AND PERSPECTIVES Are lipid rafts central stages where multifactor signaling complexes are assembled, or are they platforms that carry the signaling apparatus where it is required, largely dependent on protein interactions, as recent advances seem to suggest? If so, what role is performed by the lipid content of rafts; is the lipid environment essential for signal transduction or is it merely the result of random or facilitated aggregation of proteins that are covalently connected to the plasma membrane? Development of more refined techniques that allow a better understanding of protein movements within the plasma membrane is thus required. The development of new sets of fluorescent lipids, such as polyene-lipids that exhibit a similar distribution in respect to the membrane ordered and disordered liquid

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phases as their natural counterparts, will make it possible to trace the movement 48 of individual proteins in membrane microdomains of diverse composition . With the appropriate tools, the concepts of the immunological synapse and clustering of proteins in lipid rafts microdomains will undoubtedly be assessed.

7. ACKNOWLEDGEMENTS This work was supported by grants from Fundação para a Ciência e a Tecnologia (FCT) and FEDER-POCTI. RJN receives a studentship and MAAC a fellowship from FCT.

8. REFERENCES 1.

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11 ROLE OF LIPID RAFTS IN ACTIVATIONINDUCED CELL DEATH : THE FAS PATHWAY IN AGING 1

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Anis Larbi , Elisa Muti , Roberta Giacconi , 2 1 Eugenio Mocchegiani and Tamàs Fülöp 1. INTRODUCTION Apoptosis is a complex process that involves a variety of molecules depending 1 on the receptor elicited . Following T-lymphocyte activation the immune system downregulates the proliferative response to avoid any uncontrolled inflammation. The immune system has evolved to eliminate T-lymphocytes by activation-induced cell death (AICD), which is a programmed cell death to control 2,3 cellular homeostasis in the periphery . T-lymphocyte activation and clonal expansion via TCR/CD28 ligation is thus controlled by immune response downregulation with AICD commitment. In one death receptor, which is able to initiate T-lymphocyte apoptosis, the Fas as well as its ligand, Fas-L, are overexpressed following TCR/CD28 activa4,5 tion . Initiation of AICD is controlled by membrane proximal events that in6 volve special membrane microdomains called “lipid rafts” . The formation of the death-inducing signaling complex (DISC) depends on lipid rafts in T7 lymphocytes . It has been reported that aging is accompanied with a change in apoptosis susceptibility. We already reported that lipid rafts in T-lymphocytes 8 undergo changes during aging that interfere with their functions . Hence, dysfunctionnal lipid rafts may play a role in immunesenescence and age-related 9 dysfuntions such as apoptotis susceptibility . In this chapter, we will review AICD in T-lymphocytes, the role of lipid rafts in T cell functions as well as age-related changes in T cell signaling with recent data from our group showing the relation between lipid rafts, AICD, Fas, and aging.

1

Research Center on Aging, Immunological Graduate Programme, Department of medicine, Department of Biochemistry, University of Sherbrooke, Sherbrooke, J1H 4C4, Québec, Canada. 2 Section Nutrition, Immunity and Aging, Immunology Centre, Research Department INRCA, 60121, Ancona, Italy.

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2. ACTIVATION-INDUCED CELL DEATH 2.1. Apoptosis There are two ways that cells can die — by necrosis (which induces an inflam10 mation) and by apoptosis (which is a programmed and controlled cell death). Necrosis is characterized by the rupture of cell membrane, which allows cellular content release responsible for the inflammatory process. In contrast to this necrotic death, apoptosis is programmed in order to avoid any inflammatory reac11 tion . Apoptosis seems to be the most common way to eliminate cells from the periphery. Apoptosis is defined by morphological changes including chromatin 12 condensation , nuclear fragmentation, apoptotic body formation, and cell shrinkage that culminate in cell suicide. The early phase includes loss of mem13 brane potential . The content of the apoptotic bodies is immediately removed by 14 macrophages, which recognize the externalized phosphatidylserines . This is an important characteristic of apoptosis since no inflammation will occur in the surrounding tissues. The immune response is controlled by cell–cell communication, cytokine levels, and chemokine concentrations. The induction of the immune response is rapidly ordered; cells from the innate part of the immune system such as neutrophils are the first barrier to an aggression. These cells have a very short lifespan (24 hours), as they die by spontaneous apoptosis, which can be delayed by pro15 inflammatory (anti-apoptotic) factors, such as the GM-CSF . The cells from the adaptive part are long-lived cells, such as lymphocytes. Cells being part of the immune system need a very strictly regulated mechanism to provide a tightly regulated immune response, mainly regarding intensity and duration. Apoptosis or programmed cell death is a very efficient way to control cellular homeostasis. Cells can undergo apoptosis at different levels of cell development — sometimes at very high levels such as during positive selection in the thymus where 95% of T cells are eliminated. Cellular homeostasis in the periphery is directed by cell renewal and programmed cell death. Apoptosis plays also a major role in shutdown of the immune response against an antigen, as shown in Figure 1. 2.2. Activation-Induced Cell Death in T Cells Apoptosis is not restricted only to selection but also to cell activation. Response to an antigen implies cell expansion; however, the immune response should be shut down when the antigen load has been controlled. Following the peak of activation and clonal expansion, there is a drop in specific T cell number due to 2 growth factor deprivation and activation-induced cell death (AICD) . The very last event in the immune response to an antigen is the maintenance of memory 16 cells, which is the result of controlled apoptosis . Cell death is determined by integration of the signal from death receptors. The stimulation of T-lymphocytes with anti-CD3 mAb is known to induce cellular activation, signaling, IL-2 pro17 duction, and clonal expansion .

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T-cell apoptosis T-cell expansion Pathogen killing Pathogen expansion

Figure 1. The role of apoptosis in the downregulation of the immune response. Following pathogen entry, T cell activation will induce their proliferation. Pathogen killing by effector cells is followed by a severe diminution of T cell proliferation and the beginning of T cell elimination by apoptosis. This phenomenon allows regulation of homeostasis.

Activation-induced cell death can be achieved in activated T cells via the T cell receptor and its CD3 complex concomitantly to CD28. While the expression of the TCR and MHC is stable in circulating lymphocytes, expression of the death receptor ligand, Fas-L, is very low. Thus, TCR ligation can lead to AICD, which contributes to downregulation of the immune response. Susceptibility to 18 apoptosis is increased by factors such as interleukin-2 (IL-2) . 2.3. Fas/Fas-L Death Receptor Death receptors are part of the superfamily of the Tumor Necrosis Factors (TNFs) involved in many cellular processes including differentiation, prolifera19 tion, and apoptosis . Death receptors are type I receptors with a conserved extracellular domain containing two to four cysteine-rich pseudo-repeats, a single transmembrane region, and a conserved intracellular domain about 80 amino 20,21 acids in length that binds to adaptor proteins and initiates apoptosis . Death receptor ligation induces receptor trimerization. Fas (CD95/Apo-1) is one of 22 these receptors that triggers cell death through the presence of a death domain in its cytoplasmic portion after receptor engagement with Fas-L or agonistic 23,24 anti-Fas antibodies . Other receptors belong to the TNF superfamily receptors, 25 including TNFR1 and TRAIL (TNF-related apoptosis-inducing ligand) recep26 tor . Fas/Fas-L play a major role in T cell AICD. Fas trimerization activates a sphyngomyelinase that is responsible for cera27,28 mide formation . Increased ceramide levels will initiate an intracellular signalization. Molecule recruitment to the death receptor is a critical step in Fasdependent AICD. A Fas-associated death domain (FADD) contains a death domain and functions as an adaptor protein that recognizes and binds with the

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Figure 2. Fas-induced apoptosis in T cells.

29

corresponding death domain of the intracellular part of Fas . FADD is important for recruitment of the components of the death-inducing signaling complex 2 (DISC) . The initiation of apoptosis via DISC formation involves the caspases (caspases-8 and -3) by the interaction of the death effector domain (DED) of the 30,31 caspase and of FADD . Then, pro-caspase-8 is cleaved and activated, which 32,33 allows caspase-3 actions for apoptosis commitment as shown in Figure 2. Fas-dependent apoptosis is facilitated by the increase in Fas expression at the cell surface of activated T cells while the role of soluble Fas-L is still a mat34-36 ter of debate . Alternative splicing, protease cleavage, and Fas sequestration are used to mediate AICD. While eight variants of Fas have been demonstrated, only one is functional. Concerning Fas-L, it is generated by posttranslational modification of Fas and metalloproteinases (MMP-7), an action that can lead 37 to variants . The modulation of Fas and Fas-L expression has been implicated 38 39 in several models of resistance to apoptosis and tumor progression . AICD 40,41 induced by TCR ligation is mediated by Fas/Fas-L expression . There is clear evidence that indicate the role of Fas/Fas-L in TCR-induced and IL-2induced AICD.

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3. ROLE OF LIPID RAFTS IN T-LYMPHOCYTES 3.1. What Is a Lipid Raft? Lipid rafts are membrane microdomains involved in T cell activation and are 7 critical for T cell signaling . Enriched in cholesterol and having the ganglioside M1 as a marker, these domains are mobile through the membrane. This ability to move is due to their difference in cholesterol/sphyngolipids packing compared to the rest of the membrane. The liquid ordered phase (Lo) of lipid rafts face the 42 liquid disordered phase (Ld) . Lipid raft heterogeneity is an idea gaining credence in the concept of plasma membranes. Actually, two interpretations of lipid 43 rafts have been posited . In the first one, lipid rafts may be small entities (<100 nm) that are composed of several different molecules, and that following stimulation all rafts are polarized to the site of stimulation; then the right protein– protein interaction will occur. In the second one, lipid rafts are composed of one kind of molecule and following stimulation all the rafts will form the signaling 43 platform, called a signalosome . This second interpretation might be closer to reality since molecule sequestration in different lipid rafts avoids activation, 44 which is possible only upon TCR/CD28 ligation . Lipid rafts have been shown to be involved in many processes, but mainly in signal transduction in T-lymphocytes. It has been shown that lipid rafts are not activated following CD3 activation alone. Both TCR and CD28 ligation are 45 required to reach the full state of activation of T-lymphocytes . In this connection, it has been shown that IL-2 production is dependent on CD28 activation. These data link T-lymphocytes activation, lipid rafts, and CD28. The signaling cascade that is provoked is involving a myriad of kinases and adaptors. By suc2+ cessive protein phosphorylation and the increase in free Ca , several nuclear factors translocate to the nucleus to allow protein expression and cellular re44,46 sponse . 3.2. T-Lymphocyte Signalling 2+

The mobilization of Ca as well as the activation of protein tyrosine kinases 47 lck (PTK) are the earlier events in TCR signaling . p56 , a member of the src family of PTK, which is regulated by CD45, is a crucial target of immunoreceptor tyrosine-based activation motifs of the CD3 complex and the ζ-dimer. ZAP-70 lck is recruited to the ζ-dimer and phosphorylated by p56 . Activated ZAP-70 phosphorylates the linker of activated T cells (LAT), which becomes a scaffold protein for the recruitment of multiple partners, including the adaptors Gads and Grb2, and the enzymes of phospholipid metabolism, phosphatidylinositol-348,49 kinase (PI3K) and phospholipase-Cγ1 . LAT-associated Gads bring SLP-76 to the plasma membrane, where it becomes phosphorylated, allowing its interaction with the exchange factor Vav, which provides a link between T cell activation, upregulation of integrin affinity/avidity, and reorganization of the cell cy50 toskeleton . Although the engagement of the TCR provides an essential signal

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to T cells, commitment to proliferation and differentiation will not occur unless a secondary signal is provided by ligation of CD28. CD28 signaling is still unclear; however, it has been shown that PI3Kinase is a cornerstone in CD2851 dependent activation . Lipid rafts have been shown to be involved in the earliest events of Tlymphocyte activation. For instance, src PTK, LAT, protein kinase C (PKC)-θ, Gads, WASP, and a host of other proteins are recruited to lipid rafts following 52 TCR ligation. The T cell receptor and CD28 are also localized to lipid rafts . Lipid raft polarization is visible by fluorescence microscopy. Many reports now have shown that lipid rafts can be isolated from plasma membranes using sucrose gradient ultracentrifugation of a lysate containing non-ionic detergent such 53 as Triton X-100. Lipid rafts float in the upper part of the gradient . After isolation, lipid rafts are loaded for SDS-PAGE analysis. Using electrophoresis and microscopy analysis, many studies have shown that signaling molecules are associated to lipid rafts or can be recruited to these membrane microdomains to increase signaling intensity. 3.3. Lipid Rafts and Apoptosis Fas trimerization induces ceramide production by sphingomyelinases, which plays a role in signal transduction and regulation of cellular differentiation, activation, survival, and apoptosis. Ceramides are sphingosine-based lipid second messenger molecules. Ceramide formation also changes membrane fluidity and permeability, which may interfere with membrane fusion processes 28 and the function of lipid rafts . Thus, ceramide can directly interact with signaling proteins or can facilitate the formation and trafficking of signal 54 transduction complexes . It has been shown that ceramide is essential for clustering of CD95 in sphingolipid-rich membrane rafts and for induction of 27 apoptosis . A defect in ceramide production as well as lipid raft disruption will 55 cause CD95 clustering and apoptosis . Recent studies have shown that 56 multidrug resistance is accompanied by changes in lipid raft composition . Many molecules relocate subcellularly in cells undergoing apoptosis. It has been shown that the pro-apoptotic molecule Bad localizes in lipid rafts in IL-4stimulated thymocytes. The disruption of lipid rafts by methyl-β-cyclodextrin (MBCD) induces segregation of Bad from lipid rafts and initiates apoptosis. It is 57 shown that Bad actively interacts with lipid rafts . More recently, it has been shown that Fas-induced apoptosis in CD4+ T-lymphocytes was inhibited by MBCD. Lipid raft disruption and disorganization interfere with commitment to apoptosis. This study also shows for the first time that Fas is associated to lipid rafts following Fas ligation. FADD and procaspase-8 were also present at higher 7,58 levels in the raft fraction isolated from Fas ligand treated T-lymphocytes . Another study has shown that upon TNFα binding TNFR1 translocates to lipid rafts, where it associates with the adaptor proteins TRADD and TRAF2, forming a signaling complex. By interfering with a lipid raft, the authors have

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shown a shift of TNF signaling from NF-kappaB activation to apoptosis . One 60 study used a polyphenol to test apoptosis resistance in tumor cells . All these studies point to the importance of lipid rafts in the apoptotic process in T cells. 3.4. Fas in Lipid Rafts It was shown that resveratrol did not modulate the expression of Fas and Fas-L but induced clustering of Fas and its redistribution in lipid rafts. This redistribution is associated with the formation of a death-inducing signaling complex 61 (DISC) including FADD and procaspase-8 . TCR ligation triggers Fas-L synthesis but also sensitizes activated T-lymphocytes to Fas-mediated apoptosis. Muppidi et al. have shown that TCR restimulation of activated human CD4+ T62 lymphocytes resulted in Fas translocation into lipid rafts . After Fas-L binding, these cells became sensitive to apoptosis. Using MBCD, they showed a reduced sensitivity to Fas-mediated apoptosis after TCR restimulation. Thus, the redistribution of Fas and other tumor necrosis factor family receptors into and out of lipid rafts may dynamically regulate the efficiency and outcomes of signaling by 62 these receptors . Another study supports these results in Jurkat and peripheral T cells, where caspase-3 was shown to localize in lipid rafts of untreated cells. Caspase-3, caspase-8, and FADD are further recruited to lipid rafts of Jurkat cells following anti-Fas treatment. Caspase-3 activity is shown to be essential 63 for caspase-8 processing . It has also been demonstrated that the pro-apoptotic member of the Bcl-2 family, Bad, segregates from lipid rafts during initiation of 64 apoptosis . Most of the studies used MBCD for lipid raft disruption. MBCD extracts 65 cholesterol from the plasma membrane and perturbs lipid raft organization . Cholesterol is an important component of lipid rafts that stabilizes their struc6 ture . A change in cholesterol content would have critical effects on lipid raftdependent processes. For example, TCR signaling is abolished following 65 MBCD treatment . Lipid raft disruption can also be attained by using filipin, cholesterol oxidase, or statins. This leads to clustering of Fas in the non-raft domains but still along with formation of Fas–FADD complexes, activation of caspase-8, and apoptosis. Following the second interpretation of lipid rafts evoked previously, lipid raft-associated Fas is sequestrated in lipid rafts and excluded from the other components of the DISC, avoiding commitment to 66,67 68 apoptosis . These data were also shown to be true in the case of neutrophils . How the DISC is formed depends strongly on the cytoskeleton. The actinlinking proteins ezrin, moesin, RhoA, and RhoGDI were recruited to lipid rafts in drug-treated leukemic cells. This indicates a major role of the actin cytoskeleton in DISC formation. This also highlights the crucial role of the grouping of 69 signaling molecule-enriched rafts in apoptosis . One study showed that membrane FasL-induced Fas clusters were formed in caspase-8- or FADD-deficient cells or when a cytoplasmic deletion mutant of 70 Fas was used . This strongly suggests that cluster formation is independent of

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the assembly of the downstream signaling complex. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-β-cyclodextrin, whereas Fas aggregation by soluble Fas-L was blocked. These data suggest that the extracellular domains of Fas and FasL are sufficient to drive formation of Fas–FasL complexes. On the contrary, soluble FasL-induced Fas aggregation is dependent on lipid rafts and mecha70 nisms associated with the intracellular domain of Fas . Altogether, experimental evidence suggests that lipid rafts may play a critical role in Fas-mediated signal transduction for induction of death of activated T cells (Figure 3). The physical association of Fas/FasL to lipid rafts with their effector molecules is essential for an ordered and effective AICD. Thus, as lipid rafts are essential for activation, they are also essential for death, which is a continuation of the same phenomenon.

Fas trimerization

Lipid rafts A SM

Bad

ASM

DISC Lipid rafts

ASM

Bad

Apoptosis Figure 3. Role of lipid rafts in activation-induced cell death. Fas trimerization initiates ASM (sphyngomyelinase) translocation to lipid rafts and ceramide production. This induces a change in lipid raft composition that facilitates Fas recruitment into lipid rafts. Trimerized Fas will induce the formation of the death-inducing signaling complex (DISC). Lipid raft-associated Bad is unphosphorylated, and during apoptosis Bad is phosphorylated and segregates from lipid rafts to accelerate apoptosis commitment.

4. THE AGING IMMUNE SYSTEM 4.1. Immunosenescence With aging, T-lymphocytes lose their capacity to produce IL-2 and to prolifer71 ate . T-lymphocyte senescence is part of the immunosenescence that has been demonstrated to exist in most of the other cells of the immune system. Neutrophils, macrophages, and NK T cells were all shown to lose some of their func-

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tions, while other functions are enhanced . It has been proved that the number of cells in the immune system remained unchanged; however, the ratio of these cells could fluctuate between cell types and cell subsets. For example, the num74 ber of NK T cells is significantly increased concomitant with the number of memory T cells (CD45RO+), while the number of naive T cells (CD45RA+) decreased. Thus, the immune system adapts itself to aging. The loss of T-lymphocyte function in aging has been intensely studied. However, the exact causes are still unknown. Several hypotheses have been put forward in the literature. Thymic involution has been observed during adult75 hood . This phenomenon causes shrinking of T-lymphocyte renewal while in the periphery the number of T-lymphocytes should remain constant. As a consequence, T-lymphocyte homeostasis is maintained by the accumulation and persistence of anergic T-lymphocytes in the periphery. These anergic T-lymphocytes are in replicative senescence since they do not respond to stimulation 76 or to apoptosis commitment . Aspinall et al. have established a protocol to counteract thymic involution in mice using the cytokine IL-7 and actually 77 78 achieved optimistic results . One other theory is telomere shortening . It is now known that during senescence telomeric length is diminished due to successive cellular division. Once the limit of cellular division and telomeric length are achieved, the cells enter into a “crisis” state that will lead to senescence or to cellular transformation. This is the Hayflick’s theory of cellular senescence. Several groups have demonstrated a decrease in telomere length in the T79 lymphocytes of elderly people . Surprisingly, telomeric length was correlated to 78 CD28 expression in T-lymphocytes . There is also a correlation between telomeric length and the state of replicative senescence. 4.2. T-Lymphocytes Functions during Aging Several functions of T-lymphocytes are hypothesized to change during aging. The reductions in IL-2 production, in proliferation, and in phosphorylation of molecules during the T cell receptor signaling cascade are hallmarks of T71 lymphocytes dysfunction during aging . The causes of these age-related defects are multifactorial, and several hypotheses have been tested, including the effect of oxidative stress, the levels of circulating cytokines, the effect of diet (vitamins 80 81 C, E, and A) , as well as chronic stimulation of T-lymphocytes (viruses) . Although the causes are still a matter of debate, there are clear-cut consequences affecting T-lymphocytes response to stimulation. Thus, following TCR and CD28 stimulation the signaling cascade was shown to be severely impaired, 82 which might explain defects in the T-lymphocyte response to stimulation . It was shown that the number of cells able to form an immune synapse declines 83 with age in mice . Also, a decline in the levels of heavily glycosylated CD43 is observed in aging. Thus, the cytoskeleton-dependent relocation of CD43 is im84 paired with aging . We have documented several years ago the defects in T cell signaling with aging in humans. As described in the §§3.1 and 3.4, lipid rafts

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may play a major role in T-lymphocyte signaling and in apoptosis commitment, respectively. In this connection, we have shown that impaired lipid raft functions are in part responsible for the defects in T-lymphocyte response under 8 TCR and CD28 stimulation . Thus, several aspects of T-lymphocyte activation are changed when lipid raft composition and function are modulated. We have shown that lipid raft disruption by cholesterol-extracting agents, such as methyl65 β-cyclodextrin (MBCD), led to severe impairments of T-lymphocyte function . We have observed that signaling molecule association to lipid rafts was completely abolished, leading to alterations in function, such as proliferation, which was inhibited. Following these results, it is suggested that many processes involving lipid rafts would be altered in T cells during aging. 4.3. Apoptosis during Aging Aging is accompanied by a shrinking in the T-lymphocyte repertoire mainly due to thymic involution. However, the organism should maintain a constant number of circulating cells and with aging we observe an increase in anergic circulating lymphocytes. These T-lymphocytes are in a senescent state, i.e., they do not respond to stimulation or to death signals. We described in the last section that T-lymphocyte signaling is impaired with aging, which could be explained by 85 this senescent state . Therefore, it could also be possible that the sensitivity of T-lymphocytes to apoptosis is changed with aging to compensate for thymic 86 involution . Such compensation could lead to a shrinking in the T-lymphocyte repertoire, thus explaining the increased susceptibility to infection. Data from the literature guide us to two possibilities. The first is that T86 lymphocyte susceptibility to apoptosis is increased . In this case, it was ob87 served that the expression of Fas is increased with aging in T-lymphocytes . Following Fas ligation, T-lymphocyte apoptosis is enhanced more importantly in the case of old donors. These data were confirmed at the protein level using flow cytometry and Western blotting, and at the mRNA level using quantitative 88 PCR . These cells also tend to spontaneous apoptosis. This tendency is explained by an increased expression of pro-apoptotic molecules, such as Bax/Bad, while anti-apoptotic molecule expression (e.g., Bcl-2/Bcl-xL) is not maintained. Furthermore, there was an increase in the activity of caspase-8 and caspase-3 compared to younger controls. Aggarwal et al. also demonstrated that protein expression of FADD, caspase-8, and caspase-3 was observed in quiescent lym89 phocytes from aging humans with their experimental conditions . With aging we observed an increased susceptibility to Fas-induced apoptosis due in part to the increase of pro-apoptotic signaling molecules and receptors. This could ultimately lead to a higher-potency DISC formation and apoptosis commitment. However, these data are still controversial since not much change in intracellular protein expression was described. Moreover, protein expression levels were assessed in Fas-induced cell death, and not during activation-induced cell death. Depending on the stimuli, T-lymphocytes could switch from susceptibil-

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ity to resistance to apoptosis . Also, it was recognized that these differences 91 could be the consequence of membrane function . The second hypothesis is that T-lymphocytes from elderly donors lose 91 apoptotic susceptibility due to a decrease in signaling functionality , also known 92 as apoptotic resistance . This could be explained by the increased number of replicative senescent T-lymphocytes that did not respond to stimulation because 93 of loss of functionality . We described in §4.2 that T-lymphocyte functions are altered with aging. These alterations could be explained by changes in lipid raft composition and function. In Figure 4 we depict Fas and Fas-L expression in stimulated T-lymphocytes. T-lymphocytes were activated with anti-CD3 and anti-CD28. After IL-2 supplementation for three days, T-lymphocytes from young and healthy elderly donors were analyzed by Western-blotting for death receptor expression. In Figure 4A we can clearly see a higher expression of Fas in resting T-lymphocytes from elderly donors, as measured in Figure 4B by densitometry. These data were confirmed with flow cytometry experiments (data not shown). Following activation, T-lymphocytes increased Fas expression in both groups of donors; however, it is significantly more pronounced in the case of young donors. When stimulation was enhanced concomitant with lipid raft disruption by MBCD, we can observe an increase in Fas expression at the protein level. However, this was significantly lower compared to non-treated cells. Alteration of lipid raft structure led to an alteration of activation-induced cell death. This was true in the case of both groups of donors. T-lymphocytes from young donors treated with MBCD could increase Fas expression to the same level as untreated T-lymphocytes from elderly donors. Fas-L expression is much lower when compared to Fas and very low in non-stimulated cells (Figure 4C). When stimulated with anti-CD3 and antiCD28 in addition to IL-2, T-lymphocytes increased Fas-L expression. We were not able to find any significant difference between young and elderly donors. However, MBCD-treated T-lymphocytes were completely inhibited toward FasL expression (Figure 4D). These results corroborate the fact that isolated lipid rafts during AICD contain many more Fas molecules in T-lymphocytes in elderly donors compared to young donors. Fas shifted to lipid rafts during AICD, while this movement was significantly decreased with aging (data not shown). These data are in accor95 dance with those obtained by Yokoyama et al. Activation-induced cell death is defective in T-lymphocytes from elderly 94 donors . In our experimental conditions we stimulated T-lymphocytes with antiCD3 and anti-CD28 to mimic antigen presentation even if this does not reflect the whole reality. At the end of the stimulation we observed a lower expression of Fas in T-lymphocytes from elderly donors. This difference could be explained by the defects in TCR/CD28 signaling that are known to be impaired with aging. Thus, activating signals could not properly be transmitted for a full activation

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Figure 4. Activation-induced cell death in aging. T-lymphocytes are separated routinely in our laboratory. CD3+ T-lymphocytes were stimulated for 72 hours with IL-2 and then stimulated with antiCD3 and anti-CD28 (S) for 24 hours. Alternatively, T-lymphocytes were stimulated concomitantly to treatment with methyl-β-cyclodextrin (C = 0.5 mM). The cells were then lysed and 25 µg of protein was sized on SDS-PAGE. Membranes were probes with anti-Fas and anti-Fas-L antibodies. (A) T-lymphocytes increased Fas expression following stimulation while cyclodextrin treatment reduced Fas expression. (B) Densitometry was performed on results obtained from three different donors of each group (*, p < 0.05). The same experiments were performed for Fas-L expression in C with the densitometry in D.

state. In this case, T-lymphocytes may not be activated to a sufficient level to accomplish cell death by AICD. Lipid rafts are the structures involved in impaired T-lymphocyte signaling and immunosenescence. Here we disrupted lipid rafts with MBCD and found that Fas expression (Figure 4) and apoptosis commitment were altered (data not shown). When lipid rafts were disrupted, we observed that the level of Fas expression was similar to that of T-lymphocytes from elderly donors. Since lipid raft composition and function are altered with aging, we can suppose that the defect in AICD commitment with aging could be explained by lipid raft differential composition with aging. We demonstrated that lipid raft cholesterol content was increased twofold with aging. The subsequent decrease in membrane fluidity is partly responsible for decreased protein movement and recruitment to the membrane as well as impaired T-lymphocyte functions.

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5. CONCLUSIONS T-lymphocytes are key players in the immune response. An imperfection in Tlymphocyte functions could lead to a variety of pathologies. Aging is not a disease, but a normal process that induces several changes and adaptation. Among them we can spell out thymic involution that leads to T-lymphocyte repertoire shrinkage. Another adaptation to thymic involution is the maintenance of nonresponding T-lymphocytes to balance cell number for homeostasis maintenance. The maintenance of the number of circulating T-lymphocytes could be possible by apoptosis resistance. Activation-induced cell death is delayed in T-lymphocytes from elderly donors by impaired Fas expression following stimulation. As shown by MBCD experiments, lipid rafts may play a role in these age-related events of apoptosis commitment and defect. Since Fas was not localized to lipid rafts with aging during AICD, we can easily hypothesize that the formation of the Death-Inducing Signaling Complex is also impaired. Ongoing studies in our laboratory could help us to understand the mechanism by which T-lymphocytes modulate apoptosis sensitivity. If confirmed, the modulation of lipid rafts could lead to several important clinical applications.

6. ACKNOWLEDGMENTS This work was partly supported by a grant-in-aide from the Canadian Institute of Health Research (No, 63149), by the Research Center on Ageing and the Immunological Programme, the Research Department of INRCA and the ZINCAGE project (FOOD-CT-2003-506850).

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12 T CELL RESPONSE IN AGING: INFLUENCE OF CELLULAR CHOLESTEROL MODULATION Tamas Fulop,1,2,3 Gilles Dupuis,2,4 Carl Fortin,1,2 Nadine Douziech1 and Anis Larbi1,2

1. INTRODUCTION Membrane lipids play a major structural and functional role in the cell membrane, where they can influence the conformation, function, and regulation of receptors and signaling pathways. Cholesterol is an essential component of the 1,2 cellular plasma membrane, especially in T cells . The recently discovered lipid rafts reinforced this well-known fact. Lipid rafts are specialized membrane domains that are mainly composed of cholesterol and sphingolipids, playing a fun3-5 damental role in signal transduction and immune synapse formation . Many signaling molecules are constitutively associated or recruited to lipid rafts including src kinases, LAT, and Csk, while others are definitively or temporarily 4,5 excluded from it . This will assure a specific protein–protein and protein–lipid 6 interaction . However, the specific effects of the selected lipid components are 7 still unknown and controversial . The metabolism of cellular cholesterol is very complex and depends on uptake by LDL receptors, on intracellular cholesterol synthesis by HMGCoA reductase, and on cholesterol extraction. This should be tightly synchronized, as a dysregulation in any of these steps could result in alteration in membrane cholesterol levels, resulting in cellular dysfunction. With aging we assist to an alteration in T cell functions, such as proliferation and interleukin-2 (IL-2) production, partly resulting from altered TCR and CD28 signaling due to a twofold increase in the cholesterol content of T cell 8 membranes, especially of that of lipid rafts . Thus, a modulation of the cholesterol content of T cell membranes could have important functional

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Research Center on Aging, 2Immunological Graduate Programme, 3Department of Medicine, 4Department of Biochemistry, University of Sherbrooke, Sherbrooke, J1H 4C4, Québec, Canada

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repercussions. In the following we will review how membrane cholesterol modulation of T cells can affect their functions and present some new results obtained on T cells and Jurkat cells that can be related to the immunosenescence.

2. MODULATION OF CHOLESTEROL CONTENT IN T CELL MEMBRANES BY CYCLODEXTRIN Membrane cholesterol has been shown to be involved in the activation or inactivation of various regulatory proteins, to influence membrane protein interactions, and to affect the functions of several membrane receptors, including CCK, 9 acetylcholine, and TCR . Thus, cholesterol should be maintained in a very narrow range. Consequently, cellular cholesterol homeostasis is a tightly regulated process. The maintenance of cell membrane cholesterol content depends on cho10,11 lesterol uptake mainly from circulating LDL or by de novo intracellular syn12 thesis . When LDL enters the cells taken up by its high-affinity LDL receptors, endogenous cholesterol synthesis is suppressed and the LDL receptors on the 13 cell surface are downregulated . This ensures that no cholesterol excess occurs inside the cells, which could render them dysfunctional. If for any reason cholesterol would increase in the cells, this could be eliminated by extraction of the excess cholesterol. This process is called reverse cholesterol transport, mainly 14,15 mediated by HDL . Thus, the cells, including T cells, rely on a tight regulation of their cholesterol content. The demonstration that lipid rafts, which are mainly composed of cholesterol and sphingolipids, are crucial for TCR and CD28 signal transduction 7 initiated a renewed interest in cholesterol . The modulation of cholesterol in lipid rafts has been demonstrated to affect T cell signaling and consequently their functions. Thus, TCR and CD28 receptors may be very sensitive to the plasma membrane lipid environment. Recently, several means have been developed to either extract or enrich the T cell membrane in cholesterol (Figure 1). 2.1. Extraction The most used means to extract T cell membrane cholesterol and modulate the early signaling events is to use cyclodextrins (methyl-β-CD). This substance in various concentrations is able to extract cholesterol specifically from the exo16,17 facial layer of the membrane . The effect depends on its concentration. In high concentrations (10–20 mM) MBCD is able to perturb definitively the TCR signaling by inhibiting lipid raft functions and immune synapse forma18 tion . In smaller concentrations (0.5–1 mM) it is able to only disrupt TCR signaling and disorganizing T cell functions. However, it was recently also demonstrated that MBCD has some other effects that interfere directly with the TCR signaling, such as increasing per se tyrosine phosphorylation of

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Figure 1. Cholesterol extraction/loading in T cells. T cells were treated with hydroxypropyl-βcyclodextrin (HβCD) alone to extract cholesterol. Cholesterol (3β-hydroxy-5-cholestene) in EtOH was incorporated into HβCD molecules (120 mM in PBS), forming cholesterol–HβCD complexes before loading the cells. Cells were loaded with 5 to 100 µl of cholesterol-HβCD to 5 x 106 cells in 1 ml of RPMI. Cells were incubated at 37°C for 15 min, washed with PBS, and resuspended in PBS for flow cytometry analysis. To assess cholesterol content in T cell membranes, cells were incubated with filipin, which is recognized for its particular fluorescence. Filipin binds to cholesterol, and after washing cells were analyzed for fluorescence intensity. The dashed line indicates the fluorescence of filipin in non-treated cells. The gray line indicates the HβCD-treated cells as confirmed by the shift of fluorescence, while the dark line represents HβCD-cholesterol-treated cells, which clearly shows an increase of fluorescence and incorporation of cholesterol into the T cells.

19,20

various src kinases . Other means to extract lipid raft cholesterol is to use fili21 pin, niacin, and other similar substances . The experimental results suggest that extraction of cholesterol from the lipid rafts, by MBCD, can profoundly influence T cell signaling and conse22 quently T cell functions . However, MBCD could have an effect also by other means to influence TCR signaling. In the case of T cells of an elderly subject, the cholesterol content is increased twofold, resulting in altered early TCR sig23 naling, leading to altered T cell functions . By extracting cholesterol with MBCD from elderly T cell membranes we could restore some T cell functions, 24 such as proliferation . This further supports the contention that cholesterol is one of the major regulators of TCR signaling and that an increase in cholesterol content alters it, but plasma membrane cholesterol content restoration helps to nearly normalize T cell functions. Recently, we explored new ways to extract cholesterol from the lipid rafts 25 that contain the “fast extractable” cholesterol pool, corresponding to lipid rafts . The “slow extractable” cholesterol pool corresponds to the non-raft portion of the membrane. These various cholesterol pools have been demonstrated in Jur7 kat cells as well . This extraction can also be realized by the use of HDL, medi14,15 ating reverse cholesterol transport . The extraction of lipid raft-cholesterol will allow extraction of cholesterol from the other domains of the plasma membrane. Our results show that HDL is indeed able to extract the cholesterol very rapidly (60–90 minutes) from lipid rafts, which has functional effects on T cells (unpublished data).

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Thus, modulating the cholesterol content of T cells by extracting cholesterol from lipid rafts has dramatic effects on TCR signaling and on T cell functions depending on the cholesterol levels in the lipid rafts. In normal conditions it has a disrupting effect, while in pathological conditions (i.e., cholesterol content increase) it can normalize it. Nevertheless, it should be mentioned that the exact role of cholesterol in T cell signaling and immune synapse formation is passion7 ately debated and questioned . 2.2. Addition Much less is known in relation to T cell functions related to an increase in cholesterol content. It is at least as important to study the role of cholesterol content increase as that of a decrease. Now, there exists the possibility not only of extracting but also to adding cholesterol to the membrane by using another cyclo26 dextrin: hydroxypropyl-β-cyclodextrin . Addition of cholesterol to the membrane in this manner has been shown to have dramatic effects on T cell 26 functions. Nguyen et al. have demonstrated that enrichment of T cells with cholesterol induced significant inhibition of intracellular calcium mobilization by anti-CD3. Moreover, they have also shown that cholesterol-loaded peripheral T cells were completely unresponsive to anti-CD3/anti-CD28 stimulation, resulting in blunted IL-2 production. These results fit perfectly with those found in T cells of elderly subjects where an identical cholesterol content increase oc23 curs . Identical results were found in the case of the CCK receptor, where the 9 increased cholesterol content resulted in inactive, uncoupled receptor status . Furthermore, an increase in the cholesterol content was also found in neurons of the elderly in relation to Alzheimer’s disease, and this increase will determine whether the processing of amyloid precursor protein will be in favor of insoluble 27 β-amyloid or the soluble physiological molecule. The increased cholesterol may induce changes affecting receptor conformation, binding, or interaction with their effector signaling proteins. These results collectively reinforce the role of cholesterol in T cell signaling leading to specific functions. Moreover, as a similar situation, the dysregulation of cholesterol homeostasis with aging could contribute to altered T cell func8 tions, leading to the immunosenescence phenomenon .

3. EFFECTS OF CHOLESTEROL CONTENT MODULATION IN T CELL MEMBRANE: MEASUREMENT OF T CELL SIZE AND INTERACTION WITH COATED BEADS We intended to further explore the effects of cholesterol loading on the physicochemical status of T cells, as a surrogate for changes in T cells with aging. As mentioned earlier, the signaling of TCR and CD28 is altered by cholesterol enrichment, and this results in decreased function. There are only a few data to

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explain by which means the cholesterol content modulation may exert these effects. 3.1. Size of Cholesterol-Enriched T Cells Cholesterol enrichment may also induce morphological changes, as a result of 28 cholesterol on lipid dynamics, membrane curvature, and tension . We measured the size and granularity of human T cells at basal status, after cyclodextrin treatment and after cholesterol loading. While the HβCD treatment did not influence significantly these T cell parameters, enrichment by cholesterol induced a significant increase in T cell size and granularity (Figure 2). It is known that 29 the increase in cell size specifically influences cell functions . Thus, definitively, cholesterol enrichment increases cell size, leading to disorganization of the membrane structure inhibiting correct functioning. 3.2. T Cell-Bead Contact Formation: Mimicking the APC–T Cell Interaction Activation of T cells necessitates their interaction with antigen-presenting cells 30 (APC) . This occurs through TCR and co-receptor activations by the APC counterpart molecules. The changes in T cells following modulation of cholesterol membrane level could interfere also with their interactions with APCs. For mimicking this interaction we used beads coated with anti-CD3 and anti-CD28. Thus, we further explored the effects of changes in membrane cholesterol by performing experiments with beads, coated with anti-CD3 and anti-CD28 mAbs, and Jurkat cells, during 6 hours, and measuring their interactions (Figure 3). Using flow cytometry we are able to assess the amount of cell–bead contact formation. When resting Jurkat cells (A) were incubated with beads (B) we can observe the changes in FSC that correlated with cell–bead association. When Jurkat cells were treated with methyl-β-cyclodextrin (D) there was a complete shift in the FSC parameter. This was visualized by the loss of Jurkat cell–bead contact formation since no changes between stimulated and unstimulated cells could be observed. This means an inhibition of the interaction between Jurkat cells and specifically coated beads due to cholesterol extraction. This signifies that perturbing the cholesterol content inhibits the ligand/APC and Jurkat cell interaction, leading to altered signaling and T cell functions. Altogether, the changes in T cell cholesterol content induces important physicochemical alterations, including changes in their size and granularity, leading to an altered interaction between coated beads and Jurkat cells, ultimately mimicking the T cell–APC interaction. This might explain why changes in cholesterol content, i.e., a decrease or increase such as what happens in aging, may lead to functional defects in T cells.

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Figure 2. Cholesterol enrichment of T cells by HβCD. Resting T cells (A) were treated with cyclodextrin alone (B) or in combination with cholesterol (C) as described in Figure 1. Microscopy analyses (left panel) show T cell shape concomitant with flow cytometry (right panel). The cyclodextrin treatment did not change T cell size, while cholesterol enrichment increased their size.

4. FUNCTIONAL EFFECTS OF CHOLESTEROL MODULATION ON JURKAT CELLS T cell functions are altered with aging, mainly in terms of proliferation and IL-2 31-33 production . The exact cause is not known; however, alteration of TCR and 34,35 CD28 co-receptor signaling could contribute . This alteration in signaling 23 could be related to an increased cholesterol content . Previously, we have shown that modulation of plasma membrane cholesterol in T cells by extraction 24 influences the TCR and CD28 co-receptor signaling through lipid rafts . In the present paper we have shown that cholesterol addition as well as extraction has a significant effect on T cell physicochemical properties and coated bead–Jurkat cell interactions.

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Figure 3. Cholesterol extraction by MBCD and its effects on the capacity to form bead complexes. Jurkat cells were left untreated (A) or stimulated with anti-CD3 and anti-CD28 coated beads (B). While C represents MBCD-treated Jurkat T-cells, panel D shows the defect in association of these cells when incubated with anti-CD3 and anti-CD28 beads, as performed in B.

We further investigated the direct effect of plasma membrane cholesterol modulation on Jurkat cell proliferation (Figure 4). We found that after 24 hours of culture there was no difference between anti-CD3 and anti-CD28 stimulated Jurkat cell proliferation and cholesterol-enriched Jurkat cell proliferation. As a control we used other cholesterol-modulating agents — such as oxLDL and lovastatin. OxLDL induced a slight but significant decrease in Jurkat cell proliferation, while lovastatin, an HMG-CoA reductase activity-inhibiting statin, induced massive Jurkat cell death. After 72 hours of culture we assist to a significant Jurkat cell proliferation under anti-CD3 and anti-CD28 stimulation (p < 0.01) in comparison to the baseline state. The enrichment by cholesterol induced a dramatic decrease in Jurkat cell proliferation. This demonstrates that the increase in membrane cholesterol leads to altered T cell function, namely, proliferation. These results correlate well with the decreased IL-2 production follow26 ing T cell membrane cholesterol enrichment performed by the group of Taub . 23 Identical effects were seen in the T cells of elderly subjects . oxLDL and lovastatin induced cell death in culture. This cell death occurred by apoptosis as

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measured by Annexin V expression (data not shown). This demonstrates that either an oxidative stress by oxLDL or inhibition of endogenous cholesterol synthesis by a statin leads to the death of T cells. Similar effects were observed on 36 prostate cancer cells by simvastatin . These data reinforce previous results underscoring the importance of cholesterol for T cell function. 37 Statins have been shown to have powerful immunomodulating effects . Cholesterol enrichment was shown in xenograft tumors to increase tyrosine 36 phosphorylation, while statins decreased it . We further investigated the effect of lovastatin on tyrosine phosphorylation by using FACScan (Figure 5). We observed earlier that Jurkat cells died even after 24 hours of culture. During short-time incubation (60 minutes) lovastatin, in various concentrations, did not influence the tyrosine phopshorylation induced by antiCD3 and antiCD28 stimulation. After 18 hours of stimulation we observed a further significant increase in tyrosine phosphorylation in Jurkat cells (p < 0.05) compared to basal and 60-minute stimulations. In contrast, in the presence of lovastatin, independent of its concentration, tyrosine phosphorylation was completely inhibited, which corroborates earlier results on the possible immunosuppressive ac36 tivities of statins . Altogether, these data reinforce the hypothesis that enrichment of membrane cholesterol synthesis leads to decreased proliferation, while inhibition by lovastatin of endogenous cholesterol synthesis leads to Jurkat cell death by blunting the tyrosine phosphorylation induced by TCR and CD28 co-receptor stimulation. It is well known that statins have very powerful immunomodulating effects, and this is used in their therapeutical effects to decrease the 38 atherosclerotic inflammatory process . However, the fact that they can induce the death of Jurkat cells in stimulatory conditions, and taking into account their use as preventive agents, should induce new studies to exclude that they may cause an immunosuppressive status even in physiological conditions.

5. CONCLUSION It has been proposed that the lipid environment is critical for the molecular in1-6 teractions in the plasma membrane to occur . The recent discovery of lipid rafts, which are composed essentially from cholesterol, generates a new interest in cholesterol as cell function-modulating molecules. There are also lipid rafts in T cells, and thus cholesterol may also be important for T cell function. With aging we assist to a decrease of T cell function related to decreased TCR and CD28 co-receptor signaling. An increase in cholesterol content in T cell lipid rafts has been shown with aging. The studies extracting cholesterol from T cell membranes by MBCD leave little doubt that cholesterol is essential for TCR and

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Figure 4. Proliferation of Jurkat T-cells modulated by lipids and the HMG-CoA reductase inhibitor lovastatin. (A) Jurkat T cell proliferation was modulated using HβCD (upper right panel), oxidized LDL (lower left panel), or lovastatin (lower right panel) for 24 hours. The same experiments were performed after 72 hours of incubation. (B) Microscopy photographs show the extent of cellular proliferation.

CD28 signaling by maintaining membrane physicochemical integrity and protein signaling molecule segregation. Nevertheless, this generated much controversy either because this is an indirect proof or because the results are contradictory. We showed here that the enrichment of T cells or Jurkat cells with cholesterol induces similar alterations that we observed during aging, such as

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Figure 5. Intracellular phosphorylation is modulated by long exposure to lovastatin. T cells were treated with lovastatin for short (1 hour) and long (18 hours) periods of time. Then the cells were stimulated with anti-CD3 and anti-CD28. Intracellular phosphorylation was assessed using Flow cytometry. Cells were permeabilized and fixed for FACS analysis as already described23. Resting cells (A) were stimulated for 5 minutes (B). When lovastatin was used to pretreat the cells for 1 hour at 1 (E) and 10 µM (F), there were no differences in phosphorylated molecule levels after stimulation. A long exposure to lovastatin (G and H) led to a decrease in the levels of phosphorylated molecules when compared to untreated stimulated cells (D) and resting cells (C).

increased cellular size, decreased T cell/APC interaction and proliferation. Furthermore, lovastatin decreasing endogenous cholesterol synthesis has deleterious effects, as it induces cell death by blunting tyrosine phosphorylation induced by TCR and CD28 co-receptor stimulation. These data further reinforce the necessity of a tight metabolic control of membrane (lipid raft) cholesterol content for optimal T cell function. Either an increase or decrease perturbs T cell function,

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leading to an inappropriate cellular response. Moreover, the increased cholesterol content of T cells with aging could be a target for modulation to restore normal T cell function. Future studies are needed to explore this aspect of immunosenescence mainly by the potential effect of HDL, which is a natural cholesterol transporter, on lipid raft cholesterol content.

6. ACKNOWLEDGMENTS This work was partly supported by a grant-in-aide from the Canadian Institute of Health Research (No, 63149), through the Research Center on Aging and the Immunological Programme.

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29. A. Bellacosa, C.C. Kumar, A. Di Critofano and J.R. Testa, Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res 94, 29–86 (2005). 30. T.M. Razzaq, P. Ozegbe, E.C. Jury, P. Sembi, N.M. Blackwell and P.S. Kabouridis, Regulation of T-cell receptor signalling by membrane microdomains. Immunology 113(4), 413–426 (2004). 31. M.M. Zatz and A.L. Goldstein, Thymosins, lymphokines, and the immunology of aging. Gerontology 31(4), 263–269 (1985). 32. M.P. Chang, T. Makinodan, W.J. Peterson and B.L. Strehler, Role of T cells and adherent cells in age-related decline in murine interleukin 2 production. J Immunol 129(6), 2426–2434 (1983). 33. N. Douziech, I. Seres, A. Larbi, E. Szikszay, P.M. Roy, M. Arcand, G. Dupuis and T. Fülöp, Modulation of human lymphocyte proliferative response with aging. Exp Gerontol 37(2-3), 369–387 (2002). 34. A.A. Sadighi-Akha and R.A. Miller, Signal transduction in the aging immune system. Curr Opin Immunol 17(5), 486–491 (2005). 35. G. Pawelec, K. Hirokawa and T. Fülöp, Altered T cell signaling with aging. Mech Age Dev 122(14), 1623–1637 (2001). 36. L. Zhuang, J. Kim, R.M. Adam, K.R. Solomon and M.R. Freeman, Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. J Clin Invest 115(4), 959–968 (2005). 37. F. Mach, Satatins as Immunomodulatory agents. Circulation 109(21 Suppl 1), II15– 117 (2004). 38. J.C. Plana and P.H. Jones, The use of statins in acute coronary syndromes: the mechanisms behind the outcomes. Curr Atheroscler Rep 3(5), 355–364 (2001).

13 LYMPHOCYTE SIGNALING AND THE TRANSLATABILITY OF MRNA Suzanne Miyamoto

1. INTRODUCTION Translation is regulated during the mitogenic stimulation of lymphocytes, and this regulation is suggested to be dependent on global and specific translation 1 mechanisms . Numerous laboratories have focused their research on the regulation of translation necessary for growth and proliferation (reviewed by Mathews, 2,3 4 5 Sonenberg and Hershey , Morris , and Pain ), and many of these regulatory mechanisms may also apply to lymphocyte signaling. Translational control has also been found to be important in developmental biology, insulin signaling, synthesis of hemoglobin in reticulocytes, synthesis of proteins in viral and phage-infected cells, and synthesis of proteins in mammalian cells responding to heat shock, hormones, starvation, and growth-controlled receptor-mediated 6 events . Global mechanisms regulate the overall growth-related increase in 7-9 translation initiation and possibly translation elongation , whereas specific translation mechanisms target the selective recruitment or increased efficiency of the translation of individual mRNAs. Regulatory elements located in the unique sequence or structure of the 3′ and 5′ untranslated regions of the transcribed mRNAs may actually control the stability, recruitment, and efficiency by which each mRNA is translated. Proteins are synthesized from mRNAs through a complex process called translation. This process of translation involves the participation of a large number of eukaryotic initiation factors (eIFs), transfer RNAs (tRNAs), ribosomal 2,5,10 subunits, and translation elongation factors . This review will highlight our current knowledge of the mechanisms of translation in the lymphocytes, describe how translation mechanisms might be regulated during the early activation process of the lymphocytes, and speculate about the importance of this

Division of Hematology/Oncology, University of California Davis, Cancer Center, Sacramento, CA 95817, USA

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regulation to the process of lymphocyte activation. It will also include a brief discussion of how dysregulation of signaling pathways might involve changes in translation of specific mRNAs that likely contribute to the onset of leukemia and lymphoma. Indeed, recent microarray-based studies show that response to oncogenic Ras and Akt signaling can rapidly alter the pattern of existing mRNAs recruited to the polysomes, even before any measurable transcriptional change. So it is entirely possible that it is translation, and not only transcription changes, which result in the immediate gene expression changes. These protein expression changes could play a significant role in the onset of diseases like cancer.

2. BASIC MECHANISMS OF THE INITIATION OF TRANSLATION AND FACTOR ACTIVITY It is important to review our current model of translation initiation, with emphasis on the role of the initiation factors, and indicate where the potential areas of 11 regulation might be located (reviewed in detail by Merrick and Hershey ). Our current model of the basic mechanism of translation initiation is based on early in vitro reconstitution studies conducted with purified proteins and ribosomes isolated from rabbit reticulocyte lysates. A simplified schematic of the stages of translation initiation is presented in Figure 1. Initially, (A) the 80S ribosome dissociates into its 40S and 60S subunits. There is evidence indicating that eIF3 might be involved in this process. (B) A ternary complex forms consisting of translation initiation factor eIF2 (a complex of three subunits, α, β, γ), GTP, and methionyl-tRNAi, the transfer tRNA that contains the first amino acid (methionine) of every protein. (C) An important regulatory step in lymphocyte activation, insulin signaling, and other signaling systems is phosphorylation of the eIF4E 12-19 binding protein 4E-BP1/2 (also know as PHASI/II) . Phosphorylation of 4EBP1 is an independently regulated step that is connected to formation of the eIF4F complex. In quiescent G0, resting peripheral blood lymphocytes, most messenger RNAs (mRNA) coding for cellular proteins contain an m7GTP cap structure and a polyadenylated (polyA) tail. The m7G-cap structure is recognized by the small translation initiation factor eIF4E, often referred to as the cap-binding protein. In resting T lymphocytes and other physiological systems, 18,20,21 eIF4E is bound to 4E-BP1/2 . Phosphorylation of 4E-BP1/2 causes the release of eIF4E, which is now able to bind mRNA and to form the eIF4F complex (composed of eIF4E, eIF4G, and eIF4A). Through eIF4E, eIF4F recruits capped mRNA to the 40S ribosomal subunit. (D) The 43S ribosome is formed with the ternary complex containing eIF2, eIF3, eIF1A, eIF1, and the 40S ribosome subunit. eIF3 is the largest of the initiation factors needed for translation 22 and consists of at least 11 subunits, (eIF3a-k) , which are somewhat depicted in this diagram. The mechanism and timing of the addition of these factors is still not well understood. (E) The 48S pre-initiation complex is formed from the recruitment of mRNA through the interaction between subunits of eIF4F

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Figure 1. Stages of translation initiation. An abbreviated rendition of the stages that occur during the initiation of translation: (A) dissociation of 80S ribosome into 40S and 60S ribosomal subunits; (B) formation of the ternary complex with eIF2, GTP, and methionyl tRNAi (Met-tRNAi); (C) phosphorylation of 4E-BP1, release of eIF4E, formation of eIF4F, and binding of the m7G-capped mRNA; (D) formation of the 43S ribosome containing eIF3, ternary complex, eIF1A, eIF1, 40S ribosomal subunit; (E) formation of the 48S ribosome containing mRNA; (F) formation of the 80S ribosome; (G) eIF2B exchange activity with eIF2. PABP is poly A binding protein, m7G presents the cap structure of mRNA; hatched borders represent mRNA, and AAAA represents the poly A tail.

(eIF4G) and eIF3. eIF4B is thought to associate with eIF4F and together with the eIF4A of the eIF4F complex enhances eIF4A helicase that has the ATPase activity needed to melt mRNA secondary structure. The initiation complex on the 48S subunit scans mRNA until the initiator AUG is recognized. At this point, initiation factor eIF5 promotes the hydrolysis of GTP bound to eIF2, and initiation factors are ejected. (F) The 60S subunit joins to form an 80S initiation complex, which is now capable of synthesizing the rest of the protein from its mRNA. (G) Because eIF2 is ejected as a stable GDP binary complex, its bound GDP must be exchanged for GTP in a reaction promoted by the guanine nucleotide exchange factor, eIF2B. More detailed descriptions of initiation factors and 5,11, 23-25 the process of initiation can be found in published reviews .

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3. TRANSLATION STUDIES IN T LYMPHOCYTES Studies conducted on peripheral blood lymphocytes isolated from human or pig sources in the late 1970s reported that protein synthesis increases at least 10 to 20-fold during the first 24 hours of activation. This increase in protein synthesis rates, also known as translation rates, occurs long before DNA synthesis is initi26, 27 ated . Cooper et al. observed that in the initial 10 hours of stimulation in lymphocytes there was a rapid conversion of inactive free ribosomes (70% of all 28 ribosomes) to active polysomes (80% of all ribosomes) . This dramatic shift in ribosomes coincided with an increase in protein synthesis rate with little observ29 able change in total RNA or DNA levels . Interestingly, mRNA could be isolated from resting G0 T cells, but somehow this mRNA was not being trans30 lated . John Kay and his coworkers prepared cell-free lysates from unstimulated and phytohemagglutin (PHA) stimulated pig lymphocytes and found that lysates from the stimulated lymphocytes were more active at synthesizing protein than 26 lysates obtained from unstimulated lymphocytes . They also determined that the rate of formation of the intermediate 40S initiation complexes was lower in lysates from the unstimulated cells. They concluded,, however, that activities associated with formation of the 40S subunit complex could only partially account for the low rate of protein synthesis in these cells. Distinctive changes in the 40S subunit of lymphocytes could be observed during the early period (2 hr) of acti31 vation . Because eIF3 and eIF2 are factors normally associated with the 40S 32 ribosome subunit (Figure 1), the 40S initiation complex formation was examined after addition of these purified factors to the unstimulated lymphocyte cellfree lysates. Addition of purified eIF3 and eIF2, but not eIF-1 to cell-free systems from unstimulated lymphocytes greatly increased 40S complex formation suggesting their importance to this process. Interestingly, when eIF2 and eIF3 were added together, their effect on 40S complex formation was greater than expected, suggesting a synergistic effect. However, it was also observed that addition of these two factors was not sufficient to promote maximal formation of the 40S complexes, indicating that additional factors or activities were required for the next step in protein synthesis. Unfortunately, the Kay laboratory did not continue these studies, and many questions remain unanswered. As mentioned earlier (Figure 1), formation of the 48S ribosomal complex is predicted to contain eIF3, eIF4B, eIF4F, eIF2, eIF1A, eIF1,eIF5, mRNA, and 24,25,33 the 40S subunit . This complex appears to undergo some dramatic changes 31 during activation of lymphocytes . Although we know that the large eukaryotic initiation factor complex eIF3 is required for initiation, little is known of how it actually interacts with the other factors — eIF4F, eIF1, eIF1A, eIF5 — and with mRNA, and how these interactions support the initiation of translation. Recent reports indicate that eIF3 itself might be involved with hepatitis C virus mRNA 34 translation , while not requiring eIF4F and some of the other factors, but still does require eIF2. Therefore, studying eIF3 in T lymphocytes under physiological conditions might shed new light on its role during formation of the preinitia-

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tion complex and the process of translation initiation, during growth and proliferation of the activated lymphocyte. eIF3 appears to have a role in the stabilization and recruitment of the eIF2–Met–tRNAi ternary complex, and mRNA binding to 40S subunits, possible through its interaction with eIF4F (Figure 1). It also has an anti-association function since it appears to keep the 60S ribosome from interacting with the 40S ribosome, thus preventing premature formation of the 80S ribosome. Physical studies show that eIF3 is located in an area extending from the protuberance of the 40S ribosome, with the shape of a flat triangu35 lar prism, attached by its triangular base to the ribosomal surface . Recognition 36 37,38 39 38,40 41,42 that eIF3 interacts with eIF4G , eIF2 , eIF4B , eIF1 , and eIF5 from work conducted in yeast and mammalian cells reinforces the possibility that it has a critical role in translation initiation and is a possible target for regulation by growth-promoting conditions. Our recent study of eIF3 expression and complex formation during the activation of lymphocytes showed that the small eIF3j subunit, recently reported to 43 be a stabilization factor in the formation of the preinitiation complex in yeast , is not part of a larger eIF3 subunit complex in unactivated lymphocytes, but upon activation with the combination of ionomycin and the phorbol ester, PMA (I+P), a larger complex is formed that is now capable of associating with the small 40S ribosomal subunit. This process can be interrupted by treatment with the mTOR inhibitor rapamycin and the PI3K inhibitor wortmannin, suggesting that this translation activity is somehow linked to these signaling pathways and possibly regulated by lymphocyte signaling events (see below).

4. SIGNAL PATHWAYS THAT CONTROL TRANSLATION IN LYMPHOCYTES Translation in lymphocytes appears to be regulated by the same signaling path44,45 ways that also regulate transcription suggesting that the same set of signals leads to stimulation of both processes. Interrupting these signals would therefore lead to disruption of translation as well as transcription. The outcome of altered signaling could be a reduction in immune response and proliferation or lead to uncontrolled lymphocyte proliferation, as occurs in many types of leukemia. The classical example of the interruption of signals in lymphocytes is treatment of lymphocytes with the immunosuppressive drugs FK506 or rapamycin. Interest46 ingly, both these drugs reduce protein synthesis at the same time they reduce 47,48 immune function . It also has been reported that three signaling pathways — MAPK, PI3K through Akt, and the mTOR pathways — are all needed for 1 maximal stimulation of protein synthesis rates in lymphocytes . These same sig49-51 naling pathways also appear to be associated with some forms of leukemia . Our current knowledge about signal transduction pathways that regulate 44,45 global eukaryotic protein synthesis reveals the enormous complexity of these systems. The best-characterized effecter of protein synthesis to date is insulin.

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Insulin activates receptor tyrosine kinases that then activate the intermediate signaling molecules Ras, raf-1, PI3-K, PDK, PKB (Akt), mTOR (FRAP), and 45 p70S6K (see Figure 2). The translation initiation factors thus far that have 52 1, 7, 53 7, 54 been identified as potential targets are eIF2α , eIF2Bε , eIF4E , eIF4G, S6, 45 eEF1, and eEF2 . In lymphocytes there is considerable knowledge about signal pathways activated through the T cell receptor. Quiescent T lymphocytes be55 come activated by engagement of the T cell receptor with an accessory cell (monocytes). Receptor tyrosine kinases become stimulated, which in turn stimulates phospholipase C activity (PLC). PLC then cleaves phosphatidylinositol (4,5)-bisphosphate, yielding diacylglycerol (DAG) and inositol (1,4,5)triphosphate (IP3). DAG activates protein kinase C (PKC) and IP3 mobilizes ++ internal and external calcium (Ca ). PKC activates raf and downstream MAPK 56 2+ targets, MEK1 and ERK1/2, in T cells . Ca activates the phosphatase calcineurin, which dephosphorylates the transcription factor NFAT (nuclear factor of T cells), thereby allowing its translocation into the nucleus, where it activates 57 transcription of IL-2 and other cytokine genes . Peripheral blood T lymphocytes in culture can be activated by a number of compounds that include the combination of anti-CD3, anti-CD28, phytohemagglutinin (PHA), the calcium ionophore ionomycin or A23187, phorbol ester phorbol myristate acetate (PMA, also 58,59 known as TPA), or phorbol 12,13-dibutyrate . All of these compounds can cause substantial increases in protein synthesis and translation rates. The combi2+ nation of ionomycin + PMA (I+P) will stimulate PKC and Ca signaling pathways necessary for activation of IL-2 gene expression, induction of DNA syn58,60,61 thesis, and cellular proliferation . These conditions bypass the need for surface receptor interactions in T cells. However, when used alone PMA is not 62 mitogenic and will not cause proliferation , but does weakly stimulate transla1,63 tion . Activities of translation initiation factors, their associated proteins, or translation initiation are all potential downstream targets of signaling in lymphocytes. Some of these translation activities have already been investigated in lymphocytes, including eIF2α phosphorylation, ternary complex formation, eIF4E phosphorylation, eIF2B exchange activity, 4E-BP1 phosphorylation, and release 1,7,52,54 of eIF4E . A number of these translation activities can be linked to specific 1,7 signaling pathways associated with activation of lymphocytes (Figure 2). However, it is clear that not all translation initiation factor activities have been rigorously investigated, and evidence from the few studies conducted in T lymphocytes indicates that other translation activities are likely to be involved and are currently being investigated.

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Figure 2. Signal transduction pathways that regulate translation initiation activities in lymphocytes. A schematic of signaling pathways activated by the combination of ionomycin + PMA or PMA alone. Inhibitors FK506, SB203980, PD98059, wortmannin, and rapamycin for each pathway are indicated by arrows. Linkages between signaling pathways and translation initiation factors are shown and described in the text.

5. TRANSLATION OF INDIVIDUAL MRNAS IN LYMPHOCYTES It is also important to consider the specific translation of individual mRNAs when attempting to study translational control mechanisms in lymphocytes. In order to study specific translation of mRNA a system is needed that is independent of transcription and other nuclear events like splicing and transport. Since translation rates change dramatically with mitogenic stimulation of lymphocytes, this system is ideal for also studying translational regulation of specific mRNAs. As mentioned earlier, studies in lymphocytes have already identified the connection between certain signals and the corresponding response in imme64 diate and early intermediate gene expression . Other, more recent studies using microarray analysis have now identified mRNA expression for large numbers of 65,66 genes being expressed in lymphocytes . These systems can identify the presence of mRNAs for individual genes, but microarray analysis cannot predict if protein is actually being synthesized from these mRNAs. Analysis of protein 66 expression (proteomics) in lymphocytes has proved to be difficult , and often

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the microarray data and proteomic data do not correlate. A complicating factor with proteomic analysis of the total protein complement of cells is the presence of stable, abundant housekeeping proteins that often mask the rare, lower abundant growth factors or regulatory signaling proteins. New, improved mass spectrometry methods are now available to directly identify proteins and greatly improve our ability to identify proteins, but these methods still suffer from the presence of the highly abundant proteins that continue to obscure the presence of the lower abundant proteins. Signals in lymphocytes are transmitted rapidly in the stimulated lymphocyte and the synthesis of proteins also responds quickly to these signals. This rapid change in translation of mRNAs can be detected by in-vivo labeling of proteins 35 in treated lymphocytes with radioactive methionine ( S-methionine). Translation rates can be measured by precipitating proteins after lysis of cells and measuring the amount of incorporated radiolabeled amino acid and analyzing the cell lysate directly by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). An example of such an analysis is shown in Figure 3. Human T lymphocytes were activated with two conditions: (1) ionomycin and PMA (I+P), which promotes proliferation, and (2) PMA alone, which does not promote proliferation. Lymphocytes were stimulated by each condition in the absence or presence of immunosuppressant FK506 or rapamycin, in the absence or presence of inhibitors of the MAPK (ERK1/2 or HOG/p38), PKC, or PI3K pathways. Translation rates were measured (Figure 3, top of figure) and found to correspond to the amount of newly synthesized proteins (Figure 3, bottom). Also included in this figure is the Coomassie stain of the gel that shows the total proteins present (proteome) in each sample (Figure 3C,D). A quick glance at the Coomassie-stained proteins demonstrates the difficulty of trying to identify significant protein changes in total proteins after treatment of lymphocytes with the immunosuppressant or with inhibitors of signaling pathways. In contrast, analyses of the newly synthesized proteins (Figure 3A,B) reveal quite distinctive changes that were not detected in the Coomassie-stained gels. The PKC inhibitor Ro3290430 totally reduced protein synthesis in both conditions of activation (Figure 3A,B, lanes 4, 8, and 12), whereas the MAPK inhibitors PD98059 (Figure 3A, lanes 3, 7, and 11) or SB203980 (Figure 3A, lanes 5, 9 and 13) did not significantly reduce protein synthesis after I+P stimulation. However, significant reduction of protein synthesis by rapamycin was observed when T lymphocytes were activated with PMA alone (Figure 3B, lanes 11 and 13). Closer examination by laser scanning of the protein gels (not shown here) demonstrate changes in the synthesis of individual proteins depending on the treatment of the lymphocytes, suggesting that translation of individual mRNAs might be independently regulated. There have been recent reports suggesting that perturbations in 67 68 signaling pathways (Ras/Akt or PDK1 ) may lead to translation changes that in turn cause changes in select mRNA populations important for cell growth and proliferation.

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Figure 3. Reduction of translation in lymphocytes by immunosuppressants and/or signaling inhibitors. Human T lymphocytes, untreated or treated with immunosuppressant FK506 (1 nM), rapamycin (1 nM) in the absence or presence of MAPK inhibitor PD98059 (50 µM), PKC inhibitor Ro320430 (10 µM), p38/HOG inhibitor SB203980 (10µM), or PI3K inhibitor wortmannin (10 µM) for 8 h after being stimulated with ionomycin + PMA (I+P) or PMA alone. Lymphocytes were incubated with 35S-methionine 1 h before harvest, cell lysates prepared, and a sample was precipitated with 10% trichloroacetic acid and the precipitated protein counted, or analyzed by 12% SDS PAGE and autoradiography. The top of the figure is the graph of data as % inhibition of translation, calculated as the %CPM of the I+P stimulated lymphocyte. The bottom of the figure shows results from the SDS PAGE analysis. (A) I+P activated autoradiograph showing newly synthesized proteins; (B) PMA alone activated, autoradiograph; (C) Coomassie stain of the I+P activated and (D) Coomassie stain of the PMA alone activated.

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A novel approach has been undertaken that attempts to examine the transla69 tion of individual mRNAs in mitogenically stimulated lymphocytes . This approach uses in vitro translation assays of native mRNAs that are isolated directly from lymphocytes and still retain their associated, potentially regulatory protein factors. Proteins synthesized from the polysome-associated mRNAs or PAmRNAs translated in vitro appear to mimic the same proteins synthesized in vivo in the intact lymphocyte. For this assay, additional translation components are added through the use of rabbit reticulocyte lysate to ensure that these factors and components are not limiting in these assay reactions. A big advantage to this approach is that the nucleus is no longer present, since it is removed by sub69 fractionation procedures, so its influence is minimized . Degradation of mRNA is still a potential problem in this system, but even this possibility can be reduced by inclusion of nuclease inhibitors. Only newly synthesized proteins are detected in this system, so abundant, stable preexisting cellular proteins are eliminated from our analysis. We are thus able to closely study only the in vitro synthesis of proteins from preexisting native mRNAs. We can determine the translation rates for the synthesis of these proteins since the assay time is controlled. Finally, we can treat the lymphocytes with pharmacological inhibitors of relevant signaling molecules or acquired mutant cell lines. These in vitro procedures were used to examine protein synthesis changes associated with the ribo69 some/polysome fractions of early-activated T lymphocytes . A direct comparison between proteins synthesized immediately after activation of human lymphocytes showed not only that in vitro translation of these mRNAs in the presence of an active translation system (nuclease-treated, mRNA-depleted rabbit reticulocyte lysate, nRRL) could be accomplished, but that it may actually be a more sensitive way of directly studying the translation of individual mRNAs. Our ability to mimic the natural regulation of these mRNAs, especially repression of the translation of mRNAs in the quiescent G0 lymphocyte, appears to be due to the presence of protein factors interacting with these mRNAs since re69 moval of these proteins then allows these mRNAs to be in vitro translated . In vitro translation of proteins in the presence of biotinylated lysine/tRNA from PAmRNAs was also performed, which allowed us to quickly isolate the newly synthesized proteins using magnetic streptavidin beads. This process was scaled up to yield enough protein for mass spectrometry analysis and identification of 69 some of these proteins . The sequence for one gene product, p33, was identified as BAP37 (also known as the IgM-associated protein or prohibitin-associated 70,71 protein ). Gene expression of this gene was particularly interesting because our data indicated that there was a rapid increase and then a decrease in its protein when most or the other genes showed increased protein synthesis. Further identification of other protein products by mass spectrometry will make it possible to identify other genes that result in patterns of protein expression that are tightly regulated during activation. These genes may prove to have important regulatory roles in the proliferation of T lymphocytes.

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Kay et al. also observed the presence of untranslated mRNA in resting lymphocytes and attributed the lack of translation of mRNAs as being due to an 72 "inhibitor" of translation . They also found that addition of active nRRL did not efficiently translate these mRNAs. Only when the mRNAs were purified away from their regulatory cofactor proteins were these mRNAs efficiently translated. The explanation for this phenomenon remains to be elucidated, but might be 73 connected to some of our recent findings about eIF3 . There are several possibilities for the lack of translation of the mRNAs. In some situations, as in certain diseases like cancer, the ability of cancer cells to translate these mRNAs may be lost, thus allowing these mRNAs to be translated inappropriately, thereby causing disease. While this is indeed possible, in order to evaluate this possibility it is essential that we first understand how individual classes or "populations" of mRNAs become translated during normal cellular conditions. Two different mechanisms exist for recognition of mRNA and the start of translation initiation in eukaryotic cells. The most common, the cap-dependent 7 scanning mechanism, is dependent on the presence of the m GTP cap structure on the 5′ end of each mRNA, which is recognized by cap-binding translation initiation factor eIF4E. The second mechanism is cap-independent, but may rely on the presence of an internal ribosome entry element (IRES), a complex RNA 74 structural element that is located in the 5′ untranslated region of the mRNA . An IRES element, originally identified in viral mRNAs, allows the ribosome to be recruited to the mRNA without the need for the cap-binding protein eIF4E. Stoneley and Willis predict that up to 10% of all mRNAs are able to initiate 74 translation by an IRES mechanism . The majority of IRES-containing mRNAs identified so far are for protein products associated with the control of cell growth and cell death, such as growth factors, protooncogenes and proteins re74 quired for apoptosis . However, unlike viral IRES sequences, no mammalian IRES sequence or structure has yet been identified, and because of this few eu74 karyotic IRES binding proteins have been identified . Other potential 5′ regulatory sequences include upstream open reading frames (uORFs) and upstream AUG (uAUGs) that are in a less favorable start site context and that may utilize proposed "leaky scanning" mechanisms that 75,76 could also impact the translatability of mRNAs . The 3′UTR also has been 77 implicated in the translatability of mRNAs and it has been suggested that the 3′UTR might be influenced by or be depended on the interaction between the 5′ 77 and 3′ termini of the mRNA . Interestingly, the complexity and length of 3′UTRs has increased through evolutionary development of eukaryotic cells from the fungi to invertebrates to vertebrates to humans, whereas the 5′ UTR lengths are more consistent and change less during evolution. The reasons for this expansion of the 3′UTRs is not known, but it may be important for the regulation of translation in higher vertebrates. A number of other approaches have been taken to examine global mRNA gene expression and relate it to mRNAs that are being translated. This includes 78 the use of a "ribonomics" and ribonomic profiling , mircorarray analysis of

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mRNA directly isolated from polysomes or gene expression analysis of 80 high-resolution state array analysis and quantitative proteomics . Ribonomic profiling uses antibodies to RNA binding proteins and genomic arrays to identify mRNA subsets in mRNP complexes isolated from P19 embryonal carcinoma stem cells. These mRNA profiles were associated with different mRNAbinding proteins and were found to contain mRNAs (called gene clusters) that 78 differed mathematically from those calculated from total cellular RNA . This interesting approach still does not actually examine the translatability of these mRNAs but uses a "guilt by association" approach, suggesting functional impor81 tance . Another approach used by MacKay et al. examined protein synthetic 80 rates from a transcript as a function of its ribosomal loading . They observed that each mRNA had its own pattern of ribosome loading and found that translation rate changed in their system as the result of increased translation efficiencies of individual mRNAs. Other groups have also use mRNA association or "loading" on polysomes as an indicator of translation efficiency of subsets of mRNAs. Selective translation of mRNAs in response to Ras or Akt signals in 67 gliobastoma cells or to reduction of the signaling molecule PDK1 in embryonic 68 stem cells have been identified. However, the mechanisms by which these mRNA subsets, in response to upstream oncogenic signals that result in these 82 translational changes, still need to be identified .

6. CONCLUSIONS Protein expression of a gene is dependent on many processes in the cell that include transcription of the gene into mRNA, splicing of the mRNA, transportation of the mRNA into the cytoplasm, and finally translation of the mRNA into protein. It is important to remember that expression of the gene is a dynamic process that is usually regulated at multiple levels. Because of the complexity and dynamic nature of this process, studying only one aspect of gene expression is not likely to give a total picture, but only a "snapshot" in time. But it is necessary to dissect the entire process into multiple, separate snapshots so that we can recognize the contributions of each process to the total picture. We know that the total amount of each gene's mRNA is dependent on its transcription, splicing, transport, and stability. What is important for its translatability? We know that the translatability of mRNA is dependent not only on the amount, the pres7 ence of the m GTP cap and poly A tail, but we are now beginning to fully appreciate the influence of additional regulatory elements in the 5′ and 3′ untranslated 77,83-86 regions (UTRs) of these mRNAs . Therefore, when studying the “translatability” of specific mRNAs, all of these features need to be considered. In the future, a better understanding of the normal process and regulation of individual mRNA translatability may yield vital information about how theses systems become dysregulated in diseases such as leukemia and lymphoma.

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7. ACKNOWLEDGMENTS The author would like to acknowledge discussions and assistance from John Hershey and funding from the Children's Miracle Network.

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17. D. C. Fingar, C. J. Richardson, A. R. Tee, L. Cheatham, C. Tsou and J. Blenis, mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E, Mol Cell Biol, 24, 200–126 (2004). 18. A. C. Gingras, S. G. Kennedy, M. A. O'Leary, N. Sonenberg and N. Hay, 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway, Genes Dev., 12, 502–513 (1998). 19. N. Hay and N. Sonenberg, Upstream and downstream of mTOR, Genes Dev, 18, 1926–1945 (2004). 20. M. Rau, T. Ohlmann, S. J. Morley and V. M. Pain, A reevaluation of the capbinding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate, J.Biol.Chem., 271, 8983–8990 (1996). 21. T. A. Lin, X. Kong, T. A. Haystead, A. Pause, G. Belsham, N. Sonenberg and J. C. Lawrence, Jr., PHAS-I as a link between mitogen-activated protein kinase and translation initiation, Science, 266, 653–656 (1994). 22. K. S. Browning, D. R. Gallie, J. W. Hershey, A. G. Hinnebusch, U. Maitra, W. C. Merrick and C. Norbury, Unified nomenclature for the subunits of eukaryotic initiation factor 3, Trends Biochem Sci, 26, 284. (2001). 23. N. Sonenberg and T. E. Dever, Eukaryotic translation initiation factors and regulators, Curr Opin Struct Biol, 13, 56–63 (2003). 24. L. D. Kapp and J. R. Lorsch, The molecular mechanics of eukaryotic translation, Annu Rev Biochem, 73, 657–704 (2004). 25. T. Preiss and W. H. M, Starting the protein synthesis machine: eukaryotic translation initiation, Bioessays, 25, 1201–1211 (2003). 26. J. E. Kay, D. M. Wallace, C. R. Benzie and R. Jagus, Regulation of protein synthesis during lymphocyte activation by phytohaemagglutinin., edited by M. R. Quastel. (NYC, Academic Press, 1979), pp. 107–114. 27. R. E. Wettenhall and D. R. London, Evidence for translational control of "early" protein synthesis in lymphocytes stimulated with concanavaline, A Biochim.Biophys.Acta, 349, 214–225 (1974). 28. H. L. Cooper and R. Braverman, Free fibosomes and growth stimulation in human peripheral lymphocytes: activation of free ribosomes as an essential event in growth induction, J.Cell Physiol., 93, 213–225 (1977). 29. R. Jagus-Smith and J. E. Kay, Messenger ribonucleic acid content of phytohaemagglutinin-treated lymphocytes, Biochem.Soc.Trans., 4, 783–785 (1976). 30. K. V. Kandror and A. S. Stepanov, [Informosomes and translation activity of mRNA in eukaryotic cells, Mol.Biol.(Mosk). 22, 31–43 (1988). 31. R. Jagus and J. E. Kay, Distribution of lymphocyte messenger RNA during stimulation by phytohaemagglutinin, Eur.J.Biochem., 100, 503–510 (1979).

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14 PROTEIN ARGININE METHYLATION: A NEW FRONTIER IN T CELL SIGNAL TRANSDUCTION Brandon T. Schurter, Fabien Blanchet, and Oreste Acuto 1. INTRODUCTION Extracellular stimuli activate, via specific receptors, intracellular signals that modify cellular behavior. These intracellular signals are conveyed as cascades of biochemical modifications governing enzymatic activities, protein complexes assembly, and subcellular translocation. Dynamic signaling networks are thus generated through the interaction of various proteins, phospholipids, and nucleic acids. These interactions are mediated through a number of known protein do1 mains . It has been demonstrated that these pathways induce immediate cellular modifications, such as shape and motility, and re-program genome expression through the regulation of epigenetic modifications, co-activators, transcription and translation factors resulting in cell growth, proliferation, differentiation, and death. Studies aimed at understanding the molecular basis for receptor-mediated signal transduction have focused mostly on mechanisms based on protein and 1 lipid phosphorylation . Protein tyrosine or serine/threonine and lipid kinases and phosphatases, usually acting seconds/minutes after receptor triggering, allow proteins to become membrane-anchored, enzymatically active, or serve as scaffolds, adapters, and/or activators of other components. Because of their highly dynamic nature, cellular phosphorylation reactions allow fast and transient juxtaposition, in an orderly fashion, of protein components to drive signal initiation, propagation, and regulation. Significant gaps may exist in the cell-signaling picture because, in addition to phosphorylation, proteins can be subjected to a variety of other co- and posttranslational modifications that may impart additional functions or improve ex1 isting ones . The biological role and regulation of palmitoylation, acetylation, Molecular Immunology Unit, Institut Pasteur, Paris 75015, France. Corresponding author: B. Schurter: Molecular Immunology Unit, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, Cedex 15, France, Tel. +33-1-4061 3655; Fax +33-1-4061 3204, E-mail: [email protected].

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ubiquitination, SUMOylation and methylation, to cite a few, have begun to be studied in further detail in recent years. Methylation alters protein function by local structural changes affecting various amino acids (lysine, arginine, histidine, and carboxyl residues). Studies on histone modification during chromatin remodeling have made lysine and 2-4 arginine methylation a more popular notion , and early studies found arginine methylation associated to proteins implicated in various aspects of RNA regula5,6 tion . Recent advances implicate this modification in various cellular functions, 3,6 including signal transduction . Extracellular stimuli such as antigen, costimulators, cytokines, and chemokines activate via their receptors intracellular signals that modify lymphocyte behavior and shape the outcome of adaptive 7 immune responses . However, in spite of this immense field of signaling networks, arginine methylation has only recently been described as an additional mechanism that may regulate immunity. Here we briefly review recent advances implicating protein arginine methylation in various cellular functions including T cell activation. 2. PROTEIN ARGININE METHYLTRANSFERASES (PRMTS) Arginine methylation has been studied for 40 years. The first publication de8 scribing arginine methylation of histones appeared in 1964 , and the first crude purification of an arginine methyltransferase was performed by Paik and Kim in 9 1968 . Although arginine methylation has been studied for many years, it has received wider attention only recently and has been found to be more common and biologically relevant than initially suspected. Forty years after the initial discovery of histone–arginine-methylation, we have only scratched the surface of discovering the substrates for arginine methyltransferases in addition to fully understanding the biological role of this modification. In proteins, arginine residues can play a role in both intra- and intermolecular interactions. This residue has been found in active sites of enzymes 10 (for example, the rat choline acetyltransferase) , and its basic charge is essential for the interaction with acidic molecules (as is the case for histone interaction 11 with DNA) . While not modifying the charge, methylation of arginine can modify polypeptide interactions, disrupt hydrogen bonding, and/or create steric hin3,4,6 drance that can affect molecular interactions . To date, eight arginine methyltransferases have been identified and characterized (Figure 1). These methyltransferases have been further divided into two classes based on the symmetry of their reaction products. Each class catalyzes the formation of mono-methylarginine; however, type I PRMTs catalyze formaG G tion of asymmetric N ,N -di-methylarginine, whereas the type II PRMTs form G ’G symmetric N ,N -di-methylarginine (Figure 2). Five related type I enzymes (named PRMT 1, 3, 4, 6, 8) and two type II enzymes (PRMT5 and 7) have been identified. Although the sequence of PRMT2 is related to the other

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Figure 1. The protein arginine methyltransferase and peptidylarginine deiminase families. (A) There are eight known members of the PRMT family. Each of the members has a conserved Sadenosylmethionine binding domain (SAM B.D.) that harbors the motifs: I, post I, II, and III, in addition to the THW loop in the C terminus. PRMT 7 has a repeat of these domains. PRMT2 and PRMT3 also contain an SH3 domain, and a Zinc Finger (Z-F) domain, respectively. The number of amino acids, cellular localization, and type of di-methylation produced are also shown. (B) There are five members of the PAD family; all share a high sequence similarity. The crystal structure of PAD4 is known and amino acids required for calcium binding and/or enzymatic activity are shown with black bars. Amino acids that are conserved in the other PADs are shown with gray bars. PAD4 also contains a nuclear localization sequence in the N terminus.

arginine methyltransferases, it has yet to display enzymatic activity, and therefore has not been assigned to a class. There exists high sequence identity within several conserved domains/ motifs of arginine methyltransferases. All PRMTs have a S-adenosyl-L-methionine (SAM) binding domain, a region common to all SAM-dependent arginine me12 thyltransferases, which encompasses motif I, post I, II, and III . In addition, the PRMTs also have a conserved threonine–histidine/tryptophan loop (THW loop) in the C terminus. PRMT7 consists of a repeat of these two domains, both of 13 which are required for enzymatic activity . The crystal structures of PRMT1, 14-16 PRMT3, and Hmt1 (the yeast homolog of PRMT1) have been described , and similarities within the core structure of these enzymes exist. Historically, protein arginine methylation has been implicated in transcriptional regulation through nucleic acid (mainly RNA) binding, processing, and 3,4,6 transport . Early studies on arginine methylation described histones as

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Figure 2. Mechanism of arginine methylation. Arginine can be mono-methylated on a guanidino nitrogen atom by both type I and type II PRMTs. Type I PRMTs further catalyze the formation of asymmetric di-methylarginine by the addition of a second methyl group to the same guanidino nitrogen atom, whereas type II PRMTs form symmetric di-methylarginine by the addition of a second methyl group to the opposite nitrogen atom. S-adenosylmethionine (SAM) is the methyl donor, and is converted into SAH after the reaction. Arginine and mono-methylarginine can be converted into citrulline by peptidylarginine deiminase 4 (PAD4). To date, no aminotransferase has been discovered, which can convert citrulline into arginine.

substrates in addition to the functional role of methylation on hnRNPs (proteins 3,4,6 that bind to and transport RNA from the nucleus to the cytoplasm) . In recent years, this modification has been shown to be involved in the regulation of a number of cellular functions, including signal transduction, gene accessibility 6,17 and transcription, and nuclear import/export . PRMTs have been shown to have essential regulatory functions by methylating nuclear proteins (e.g., Histone 2A, 3, and 4, nucleolin, Poly(A) binding protein II, HuR, an AU-rich mRNA binding protein; nuclear factors such as CBP/p300, ILF3, STAT1, RNA

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Table 1. Known in vitro and in vivo substrates of PRMTs organized by function PRMT1 PRMT2 PRMT3 PRMT4

PRMT5

PRMT6

PRMT7 PRMT8 Unknown

RNA Binding/Proc. Proteins CIRP

X

X

Coilin EWS

X

Fibrillarin

X

hnRNP A1

X

hnRNP A2

X

hnRNP R

X

hnRNP K

X

X

X

X

X

X

HuR

X

LSm4

X

Npl3

X

PABP1 PABPN1

X

RBP58

X

RNA Helicase A

X

rpS2 SAF-A

X X

X

Sm B/B' Sm D1 Sm D3 TAFII68 TLS/FUS Transcription Factors/Repressors

X X X

X

X X

ILF3

X

NIP45

X

X

p300/CBP PGC-1alpha

X

STAT1

X

STAT3

X

STAT6

X

SPT5

X

ZF5 DNA Binding/Processing & Structure

X

X

X

X

Histone H2A

X

Histone H3 Histone H4

X

Mre11

X

Nucleolin

X

X X X

X X

Signal Transduction Sam68

X

X

X

Vav1 Growth Factor FGF-2

X

T Cell Development

X

TARPP Viral Protein

X

HIV Tat GPI-anchor protein p137GP1

X

Methyltransferase

X

PRMT6 Multiple functions

X

MBP Unknown Function SAMT1 No known Substrate

X X

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helicase A, and NIP45), FGF2, the adapter Sam68, and apoptosis proteins — to name a few (see Table 1 for a more detailed list of substrates). The list is rapidly growing thanks to proteomic Mass Spectrometry (MS) studies of argin17 ine methylated proteins and the use of recombinant PRMTs on protein microar24 rays .

3. PRMTS, A CLOSER LOOK PRMT1 was first described as a protein that interacts with the immediate-early TIS21 protein and leukemia-associated BTG1 protein in a yeast-two hybrid 25 screen . This interaction modulated the methyltransferase activity of PRMT1. It was later demonstrated that PRMT1 accounts for a majority of the arginine me26 thylation within a cell , and that it was required for growth and development, as mice embryos lacking a functional PRMT1 gene die before E6.5, a phenotype 27 consistent with a fundamental role in cellular metabolism . Several of the known substrates for this enzyme have been described (see Table 1), and analysis of the methylation sites in these proteins and synthetic peptides revealed a 28 methylation consensus motif of Arg–Gly (RG) or Arg–Gly–Gly (RGG) . As seen in Table 1, this enzyme has been shown to modify many nucleic acid (mainly RNA) binding proteins. Recently, however, it has been discovered that proteins with different cellular functions are also modified by this promiscuous enzyme. PRMT1 localizes to both the cytoplasm and nucleus. Although not much is known about the regulation of this enzyme, recent work has demonstrated that inhibition of cellular methylation results in its nuclear accumulation and immobility, an effect thought to be the result of the enzyme being bound 29 and retained to the nucleus by nuclear substrates awaiting modification . 30 PRMT2 was identified by sequence similarities to the yeast Hmt1 . Although no substrates have been found for PRMT2, it has been shown that this enzyme is a co-activator for the Estrogen Receptor α, and that its ability to bind 31 to SAM is required for its co-activator function . Although PRMT2 is the only 31 methyltransferase that has an SH3 domain , this domain is not required for its ability to enhance expression of genes regulated by ERα. PRMT2 has also been shown to interact with E1B-AP5, and it was suggested that this enzyme is responsible for methylating E1B-AP5 in an RGG box; however, direct evidence of 32 methyltransferase activity was not shown . 33 PRMT3 was discovered through its sequence similarity to PRMT1 . It is the only arginine methyltransferase to contain a zinc finger domain in its N terminus. Although PRMT3 mutants lacking the zinc finger are still enzymatically active in vitro, it appears that this domain is required for substrate recognition. This is evidenced by the fact that PRMT3 mutants that lack the zinc finger are 34 unable to methylate hypomethylated substrates from Rat1 cell extracts . In vitro, PRMT3 has the ability to methylate substrates for PRMT1 (e.g., glycine-rich and arginine-rich region from fibrillarin, Sam68). However, it is not a com-

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pletely redundant enzyme, as it is unable to methylate all sites recognized by 35 PRMT1 . PRMT3 also has the ability to methylate other substrates not recog36 nized by PRMT1 such as the ribosomal protein rpS2 . PRMT4, also known as CARM1 (Coactivator associated arginine methyltransferase 1), was discovered as an enzyme that associates with the nuclear 37 receptor coactivator GRIP1 . In characterizing this enzyme, it was found to me38 thylate histone H3 on several arginines . Although additional substrates for PRMT4 have been described (see Table 1), a defined methylation consensus 3,39 motif has yet to be established . Similar to PRMT1, PRMT4 is required for growth and development as mice embryos with a targeted disruption of CARM1 40 are small in size and die perinatally . CARM1 has been shown to be required for lymphocyte development because knockout mice also have a deficiency in promoting the differentiation of early thymocyte progenitors. PRMT5 was discovered in a yeast-two hybrid screen as a protein that inter41 acts with Jak2 . Initially it was named Jak2 Binding Protein (JBP1); however, as it is an arginine methyltransferase, its name was later changed to PRMT5. PRMT5 along with PRMT7 are the two type II methyltransferases catalyzing the formation of symmetric di-methylarginine. Initially it was discovered that PRMT5 can symmetrically di-methylate histone H2A and H4, and recently it has been shown to be part of an S20 complex containing pIC1n, called the “me41,42 thylosome” . This complex is responsible for di-methylating two proteins (SmD1 and SmD3) of the pre-mRNA splicing machinery, an event that targets these proteins to the SMN complex for assembly into snRNP core particles. In searching the human genome for PRMT family members, three methyltransferases have recently been identified: PRMT6, PRMT7, and PRMT8. As these are novel methyltransferases, information on substrates and cellular function is limited. PRMT6 has been shown to perform automethylation, in addition 43-45 to methylating the HIV Tat protein, and the non-histone HMGA1a . PRMT7 and PRMT8 have only been characterized in vitro, and in vivo substrates have not been described. Interestingly, PRMT7 contains two SAM binding domains 13 (see Figure 1), both are required for enzymatic activity . PRMT8 is myristoylated on a glycine in the N terminus, which targets this enzyme to the plasma 46 membrane, although the function of this localization has yet to be determined .

4. PROTEIN ARGININE METHYLATION IN RECEPTOR SIGNAL TRANSDUCTION AND LYMPHOCYTE ACTIVATION The notion that signal propagation from triggered receptors may be mediated in part by PRMT-directed modifications is fairly recent. The pioneering work by 47,48 Aletta and coworkers demonstrated that arginine methylation of several proteins is increased within minutes/hours after NGF stimulation of PC12 cells. Of particular interest was the observation that methylation of several proteins accumulated over hours and that pharmacological inhibition of cellular methyla-

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tion had a dramatic effect on PC12 differentiation (neurite outgrowth) but only marginally affected proliferation. Moreover, it has been shown that the IFNAR1 chain of the IFN-α/β receptor interacted with PRMT1 and inhibition of PRMT1 49 expression reduced IFN-α/β-mediated biological effects . Subsequent studies on estrogen receptor-regulated gene expression precisely defined an implication of 4 PRMTs in intracellular signaling . It was found that upon nuclear receptor triggering CARM1 synergistically activated transcription from a trimolecular com37 plex formed together with GRIP-1 and the histone acetyltransferase CBP/ 50 p300 . The enzymatic activity of CARM1 was required for this increase in transcriptional activity, and Histone H3 and CBP/p300 have been described as substrates for this methyltransferase. More recently, a subset of TNF- or LPS-induced NF-κB-dependent genes, involved in immune and inflammatory responses, was found to be regulated by 51 PRMT4 in combination with p300/CBP and the p160 family of co-activators . Thus, these co-activator complexes seem to constitute “endpoints” of signaling pathways that utilize arginine methylation to activate the basic transcription machinery from enhancer-bound factors and/or to relieve chromatin-mediated re4 52 53 pression . STAT1 and STAT6 were also reported to be arginine methylated, a modification suggested to regulate cytokine-directed gene expression. Recently two groups have disputed the methylation of STATs; however, these groups utilized experimental conditions different from the original publications, which 54 may contribute to the differences in observations . Finally, LPS treatment of macrophages was found to induce, within minutes, arginine methylation of the 20 mRNA binding protein HuR , a major trans-acting AU-rich element (ARE)binding protein that controls the stability, subcellular shuttling, and translation of ARE-mRNAs. While these examples provided evidence that PRMTs and their substrates are an integral part of receptor-mediated signaling pathways, until recently limited data suggested a direct implication of arginine methylation in specific lymphocyte activation and differentiation processes. Early work using pharmacological inhibition of S-adenosyl-L-homocysteine hydrolase (SAHase), which results in negative feedback inhibition of methyltransferases by S-adenosyl-Lhomocysteine (SAH), showed in vitro and in vivo inhibition of T cell re55,56 sponses , indirectly suggesting that arginine methylation may play a role in lymphocyte signaling. Lymphocyte development and differentiation are very complex biological processes implicating activation/repression of hundreds/ thousands of genes. It would therefore be surprising if PRMTs were not involved. Indeed, PRMT4-deficient mice showed partial developmental arrest of the earliest thymocyte progenitors; however, it was unclear if this defect was due to limitations in nuclear signaling consequent to pre-TCR- or growth fac57 tor(s)-receptor(s) triggering in the immature thymic populations . Studies of mature T cells were hindered by perinatal lethality of PRMT4 deficiency. It was also found that TARPP (thymocyte cyclic-AMP regulated phosphoprotein), a protein specific for immature thymocytes, is methylated by CARM1 and may

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also play a role in the development of thymocytes . Two recent studies shed new light on the implication of arginine methylation in the adaptive immune response, by demonstrating the implication of the PRMTs and their substrates in the signaling pathways triggered by the T cell antigen receptor and the costimulatory receptor CD28 (Figure 3).

5. ARGININE METHYLATION OF NIP45 AND ITS ROLE IN NF–AT ACTIVATION In T cells, nuclear translocation and activation of the calcineurin-regulated family of transcription factors, NF-ATs, are induced by TCR triggering and are 58-60 regulated by the costimulatory receptor CD28 . NF-ATs play essential roles in the expression of multiple cytokine genes that are responsible for T cell prolif58 eration and differentiation . Studies by Glimcher and coworkers indicated the capacity of NF-AT to direct the IL-4 gene, and to a minor extent INFγ gene ex23,61 pression was potentiated by the NF-AT-interacting protein 45 (NIP45) . Consistently, NIP45-deficient mice have a reduced expression of IL-4 and IFNγ genes in T helper subsets Th1 and Th2 upon T cell antigen receptor (TCR) trig23 gering . The N-terminal region of NIP45 possesses an RGG-rich sequence that was robustly methylated in vitro by PRMT1, and NIP45 was indeed found arginine 23 methylated in activated Th cells . Deletion of the NIP45 N-terminal tail abolishes its methylation, interaction with NF-AT, and, consequently, its coactivation of IL-4 gene expression. More importantly, PRMT1 was found to synergize with NF-AT to induce IL4- and IFNγ-gene reporter expression, and NIP45 was required to form a trimolecular complex implicating these three factors. Taken together, these data strongly suggest that PRMT1-mediated arginine methylation of NIP45 regulates optimal gene expression of two central cytokines that direct T cell differentiation. It is not currently known when arginine methylation of NIP45 occurs; however, a possible scenario could be that TCRCD28 stimulation allows persistent NF-AT nuclear translocation, followed by NIP45/PRMT1 binding and methylation, which in turn enables efficient association with NF-AT. This trimeric interaction (NF-AT, NIP45, and PRMT1) thus promotes co-activator function and/or local chromatin remodeling for enhanced transcriptional activity. Pharmacological inhibition of methylation by MTA, a generic inhibitor of all methyltransferases, reduced cytokine production in differentiated Th 23 cells . However, it is difficult to conclude that this was due only to reduced NIP45 methylation. Indeed, other methyltransferases as well as other key upstream activating events may be blocked by MTA (e.g., Vav-1 arginine methylation; see below). Of interest in this study was that the level of PRMT1 is

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Figure 3. T cell processes regulated by arginine methylation. (A) Upon CD28 stimulation, and to a lesser extent by the T cell receptor, Vav-1 becomes arginine methylated. A majority of the methylated Vav-1 is found within the nuclear fraction. (B) Arginine methylation of NIP45 is required for efficient interaction with NF-AT to enhance transcription of genes under the control of this transcription factor. (C) Although the function of TARPP methylation is not fully understood, it is proposed that its methylation plays a role in promoting the differentiation of early thymocyte progenitors.

increased following TCR and CD28 stimulation in Th cell precursors, a finding that may indicate a need for an increase in arginine methylation required for T cell differentiation. Finally, PRMT1-6 are all expressed in differentiated T 23 cells , suggesting that the entire repertoire of arginine methylation on many cellular substrates is required for driving T cell differentiation.

6. CD28-MEDIATED COSTIMULATORY SIGNALING AND INDUCTION OF ARGININE METHYLATION A hallmark of the differentiation process of naive T cells is the requirement for a second signal provided by the costimulatory receptor CD28. CD28 triggering amplifies the signal induced by a productive association of the TCR with peptide

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antigen/MHC . Without it, most T cell responses are of low intensity and duration, leading to cell anergy and/or death. Instead, full differentiation programs that drive effective T and B cell cooperation with consequent Ig switch and somatic hypermutation, Th1/Th2 development, establishment of central and effec60 tor T cell memory require CD28 stimulation . In principle, CD28 may provide a quantitative amplification of the TCR signal and/or activate unique signaling pathways, although the combination of the two may be more likely the case, considering the type and extent of biochemical and biological changes that CD28 stimulation induces in combination with antigen stimulation. Recent results may now shed new light for a more complete molecular understanding of a “second signal” delivered by CD28 and T cell differentiation programs. CD28 was found to be a major provider of a signal that increases 62 PRMT activity and induces arginine methylation . Using an antibody specific for mono- and asymmetric di-methylated arginines, it was found that CD28 triggering induced an increase in arginine methylation of several proteins. TCR stimulation only marginally increased this effect. Reminiscent of NGF47 stimulated PC12 cells , it was found that protein arginine methylation was not concomitant with tyrosine phosphorylation (which can be observed seconds after CD28 stimulation) but appeared several minutes after stimulation and increased even after 30 minutes of stimulation. This pattern is distinct from usual receptor-induced phosphorylations that peak early and decline to remain low, but stationary, for the duration of receptor engagement. Measurement of PRMT1 activity using a specific substrate revealed that CD28 also induced an increase of 62 enzymatic activity with similar kinetics . Among key effectors of CD28 signaling — Lck, Grb2, Itk, and Vav1 — the 62 latter two were found to become arginine methylated upon CD28 stimulation . This was true both in cells of human and mouse origin. Vav1 is a GDP–GTP exchange factor (GEF) for small GTPases that has a critical role in T cell devel63 opment and differentiation . It participates in signaling pathways implicated in actin cytoskeleton reorganization and in gene expression, by controlling calcium, Map kinases, and activation of NF-κB. Vav1 methylation was dependent 62 on the CD28 intracellular tail and Src-PTK activation , suggesting that, similar to the observation with bulk protein populations, Vav1 arginine methylation may be a relatively delayed event in signaling pathways and is controlled upstream by protein tyrosine kinases. TCR stimulation only weakly induced Vav1 arginine methylation, indicating that CD28 may play a dominant role in this pathway. Interestingly, methylated Vav1, which constitutes a small proportion (about 5%) of the entire Vav1 cellular pool, was entirely associated with the nuclear fraction. This localization is consistent with the fact that methylated 62 Vav1 was only detected 20–30 min after CD28 stimulation . Recent data have indicated that a small fraction of Vav-1 is detected in the nucleus 15–20 min after strong FcR stimulation and may be associated with various transcription 64 factors . Vav1 in the nuclear fraction was detected with an antibody that specifically recognizes phosphorylated tyrosine 174 of Vav1, a marker for the cata-

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lytically active form of this protein . A likely explanation for the above findings is that CD28 induces Vav1 activation and translocation to the nucleus where it becomes methylated by a PRMT. This is consistent with data indicating that 66 Vav1 is robustly and persistently phosphorylated/activated upon CD28 ligation (and our unpublished data). In contrast, TCR ligation alone induces only tran66 62 sient Vav1 phosphorylation and little methylation . An interesting mechanistic scenario would be that only CD28 ensures efficient Vav1 nuclear translocation (resulting in prolonged phosphorylation) and methylation. At present, no function has been assigned to the nuclear methylated Vav1; however, a pharmacological inhibitor of SAHase drastically reduced Vav1 methylation and also inhib62 ited TCR–CD28-induced IL2 gene activation . Vav1 contains multiple functional domains, including a “core” GEF region composed of Dbl-homology (DH), pleckstrin homology (PH), and cysteine-rich 67 (or Zn-finger) domains in tandem . This region is regulated by N-terminal calponin homology and acidic regions. A C-terminal SH3–SH2–SH3 module is 67 required for protein–protein interactions and for Vav1 phosphorylation . The methylated arginine residue(s) on Vav1 has (have) not been identified yet, but Vav1 contains a number of RG-containing sequences in these domains that might be the target of PRMT1.

7. REGULATION OF ARGININE METHYLATION BY PEPTIDYLARGININE DEIMINASES (PADs) For many years, it has been believed that arginine methylation is a stable modi2 fication that cannot be removed . It seemed reasonable that this modification, like most other posttranslational modifications (i.e., phosphorylation and acetylation), is reversible since it has been implicated in a number of signal transduction processes that regulate protein activation/function (as described above). To date, no known demethylase exists that can directly remove the methyl group from methylated arginines. Recent reports, however, have demonstrated that the methyl group on a mono-methylated arginine can be removed, in the form of 68-70 monomethyl-amine, by peptidylarginine deiminase 4 (PAD4) , a process called demethylimination. PAD4 is one of five members of the PAD family (PAD1–4 and 6) (Figure 1) that catalyze the conversion of protein-bound arginine to citrulline (Figure 71 2) . PAD4 has been shown to remove the methylamine group from monomethylated arginine in histone H3 and H4; however, di-methylated arginines still appear to be a stable modification, as these methyl groups cannot be re68,70 moved by this enzyme . It was also demonstrated that PRMT substrates lose 70 their ability to become arginine methylated after pretreatment with PAD4 . However, it is still unclear as to which process (prevention of methylation or removal of methylation) is more significant in regulating arginine methylation. To complicate matters, an amino transferase has yet to be discovered that can

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convert the atypical amino acid citrulline into arginine. It appears as though we may have solved one problem (removal of mono-methylation, prevention of methylation), by creating another (inability to convert citrulline back into arginine). Therefore, we have yet to fully understand the mechanism by which arginine methylation is regulated. 8. CONCLUSIONS AND PERSPECTIVES The studies summarized here are alerting us that we are on the verge of uncovering a new world of biochemical signals regulating immune responses. Thus, signals emanating from the antigen and costimulatory receptors can be propagated by arginine methylation, in addition to more conventional phosphorylation reactions. It is likely that other immune receptors will also be found to transduce 20,51 stimuli via arginine methylation . Future investigations should inform us to what extent PRMTs and arginine methylation of proteins have been adopted by lymphocytes to serve in adaptive immune functions. It is widely accepted that biological signaling acts as an on/off switch. Signal transduction pathways must be readily reversible in order for a biological response to a stimulus to be shut off after removal of the stimulus. Similarly, if a stimulus is continuously supplied, cells should possess devices to become desensitized to it in order to prevent unnecessary stress and waste to the cellular machinery. These notions, intuitively obvious and experimentally verified, seem somehow at odds with the long-lasting, quasi-irreversible nature of protein ar72 ginine methylation . However, this salient feature of arginine methylation may prove advantageous and serve unique functions in signaling networks. Arginine methylation, which is accumulated on proteins relatively late after receptor trig62,73 gering , may represent a form of storage of chemical information, possibly a “signal memory.” Thus far, arginine methylation has not been implicated in protein degradation pathways, suggesting that arginine methylation is meant to maintain signals via protein–protein or protein–nucleic acid interactions over times much longer than those allowed by reversible phosphorylations. One can readily see the advantage of this property in immunological processes, where signal integration is necessary over time and space. To exemplify, a signal received by a lymphocyte in a given anatomical compartment may be “remembered” later in a different site, thanks to a relatively long-lasting chemical mark (and function) of a protein, when the cell is ready to receive signals of a different nature. Such an accumulation may serve the purpose of directing lymphocyte differentiation programs in a stepwise irreversible fashion over time. Studies of lymphocyte signaling have considerably increased hopes for a more rational medical intervention in pathogenic infections, autoimmunity, cancer, vaccination, and transplantation. Early studies suggested that drugs that block cellular methylation by irreversibly inhibiting SAHase were effective in 56 treating autoimmune conditions in animal models . However, it is possible that these drugs did not pass the stage of development because they had unacceptable

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or hepatotoxic effects for humans due to their irreversibility and inability to discriminate between different types of cellular methylations (i.e., lysine, DNA, small molecule, etc.). Therefore, it will be interesting to see whether reversible SAHase blockers may prove less or nontoxic and whether they block key arginine methylation reactions in lymphocytes. Also, recent data have demonstrated 74 the feasibility of conceiving small molecules to specifically block PRMTs . Thus, arginine methylation seems to provide another window of opportunity for applications in clinical practice in disease prevention, diagnosis, and treatment. 9. ACKNOWLEDGMENTS Thanks to financial support from the Pasteur Institute and the Association pour la Recherche sur le Cancer (A.R.C. grant n. 3461) (O.A.), long-term E.M.B.O. fellowship (B.T.S.), and fellowships from the Ministère de la Recherche et de l'Enseignement Superieur and from A.R.C. (F.B.) 10. REFERENCES 1.

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15 IMMUNE REGULATION BY UBIQUITIN CONJUGATION K. Venuprasad, Chun Yang, Yuan Shao, Dmytro Demydenko, Yohsuke Harada, Myung-shin Jeon, and Yun-Cai Liu

1. INTRODUCTION ATP-dependent protein degradation via conjugation to a protein substrate with a 1 76-amino-acid peptide, ubiquitin, was discovered in early 1980s . It has now been well established that this conjugation process, or protein ubiquitination, is 2,3 carried out by a cascade of enzymatic reactions . The C-terminal glycine residue of ubiquitin is first activated by the ubiquitin activating enzyme or E1 to form a high-energy thiol-ester bond between the C-terminal glycine residue of ubiquitin and the active cysteine of E1. The activated ubiquitin is then transferred to one of the ubiquitin-conjugating enzymes or E2s via a similar thiol– ester bond formation. The ubiquitin ligases or E3s recruit a protein substrate and bind to the ubiquitin–E2 complex and thereby help transfer ubiquitin from E2 to the lysine residue of a substrate via a covalent isopeptide bond formation. Based on the structure of E2 binding, the E3 ubiquitin ligases can be generally divided into two families: the really interesting new gene (RING)-type E3s, and the homologous to E6-associated protein C-terminus (HECT)-type E3s. In addition to E2 binding, the E3 ligases also contain well-defined protein interaction domains, through which the E3s confer the specificity of the substrate targeting in the ubiquitin system. A substrate can be tagged with a single ubiquitin molecule to one lysine residue of a substrate, called mono-ubiquitination, or to multiple lysine residues 4 to form multiple mono-ubiquitination . A substrate can also be tagged with more ubiquitin molecules via successive conjugation of one ubiquitin to another by utilizing lysine residues of the ubiquitin to form poly-ubiquitination. Depending on the usage of different lysine residues on an ubiquitin, the poly-ubiquitin

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chain can be formed via the lysine 48 (K48) linkage, or K63 linkage. Evidence has been accumulated that mono-ubiquitinated protein occurs more often on cell surface receptors, which results in lysosome-dependent downmodulation, whereas K48-linked poly-ubiquitination leads to proteasome-dependent degradation, and K63-linked poly-ubiquitination is related to protein complex forma2,3 tion . It should be mentioned that ubiquitin conjugation is a reversible process in which ubiquitin can be cleaved off the substrate by de-ubiquitinating enzymes. In addition, ubiquitin-like molecules like SUMO (small ubiquitin-like modifier) are also tagged to the substrate in a manner similar to the ubiquitin conjugation pathway. Numerous studies have documented the importance of protein ubiquitination in regulation of the immune system, with one of the earliest findings being the proteasome-dependent degradation of the inhibitor of κB (IκB), which is a 5 critical step in activation of NF-κB and subsequent immune responses . More recent studies have been focused on characterization of novel E3 ligases and identification of their specific substrates by using biochemical approaches, which are complemented by genetic studies of E3 ubiquitin ligase-deficient mice. It becomes clear that E3 ligases are critically involved in many aspects of the immune responses, including T cell activation, differentiation, migration, 6 and tolerance induction . Here we will discuss several more recent examples to highlight the importance of the ubiquitin system in immune modulation.

2. THE E3 LIGASE ITCH IN T CELL DIFFERENTIATION Antigen stimulation of naive CD4 T cells results in differentiation into two distinct subsets of effector cells — T helper type 1 (Th1) and Th2 — and are de7 fined by their distinct cytokine profiles and their immune regulatory functions . Th1 cells provide protection against intracellular pathogens and viruses, produce IFN-γ, IL-2, and lymphotoxin, and induce isotype switching to IgG2a. Th2 cells mainly produce IL-4, IL-5, and IL-13 and are involved in humoral immunity, allergic responses, and isotype switching to IgE and IgG1. The differentiation of T cells into either the Th1 or Th2 subset is determined by several factors, such as the avidity of the TCR, the nature of the costimulatory molecules, as well as cytokine milieu. Transcription factors such as GATA-3, c-Maf, and T-bet are 8 also crucial in determining the relative ratio of Th1/Th2 responses . The initial evidence that protein ubiquitination is involved in T cell differentiation came from studies of Itchy mice. Itchy mice were originally identified from studies of mouse coat color changes in which mutation of the colordetermining agouti gene is linked to abnormal immunological responses includ9 ing skin scratching phenotype . The targeted gene was mapped as Itch, which encodes a protein containing an N-terminal protein kinase C-related C2 domain, 10 four protein-interacting WW domains, and a C-terminal HECT ligase domain . Further detailed studies of T cells from Itchy mice showed that Itch deficiency is

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linked to a biased Th2 development, with increased IL-4 and IL-5 production 11 from the mutant T cells . This finding is further supported by the observation of the increased serum IgE and IgG1 levels in the mutant mice. At the molecular level, Itch was found to bind to JunB via the WW domains of Itch and a PPXY motif of JunB. Thus, Itch promotes ubiquitin conjugation to JunB and induces its degradation. Interestingly, expression of JunB transgene in T cells leads to 12 elevated Th2 cytokine levels and JunB-deficient mice have decreased IL-4 and 13 other Th2 cytokines . Whether Itch-mediated regulation of Th2 cytokine secretion is dependent on JNK activation was analyzed recently using MEKK1 mutant and JNK1 knocout 14 –/– mice . Like Itch-deficient T cells, T cells from MEKK1 mutant or JNK1 mice are prone to Th2 differentiation, with increased c-Jun and JunB protein levels in those cells. Ectopic expression of MEKK1 in 293 T cells promoted ubiquitination of c-Jun and JunB in an Itch-dependent manner. In addition, Itch-dependent JunB degradation was enhanced by constitutively active JNK1 protein. Based on these results, it was proposed that JNK signaling modulates JunB degradation 14 and Th2 differentiation via phosphorylation and activation of Itch .

3. ROLE OF E3 LIGASES IN T CELL ANERGY Mature T cells discriminate between self and non-self foreign antigens with an exquisite degree of specificity and provide protection against a multitude of foreign pathogens. An inherent danger in recognizing many foreign proteins is the potential to respond to self-proteins, which results in autoimmune reactions. The majority of self-reactive T cells are clonally deleted in the thymus, when they undergo positive and negative selection based on the nature of their T cell receptor interactions with peptide/major histocompatibility complex. Most selfreactive T cells are deleted at this stage, but some escape into the periphery. In healthy individuals, self-antigens do not elicit a significant immune response, as 15 they are rendered tolerant to self-antigens in the periphery . One of the mechanisms for maintaining self-tolerance is via T cell anergy induction. T cell anergy is characterized by a failure to proliferate in response to subsequent delivery of activation and costimulatory signals as well as greatly 16 diminished production of IL-2 . Several recent studies have documented the 17-19 importance of E3 ubiquitin ligases in the induction of T cell anergy , in which E3 ligases Cbl-b, Itch, and Grail are upregulated during the phase of anergizing stimulation. These E3 ligases, particularly Cbl-b and Itch, then promote the ubiquitin conjugation to PLC-Ȗ1 and PKCș, two critical signaling molecules in 17,18 T cell activation . Ubiquitination of these signaling molecules results in their 17 18 degradation or functional modification , which leads to the repression of T cell signal transduction. Importantly, in an in-vivo mouse model of autoimmune arthritis, loss of Cbl-b results in the breakdown of T cell self-tolerance and the 18 development of more severe joint inflammation .

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A more recent study may shed light on the mechanisms for the upregulation 20 of E3 ligases during T cell anergy induction . It was shown that early growth response genes Egr 2 and Egr 3 are key negative regulators of T cell activation and that their overexpression can induce Cbl-b expression. Moreover, T cells –/– from Egr 3 mice had lower expression of Cbl-b and were resistant to in-vivo 20 peptide-induced tolerance . It is still unclear, however, whether Egr 2 and Egr 3 directly or indirectly upregulate Cbl-b expression. 4. ROQUIN AS AN E3 LIGASE IN CELL-MEDIATED AUTOIMMUNITY Roquin is a 125-kDa intracellular protein identified by a screen for autoimmune 21 regulators in mice . It contains an N-terminal RING finger domain characteristic for E3 ubiquitin ligases, a novel ROQ domain of unknown function, and a zinc finger domain known to have RNA binding capacity. Mice carrying a point mutation of methionine-to-arginine substitution in the ROQ domain develop systemic lupus erythematosus characterized by presence of antibodies to doublestranded DNA, focal proliferative glomerulonephritis with deposition of IgGcontaining immune complexes, necrotizing hepatitis, anemia, and autoimmune 21 thrombocytopenia . There is much greater induction of inducible costimulator (ICOS) on mutant T cells, which is reversed by reconstitution of T cells with wild-type protein. Consistent with this, it has been known that differences in ICOS expression are implicated in immune abnormalities: ICOS expression is 22 elevated in human lupus and in mice carrying a diabetes-susceptible haplotype 23 of the ICOS gene . The combination of a RING domain and an RNA-binding zinc finger in Roquin suggests that it targets RNA-associated substrates. Regulation of mRNA 24 stability and translation is emerging as an important process in inflammation . The mRNAs for ICOS contain AU-rich sequences, which is implicated in the rapid turnover of cytokine mRNAs through a mechanism dependent on ubiquiti25 nation . It is therefore hypothesized that Roquin may function as an E3 ligase for ubiquitin-dependent control of the stability of AU-rich mRNAs for ICOS and thereby regulates the function of T cells.

5. A20 IS A DE-UBIQUITING ENZYME AS WELL AS AN E3 LIGASE NF-κB is a transcription factor that plays a very important role in both innate 26 immunity and adaptive immunity . NF-κB is sequestered in cytoplasm when bound to IκB. Upon cell stimulation, IκB is phosphorylated and ubiquitinated through K48 linkage, leading to its degradation. The released NF-κB enters the nucleus and activates gene transcription. Phosphorylation of IκB is carried out by a kinase complex including three subunits of IκB kinase (IKK) (α, β, and γ). In the innate immune system, activation of the IKK complex is induced by acti-

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vation of TRAF6 through MyD88 and IRAK proteins. TRAF6 is a RING domain-containing E3 ubiquitin ligase that can promote poly-ubiquitination of 27 itself, as well as IKKγ (also called NEMO) through K63 linkage . The ubiquitinated TRAF6 activates the protein kinase TAK1, which in turn phosphorylates 28 and activates the IKK complex . A20 was first identified as a product of TNFα-inducible response gene in human endothelial cells using differentiation plaque hybridization techniques in 29 the early 1990s . It consists of a novel 790-amino-acid protein with multiple 30 zinc finger motifs. A20 is expressed with high levels in lymphoid organs and inhibits TNF-induced NF-κB activation in transfected bovine aortic endothelial 31 32 cells and reduces apoptosis in Jurkat T cells . Gene ablation studies showed that A20-deficient mice developed severe inflammation and are hypersensitive to stimulation with both lipopolysaccharide (LPS) and TNFα, and die prematurely. In addition, A20-deficient cells displayed sustained TNF-induced NF-κB 33 responses . A recent biochemical study indicated that A20 inhibits NF-κB activation by 34 dual ubiquitin-editing functions . The N-terminal domain of A20, which belongs to a de-ubiquitinating enzyme of the OUT (ovarian tumor) family, can remove the preexisting K63-linked ubiquitin chains from the activated receptor interacting protein (RIP). Then the C-terminal domain of A20, which contains seven zinc fingers, serves as an E3 ubiquitin ligase to promote K48-linked 34 polyubiquitination of RIP, targeting RIP for proteasomal degradation . Besides its negative regulation of TNF signaling, a recent study also 35 showed that A20 downregulates toll-like receptor (TLR)-mediated signaling . A20 plus TNFR1 or A20 plus TNF double-deficient mice both developed spontaneous inflammations, suggesting that A20 also regulates TNF-independent proinflammatory signals in vivo. A20 reconstituted cells were hypersensitive to LPS-induced shock. In addition, A20 deficient macrophages have enhanced NFκB activity in response to multiple TLR ligands. Thus, A20 is required for termination of TLR-induced activity of NF-κB and proinflammatory gene expression. Biochemically, A20 terminates the TLR signals by removing the K63 linkage ubiquitin chain from activated TRAF6, which is an important intermediate 35 molecule in LPS signaling .

6. REGULATION OF INTEGRIN SIGNALING T cells adhesion is an important early step in the generation of the immune re36 sponse . Integrins, a family of cell adhesion receptors, mediate the adhesion of 37 cells to other cells and to extracellular matrix molecules . Engagement of the TCR results in transduction of intracellular signaling, which causes activation of 38 integrins such as LFA-1, a process also known as inside–out signaling . It is known that the Rho-family GTPases (Rho, Rax, Cdc42, and Rap1) play an im39,40 portant role in activation of integrins .

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Rap1 has been described as a critical mediator to cause LFA-1 activation. 41 Rap1 is activated by the TCR . After activation, Rap1 recruits the effector RAPL by Ras (or Rap1) binding site to associate with LFA-1 and induce redistribution of LFA-1, which can form a "focal zone" of clustered high-affinity 42 LFA-1 . A signaling complex consisting of adaptor CrkL and C3G, a guanine nucleotide exchange factor for Rap1, is involved in Rap1 activation. In T cells, TCR signaling leads to phosphorylation of the RING-type E3 ubiquitin ligases Cbl and Cbl-b. Cbl and Cbl-b then bind to CrkL and induce ubiquitin conjugated to CrkL, preventing the association between CrkL and C3G. In response to TCR –/– stimulation, thymocytes from Cbl mice and peripheral mature T cells from –/– Cbl-b mice showed increased integrin activation, including upregulated LFA-1 43,44 clustering and adhesion to ICAM-1 . It was recently reported that Smurf1, a member of the HECT-type E3 ubiq45 uitin ligase group, regulates RhoA activation . Smurf1 specially induces RhoA ubiquitination and subsequent proteosomal degradation. After RhoA degradation, Rac and Cdc42 at the front of the migrating cell recruit Smurf1 to the PAR6 and PKCζ complex and promote downstream microtubule polarity. Thus, Smurf1-mediated ubiquitination is required in regulating RhoA localization and PKCζ-mediated actin cytoskeleton dynamics.

7. UBIQUITINATION OF CHEMOKINE RECEPTORS Chemokine receptor-mediated cell migration controls adaptive immune responses against localized infections and secondary lymphoid organ develop46 ment . They can be classified into two categories — CCR and CXCR — but all chemokine receptors belong to 7-span transmembrane G protein-coupled receptors. Binding of those receptors initiates a series of biochemical events, including their phosphorylation by G protein-coupled kinase, interaction with arrestins, and recruitment to the clathrin-coated pits where they undergo 47 endocytosis . Ubiquitination of chemokine receptors has been linked to their internaliza48 tion and endocytosis, but not recycling . There are multiple isoforms of CXCR4 49 expressed on lymphocytes, thymocytes and monocytes . It seems that a 101kDa form of CXCR4 represents the ubiquitinated form of CXCR4, caused by multiple mono-ubiquitination. Another study identified that AIP4, the human homolog of Itch E3 ligase, mediates ubiquitination of CXCR4, and recruits Hrs to CXCR4 complex, thus controlling multiple steps of CXCR4 intracellular traf50 ficking . AIP4 binds indirectly to CXCR4 through an intermediate protein, Hrs, also known as an endocytic accessory protein. AIP4 interacts directly with the PPXY motif of Hrs, and CXCR4 is colocalized with Hrs and AIP4. Both CXCR4 and Hrs are ubiquitinated by AIP4. Thus, Hrs acts as ubiquitin receptors in the surface of an endosome and gates the endocytic receptors for ubiquitinated cargo to the degradation pathway.

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8. PIAS AS A SUMO LIGASE Both type I (α and β) and type II (γ) interferons (IFNs) regulate cellular responses via activation of the JAK–STAT pathway, in which the activated STAT transcription factors are translocated to the nucleus to activate gene transcrip51 tion . Given the critical role of IFN signaling in immune responses, this pathway is tightly controlled by negative regulators, with one being the protein in52 hibitor of activated STAT1 (PIAS1) . PIAS1 directly binds to STAT1 and blocks its DNA-binding activity, and thereby inhibits STAT1-mediated gene –/– transcription. Consistent with the biochemical studies, PIAS1 mice display increased protection against viral and bacterial infections, enhanced responses to 53 IFN-β and IFN-γ treatment, and upregulation of IFN-inducible genes . PIAS1 gene-targeted mice are also hypersensitive to LPS-induced endotoxic shock. Thus, PIAS1 plays a critical role in IFN-mediated innate immune responses. The identification of the PIAS homologue SIZ1 in yeast provided the first example that SUMO conjugation requires E3-like factors for substrate sumoylation. More importantly, PIAS proteins promote SUMO conjugation to 54–56 STAT1 . However, biochemical studies did not provide a definitive clue as to how STAT1 sumoylation affects its biological function. To further complicate –/– this issue, PIAS1 cells did not reveal any difference in protein sumoylation 53 from wild-type cells nor any differences in p53-mediated thymic apoptosis . This apparent discrepancy between in-vitro PIAS SUMO ligase activity and its in-vivo biological function may be due to redundancy of PIAS family members. Alternatively, it is possible that PIAS-mediated sumoylation is secondary to the inhibiting function via direct PIAS–STAT1 interactions.

9. SLIM PROMOTES STAT UBIQUITINATION Recently, it was reported that a novel protein, STAT-interacting LIM protein 57 (SLIM), is a STAT ubiquitin E3 ligase . SLIM is a nuclear protein and contains one LIM domain at its C terminus, which consists of two tandemly repeated zinc fingers with similarity to RING fingers. Indeed, SLIM induces ubiquitination of both STAT1 and STAT4, and decreases STAT4 protein expression, suggesting that SLIM-mediated ubiquitination of STAT proteins can target them for proteosome-dependent degradation. In addition, SLIM-deficient CD4+ T cells produce a greater amount of IFN-γ compared to wild-type CD4+ T cells. SLIMdeficient mice showed enhanced inflammatory response to in-vivo challenge with heat-killed Listeria monocytogenesis. Higher levels of STAT1 and STAT4 protein expression and enhanced levels of tyrosine phosphorylation of STAT4 were observed in SLIM-deficient thymocytes and CD4+ T cells, respectively. These data suggest that SLIM regulates both the level of protein expression and the extent of STAT activation, and thereby controls the extent of STATmediated Th1 cell differentiation and subsequent IFN-γ production.

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10. CONCLUDING REMARKS It becomes increasingly evident that protein ubiquitination is closely linked to many aspects of the immune system and the E3 ubiquitin ligases play an important role in those processes via specific targeting of protein substrates. Gene database searching has indicated that there are several hundred E3 ubiquitin ligases, with the majority being RING finger-containing E3 ligases. It is conceivable that there will be very complicated interplay among the E3 ligases. Several E3 ligases may target the same substrate, or otherwise, one E3 ligase may target different substrates under diverse physiological conditions. In addition, the list of de-ubiquiting enzymes involved in immune regulation will increase in the near future. It has to be mentioned that biochemical and molecular approaches are still the main tools for identification of a substrate for a particular E3 ubiquitin ligase. The physiological relevance of such findings has to be correlated with genetic investigations. In any case, the field of research on protein ubiquitination in immune modulation will continue to be a hot topic in the years to come.

11. ACKNOWLEDGMENTS The studies in our laboratory were supported by grants from the National Institutes of Health (NIH) and American Cancer Society to YCL.

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16 CTIB (C-TERMINUS PROTEIN OF IκB-β): A NOVEL FACTOR REQUIRED FOR ACIDIC ADAPTATION Toshihiko Fukamachi, Qizong Lao, Shinya Okamura, Hiromi Saito and Hiroshi Kobayashi

1. INTRODUCTION The inside of the human body is generally maintained at near neutrality, but the surroundings of some cells are often acidic. For example, the poorly organized vasculature in tumor tissues and vessel damage in inflammation loci cause hy1-3 poxia and a shortage of nutrients . Such insufficient perfusion leads to the accumulation of metabolic wastes that acidify microenvironments around cells that 4-6 alter various cellular functions . Two kinds of findings with opposite results have been reported. The antitumoral immune response of killer cells against solid tumors was impaired in 7,8 acidic environments . Endocytosis of IL2 receptors was inhibited by cytosolic 9 acidification . In contrast, it was reported that extracellular acidification increased the cytosolic free calcium level and surface expression of CD18 in hu10 man neutrophils, resulting in activation of these cells . Since immune cells often have to work under acidic conditions, the immune response would be activated under acidic conditions or at least its defects may be minimized, even if the immune response is impaired by acidification. Investigations concerning immune responses under acidic conditions are important for the development of new anti-cancer and anti-inflammation strategies, but only a few investigations have been reported to date. We have recently found that CTIB (C-Terminus Protein of IκB-β) was required for the maintenance of internal pH and proliferation under acidic adaptation in CHO cells. Since a CTIB homologue was found in human immune cells, it would be expected to open a new field of research for immune cell functions. In this chapter, we have

Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan

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summarized recent research developments concerning the cellular response to acidic environments and have discussed roles of CTIB on cellular functions under acidic stress.

2. INTERNAL PH UNDER ACIDIC CONDITIONS The maintenance of internal pH (pHi) is the most useful way to keep cellular functions under acidic conditions. Jurkat cells proliferated in medium at pH 6.3 with only a slightly lower rate and the pHi was 7.0, while the pHi was 7.4 in 11 + + 12-14 medium at pH 7.4 . Several transporters, such as Na /H exchangers , anion 15-17 18,19 exchangers and proton translocating ATPases have been reported to regulate pHi in mammalian cells to date. However, these conclusions were based on experiments to monitor pHi recovery after artificial cytosolic acidification and the mechanism underlying the response of cells to acidic environments remains unclear. In fact, inhibitors of these transport systems showed no detectable decrease in the pHi of Jurkat cells growing in medium at pH 6.4, and the only reagent to decrease pHi was N-ethylmaleimide in Jurkat cells under such condi11 tions . An unknown system sensitive to a thiol reagent is working for pHi regulation in immune cells.

3. EFFECT OF EXTERNAL ACIDOSIS ON SIGNAL PATHWAYS In addition to immune cells, the effects of medium acidosis on signal transduction have been reported in other cells. Acidification of serum-free medium from pH 7.3 to 6.6 stimulated VEGF promoter activity in human glioblastoma cells. This activation was demonstrated to be via transcriptional factor AP-1, and to 20 require ERK1/2 MAPK . The stimulation of MAPK under acidic stress has been 21-23 reported in other cells . Since external acidification decreases the internal pH, the activation of MAPK may be induced by cytosolic acidification. Two meth24 ods for intracellular acidification activated p38 MAPK . NFATc1, a factor essential for osteoclastogenesis, was induced in a receptor activator of NF-κB ligand (RANKL)-stimulated cells via calcium/calcineurin/NFAT pathways, but 25 acidosis was shown to activate the NFATc1 independently from RANKL . External acidification of serum-free medium to pH 5.9 induced matrix metallo26 proteinase-9 (MMP-9) in mouse melanoma cells . MMP-9 is thought to participate in cell migration in injured loci that are assumed to be acidic, due to blood vessel damage. The production of tumor necrosis factor-α induced by lipopolysaccharide 27 increased with acidification of external media in macrophages . The expression of the inducible isoform of nitric oxide synthase (iNOS) gene was stimulated by 28 29 medium acidification , but alkaline pH favored production of iNOS protein . The elevated level of iNOS mRNA may be less important physiologically.

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4. DIFFERENT SIGNAL PATHWAYS ARE OPERATING UNDER ACIDIC CONDITIONS In spite of pHi regulation, the pHi decreased gradually with a decrease in external pH (pHe). Previous results suggest that immune cells are still active, even 11 when pHi decreases to a slightly acidic value ; nevertheless, it can be expected that cytosolic acidosis impairs cellular functions. It remains unclear how cellular functions remain active when the cytosol is acidified. One of the plausible explanations may be that new systems being active at acidic pHi are induced or activated to compensate for the decrease in the activity of systems functioning at around pH 7.4. Numerous signal pathways have been identified for various cellular functions. Almost all of these pathways have been examined under neutral or slightly alkaline conditions. If the above argument is true, new pathways may be found at acidic pHs or activated in acid-stressed cells. Western blot analysis with antiphosphotyrosine antibody revealed that different proteins were phosphorylated 11 in Jurkat cells cultured at acidic pH . In particular, a protein with a molecular weight of 65 to 70 kDa was preferentially phosphorylated in Jurkat cells cultured at pH 6.3. This protein remains to be identified, but interestingly, such pHdependent phosphorylation of this protein was not observed in cells derived from human B-cells. Thus, this protein might be related to signal transduction for the immune response of T-cells. These data strongly suggest that immune cells use different signal pathways at different pHi to keep cells active in a wide range of extracellular pHs. The GPCR family — consisting of OGR1, TDAG8, GPR4, and G2A — increases IP3 and cAMP. The IP3 level increased at pH 6.6 in human embryonic kidney cells containing OGR1, whereas the level was the same at pH 7.4 and 6.6 in cells expressing G2A. The elevated level of cAMP at pH 6.6 was observed in 30 cells expressing TDAG8 or GPR4, but not in cells expressing G2A . Their data suggest that TDAG8 and GPR4 have optimum activity at low pH and are working to produce a high level of IP3 or cAMP at low pH to compensate for the decrease in signal transduction mediated by G2A. Their results have been con30 firmed with knockout mice of these proteins .

5. CTIB, A NEW IκB-β FAMILY, FUNCTIONS UNDER ACIDIC CONDITIONS One of the useful ways to seek new systems functioning at acidic pH may be to isolate mutant cells deficient in the ability to proliferate at acidic pH. However, it is very hard to isolate such mutants from immune cells. It is well known that CHO cells are useful for mutant isolation. 31 Recently, Lao et al. isolated a CHO mutant that was sensitive to acidic pH and found that one clone of the cDNA library made from CHO cells conferred acid resistance partially on the mutant at acidic pH. The clone contains an open

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reading frame encoding a small protein consisting of 138 amino acids, and its deduced amino-acid sequence had a high homology to the C-terminal region of mouse IκB-β. The region around the first ATG codon of the ORF contained the 32 Kozak consensus sequence . When the DNA fragment of this clone was inserted into the upstream region of a green fluorescent protein (GFP) gene without a translational initiation start codon, a fused GFP was detected in cells harboring the resulting plasmid, suggesting that there is a translational initiation site at the 31 region of ATG that they postulated to be a start codon . Based on these results, this novel protein was designated CTIB (C-Terminus Protein of IκB-β). Western blot analysis demonstrated that CHO cells contain two proteins whose molecular weights are approximately 54 and 20 kDa, and they respond to anti-IκB-β antibody. The mutant cells of CHO that are sensitive to acidic conditions contained a small amount of CTIB. When the clone-encoding CTIB was introduced to the mutant CHO cells, the 20-kDa band was enhanced, suggesting 31 that this protein was CTIB . CTIB was difficult to detect using a polyacrylamide gel of less than 13 %. This may be the reason for the ignorance of CTIB existence in previous studies.

6. CTIB IS REQUIRED FOR MAINTENANCE OF PHI AND PROLIFERATION UNDER ACIDIC CONDITIONS Silencing a target gene using siRNA is a useful way to investigate its function. Wild-type cells of CHO proliferate in medium at pH 6.3 at a growth rate of approximately half that at pH 7.4 and pHi was 7.1 under such conditions. CTIB silencing decreased pHi to 6.7 and blocked proliferation of CHO cells completely in medium at pH 6.3. In this CTIB silencing, the CTIB level decreased dramatically, but no significant change in the level of 54-kDa protein, consid33 ered as the full length of IκB-β, was observed . These results led us to assume that IκB-β works at near neutral pH and CTIB functions at acidic pH. Interestingly, levels of IκB-β and CTIB were not changed significantly with the medium acidosis from pH 7.4 to 6.3. Since the microenvironmental pH around cells may change very frequently, cells continuing to have both proteins of the IκB-β family would have the advantage of rapid adaptation to the change in external pH, even if each of them is required at a different pHi.

7. CTIB REGULATES NF-κB FUNCTIONS An IκB family has been known to regulate NF-κB functions via binding to NFκB in cytosol that prevents migration of NF-κB into the nucleus. When cells were activated by external stimuli, IκB-β is phosphorylated and phosphorylated molecules are released from NF-κB. Then, NF-κB moves into the nucleus and 34-36 activates the expression of target genes . The C-terminal region of IκB-β has

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the domain for binding to NF-κB , leading us to argue that CTIB also regulates NF-κB functions at low pHi. IκB-β was preferentially detected in the cytosol. CTIB silencing markedly increased the nuclear level of p65 and decreased the p50 level in the nucleus; 33 both were subunits of NF-κB . It is still unclear which complex of NF-κB is the active form that induces expression of target genes. At acidic pH, CTIB silencing impaired the expression of some genes that were known to be regulated by 33 NF-κB . Immunoprecipitation analysis with anti-IκB-β revealed that CTIB is 33 bound to p65, but not p50 . These data may suggest that CTIB regulates NF-κB functions, probably by prevention of nuclear transfer of an NF-κB complex containing p65, although its precious mechanism remains unclear. 33 Interestingly, CTIB silencing decreased the MnSOD level at low pH . Su37 peroxide was reported to be increased at low pH . CTIB is likely involved in detoxication of superoxide that may impair cellular functions.

8. CTIB IN IMMUNE CELLS As described above, immune responses at acidic pH are more important for our life, leading us to argue that CTIB has an indispensable role in immune cells. Western blot analysis showed that two proteins with molecular weights of around 35 and 20 kDa reacted with anti-IκB-β antibody (Figure 1). The molecular mass of the smaller was very close to the CTIB of CHO cells. Two splicing variants of IκB-β have been reported in humans. Transcript variant 1 (ACESSION no. NM_002503) and 2 (ACESSION no. NM_001001716) encode 38- and 33-kDa proteins, respectively. The C-terminal region of an open reading frame of variant 1 encodes a putative protein consisting of 135 residues. It has 118-amino-acid residues identical to CTIB of CHO cells, and its molecular weight was calculated to be 14 kDa (Fig. 2). Thus, it may be possible that this protein is a human CTIB.

9. CTIB MAY BE A SPLICING VARIANT Is CTIB a product from a transcript variant or derived from translational variation? Multiple mRNA molecules with different molecular sizes were detected 31 with Northern blot analysis in CHO cells , suggesting that CTIB is the product encoded by one of the transcript variants. Recent analysis of the human genome revealed that many mRNA contain the second start codon, suggesting the pres38 ence of translational variants . The human genome contains only 20,000–25,000 39 protein-coding genes . If translational variation is present, a greater number of functional proteins over splicing variations may be synthesized.

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Figure 1. Western blot analysis of CTIB in CHO and Jurkat cells. CHO and human immune cells were cultured in DMEM and RPMI media at pH 7.4 with a supply of 5% FBS, respectively. Cell extracts were prepared and subjected to 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresisѽ as described previously31. CTIB was analyzed with Western blotting using anti-IκB-βҏ antibody (C-20, Santa Cruz) ѽ Lanes 1, BJAB cells; 2, Jurkat cells; 3, CHO cells.

Figure 2. Splicing variants of human IκB-β and the amino-acid sequence of CTIB of CHO cells. (A) Schematic representation of two splicing variants of human IκB-β based on the data of GenBank. (B) Amino-acid alignment of human IκB-β encoded by variant 1 (GenBank) and CTIB of CHO cells31.

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10. CONCLUSIONS After genome analyses have been completed, many people are focusing research subjects on the effect of microenvironments and cell–cell interaction. The most important factor in microenvironments seems to be the change in pH. But such studies have just begun. The data reported to date have suggested that different systems are operating at low pH to compensate for the decrease in cellular functions with acidosis. A large number of unidentified genes is likely involved in the adaptation mechanism to acidic environments. In addition to examination of genes identified already, identification of these unknown genes would be essential for dissecting the mechanism. The effect of external acidification has been examined using cells incubated at low pH for a short time in many reports published to date. Some mechanisms for acidic adaptation would be clarified under these experimental conditions because it has been suggested that some systems functioning at low pH have also been expressed at near neutral pH, such as CTIB. However, many systems functioning at low pH may require an adaptation period to induce the systems. Therefore, investigation of the immune responses of cells cultured at a low pH value for over one generation is essential to obtain novel information for acidic adaptation and the immune responses in acidic microenvironments. These two kinds of experiments would promote our understanding concerning the immune cell responses under acidic microenvironments, and help in developing new strategies for immuno- and chemotherapy.

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17 HIV MAY DEPLETE MOST CD4 LYMPHOCYTES BY A MECHANISM INVOLVING SIGNALING THROUGH ITS RECEPTORS ON NON-PERMISSIVE RESTING LYMPHOCYTES Miles W. Cloyd*, Jiaxiang Ji, Melissa Smith, and Vivian Braciale

1. INTRODUCTION AIDS is characterized by a gradual depletion of CD4 lymphocytes, which leads to declining immunocompetency. The mechanism of CD4 cell loss, however, is still not fully understood. Ever since the early discovery that HIV preferentially infects CD4 lymphocytes and uses CD4 as one of its receptors, it has been generally assumed that replication of the virus in these cells leads to their death and 1 their gradual disappearance . This could, however, involve, in addition to direct virus-mediated cell lysis, cellular or humoral HIV-specific immune killing of 2 3 productively infected cells ; induction of apoptosis by virus replication ; or induction of apoptosis in productively infected cells upon signaling of certain re4 ceptors (TCR, CD4) . However, these mechanisms do not reconcile several major enigmas in understanding HIV pathogenesis: (1) There are clear in situ hybridization data showing that very few cells in vivo produce high levels of HIV (≥250 RNA copies/cell) at any given time (ap5,6 proximately 1 in 100,000 cells) . Even if a virus quickly kills the cells it replicates in (for example, as rabies virus or picornaviruses do), a “snapshot” of the frequency of cells producing viral nucleic acids or proteins yields direct evi7,8 dence of the extent that the virus has productively infected cells in a tissue . HIV strains that are cytopathic (X4 strains) are actually relatively slow at killing

Departments of Microbiology & Immunology and Pathology. The University of Texas Medical Branch, Galveston, Texas 77555. *Corresponding author.

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their host cells , and thus, the in situ hybridization and immunohistochemistry studies examining the frequencies of cells productively infected with HIV accurately reveal the extent of cells replicating virus at any given time. Natural loss of lymphocytes occurs at much greater frequencies than the frequencies of CD4 lymphocytes producing HIV. Using sensitive in situ hybridization assays, cells possessing low levels (5–25 copies) of HIV RNA have been found at ~10-fold higher frequencies (~1 in 10,000 cells) than cells making high amounts of vi11 rus , but these cells cannot be producing many virions and would not likely be killed by replicating virus. In fact, some of these cells may be latently infected, 12,13 since such cells can produce multiply spliced HIV mRNAs . Thus, it is unlikely that elimination of productively infected cells would account for any significant loss of CD4 lymphocytes. + (2) CD4 lymphocytes in HIV subjects have been found to be dying by 14 15 apoptosis in lymphoid tissues , not in blood , and these are cells not making 16 HIV (giving the appearance of “bystander” cells dying) . (3) CD4 lymphocytes begin declining in the blood early after infection 17,18 when principally R5 (noncytopathic) HIV strains are present . (4) Finally, cell killing by replicating cytopathic HIV strains appears to be 10 primarily due to a necrotic mechanism, and not to apoptosis . Thus, most theories on how HIV depletes CD4 lymphocytes focus on the killing of uninfected “bystander” cells. These include “kiss of death” (membrane fusion) of uninfected “bystander” cells upon contacting productively infected 19 20 cells ; autoimmune killing of uninfected cells ; HIV superantigen-mediated 21 deletion of certain T cell subpopulations , or induction of apoptosis in “bystander” cells by HIV proteins (Tat, Gp120) released from nearby productively 22,23 infected cells . Many of these theories have not been supported by in vivo data. Fusion of uninfected cells with those producing HIV does not occur in primary lymphocytes — only in cell lines in vitro — and lymphoid syncytia are 24,25 rarely observed in blood or lymphoid tissues of HIV-infected individuals . + Autoimmunity against host CD4 T cells has not been a frequent observation in 26 patients , and no convincing data have demonstrated that HIV acts as a superan27 tigen . While high concentrations of exogenous soluble Tat and gp120 have been shown to induce cell death, many HIV strains (R5 strains), which make normal levels of both gp120 and Tat during productive infection of CD4 lymphoblasts, do not induce death. In fact, cytopathic R4 HIV strains, which kill their host cells when replicating in them, appear to do so by gp120-induced per10,28 tubation of the cell membrane . Thus, none of these theories of indirect mechanisms have been substantiated by convincing in vivo data.

2. WHAT IS KNOWN TO OCCUR IN HIV-INFECTED SUBJECTS The hallmark of HIV disease is the gradual loss of CD4 lymphocytes, which is generally measured in the blood. This measurement provides a reasonably good

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marker of the extent of immunodeficiency in this disease. A few studies have also measured CD4 lymphocyte levels in lymphoid tissues, but it was found that as CD4 cells disappear from the blood, their net numbers are not reduced in lymph nodes. In fact, they often increase in early disease, and only in later in 29,30 disease do CD4 lymphocytes disappear in lymph nodes (Figure 1). The ratio + + of CD4 T cells to CD8 T cells in the blood becomes inverted early and widens throughout most of the disease. However, this process does not occur in the pe29,30 + + ripheral lymph nodes , where inversion of the CD4 /CD8 ratio is found later in disease. In fact, the increasing numbers of CD4 T lymphocytes in lymph nodes in early disease appear to contribute to the common occurrence of lym+ phadenopathy in HIV individuals. In contrast, the gut-associated lymphoid system is depleted of CD4 lymphocytes very early after infection, but this is mostly 31-33 localized in effector sites (lamina propria) in the GI mucosa . CCR5+ CD4 33 lymphocytes are preferentially lost , but this is unlikely to be due to direct viral killing since the viruses are still R5 strains at these early times after infection, and R5 strains are not cytopathic. Thus, the mechanism of this early CD4 cell depletion in the gut is not clear. Furthermore, what occurs in the gut does not mirror what occurs in the blood or the degree of systemic immunodeficiency. Studies have shown that circulation of lymphocytes between gut and blood is 34 minor, and the gut seems to largely be an autonomous lymphoid system .

Figure 1. The numbers of CD4+ T cells in the blood, lymph nodes, and gut at various time points following infection with HIV.

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Studies directly quantifying the frequencies of dying CD4 lymphocytes in 15 the blood have not found elevated numbers . However, dying cells were found 14 31-33 at elevated levels in the lymph nodes and gut mucosa . These cells in the 16 lymph nodes were not producing HIV RNA and were dying by apoptosis . Thus, “bystander cells” are the cells dying in lymph nodes, and an indirect means of killing of CD4 lymphocytes appears to be the major mechanism of CD4 lymphocyte depletion in non-gut lymphoid tissues. One of the earliest immune defects, occurring before significant CD4 loss in 35,36 the blood, is a deficit of T-helper (Th) memory T cells in the blood . Some studies indicated that memory CD4 lymphocytes are more susceptible to direct 37 HIV cytopathicity , but other studies have shown that memory CD4 cells are 38 + actually more susceptible to productive infection by HIV . If CD4 lymphocytes predominantly die by indirect mechanisms, it is not clear how memory cells would be preferentially targeted. Recent studies describing the consequences of HIV contact with resting CD4 lymphocytes (which are not permissive for HIV replication) can explain the apparent preferential loss of memory cells from the blood (discussed below). There are two different outcomes of HIV infection of a CD4 lymphocyte depending on the state of the cell: (1) productive infection, which results when an already infected resting lymphocyte is activated into cell division or when an activated CD4 lymphocyte is infected, and this results in production of progeny virions; or (2) abortive infection, which results when resting CD4 lymphocytes 39 40 (comprising between 95 and 99% of the CD4 cell population ) are infected . HIV can bind to resting CD4 lymphocytes, enter them, and partially or com41 pletely reverse transcribe their RNA genomes into DNA proviruses . However, this will not integrate into the host cell genome unless the cell goes into division. The half-life of the un-integrated HIV provirus has been reported to be between 40,41 6 hr and approximately 1 week , and if the abortively infected resting lymphocytes are activated within that time, the virus can complete its replication cycle 40 and the cells become productively infected . Many studies have shown that HIV binding to its receptors on resting CD4 cells induces signals that result in 42 changes in gene expansion . In the presence of IL-2, HIV binding induces ele43,44 vated expression of L-selectin and Fas , the former important for homing to lymph nodes and the latter important for induction of apoptosis. The relative frequencies of the two types of infected cells in HIV-infected people (~10:7500 6 45 per 10 cells) for productive:abortive infection somewhat correspond to the frequencies of activated-to-resting cell frequencies (~1:100). While many HIV pathogenesis models focus on productively infected cells, the abortively infected + population should also be considered, since it comprises >99% of all HIV DNA 45 lymphocytes in vivo .

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3. A MODEL OF CD4 LYMPHOCYTE DEPLETION INVOLVING THE ABORTIVELY INFECTED OR GP120-SIGNALLED RESTING CD4 LYMPHOCYTES In general, there are three possible ways in which CD4 lymphocytes could disappear from the blood: (1) the immune system makes fewer new cells; (2) the cells die in the blood; or (3) the cells leave the blood. There is no evidence to support the second possibility, as studies examining the frequencies of dying 4,15 cells in the blood have not found higher than normal levels . Recent studies of SIV models have shown that the first cells that are infected following mucosal infections are cells in the GALT, and it is known that CD4 cells in the GALT are generally more activated. Extensive depletion of these cells occurs before 46,47 depletion occurs in the blood . Other studies examined colon biopsies of HIV patients and showed that there are higher frequencies of HIV-producing cells in 31-33 the GALT than in other lymphoid tissues and reduced numbers of CD4 cells . Since the frequencies of activated lymphocytes in GALT are much higher than in other tissues, CD4 cells that are highly permissive for HIV replication are at much higher frequencies there. A recent model of HIV pathogenesis has evolved that assumed that rapid destruction of CD4 cells by HIV replication in the 31-34 GALT leads to gradual disappearance of CD4 cells throughout the body , but this has not been proven. The normal frequencies of CD4 lymphocytes relative to CD8 cells are much less in the GALT than other lymphoid tissues. Most importantly, the extent of depletion of CD4 cells in the GALT does not mirror what occurs elsewhere, and does not mirror the levels of systemic immunodeficiency. Thus, it is questionable as to whether this model explains everything that occurs in humans regarding HIV induction of CD4 cell depletion and immunodeficiency. Various studies, as pointed out previously, have shown that there is an increase of dying CD4 cells in peripheral lymph nodes of HIV-infected humans compared to uninfected individuals, and the numbers of apoptotic CD4 cells are elevated in lymph nodes. Regarding the first possibility, renewal of T lymphocytes in adults is slow (requiring approximately one year in bone marrow transplant recipients or after 48,49 anti-CD4 antibody depletion in vivo) . If HIV impairs CD4 lymphocyte re+ + newal, then CD8 T cell counts should drop along with CD4, because CD4 and + CD8 cells arise from a common CD4/CD8-positive precursor cell. Parallel de+ creases of CD8 cell numbers do not occur; in fact, the CD8 numbers actually 50 increase above normal levels for most of the disease course . However, studies have shown that CD8 T cell increases are largely due to clonal expansion of 51 already differentiated cells , and other studies indicated that production of both + + + 52 new CD4 and CD8 lymphocytes is reduced in HIV subjects . Others suggested that CD4 lymphocyte production is nearly normal, but their survival time 53 is reduced . Thus, most studies conclude that production of new CD4 cells is not reduced much, if any — especially in the early stages of disease when the rate of CD4 reduction in the blood is greatest.

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There is much evidence now for the third possibility that CD4 lymphocytes leave the blood. HIV-abortive infection (or contact with gp120) induces resting 43,54 CD4 lymphocytes to home from blood to lymph nodes (Figure 2). HIV-induced homing of abortively infected resting CD4 lymphocytes would increase the proportion of CD4 cells migrating from blood to lymph nodes per unit time. Direct measurements of trafficking of CD4 lymphocytes present in the blood of HIV-infected subjects showed that they do, in fact, migrate twice as fast to 55 lymph nodes and axial bone marrow as those in uninfected people (Figure 3). 55 HAART treatment nearly stopped this enhanced migration . This phenomenon could explain the gradual disappearance of CD4 cells from the blood at a rate much faster than in lymph nodes. HIV was shown to induce resting CD4 mem43 ory lymphocytes, to home to peripheral lymph nodes , which could explain the 35 early deficit in Th-memory responses in blood lymphocytes . Normally, most memory CD4 lymphocytes lack L-selectin and do not migrate to peripheral lymph nodes. Presumably, most of the increase in homing CD4 lymphocytes are abortively infected or signaled by contact with producing cells or virion-coated 55 follicular dendritic cells and do not produce HIV mRNA . About one-third of them are induced into apoptosis after entering the lymph nodes, owing to secondary signals received through various homing receptors (CD62L, CD44, CD11a) 54 during trans-endothelial migration . These data can explain the observed “bystander” cell death in lymph nodes at the same time that CD4 lymphocytes grossly disappear from blood. Finally, HIV induction of enhanced homing of resting CD4 lymphocytes can explain the observed effect of HAART treatment, leading to an acute increase of memory CD4 cells in the blood. Naive CD4 cells express high levels of L-selectin, which determines the rate-limiting step for specific migration to peripheral lymph nodes, and these cells always circulate rapidly from the blood to lymph nodes and back. Most memory cells do not express L-selectin, as it is shed after activation and it only returns on ~20% of the memory CD4 cells. Thus, most memory cells generally do not home to peripheral lymph nodes and they often have other receptors specific for homing to other tissues. Upon HAART treatment, reduction of virus spread and new production of virus in cells in the lymph nodes results in much less homing, and the cells increasing in the blood will predominantly be memory cells (Figure 4). Figure 5 illustrates a model of HIV pathogenesis based on the data showing that contact of resting CD4 lymphocytes with productively infected cells or virions causes them to leave the blood and home to lymph nodes. Since ~95–99% of all CD4 lymphocytes are resting, each HIV-producing or virion-coated dendritic cell would contact many uninfected resting CD4 lymphocytes as the latter mi55 grate through the lymph system back into blood . This, in essence, amplifies the presence of the few productively infected cells. Their contact induces signals 56 57 through CD4 , and chemokine receptors , and these signals induce: (1) upregulation of CD62L, the homing receptor for peripheral lymph nodes, and an enhanced ability to home back to the lymph nodes after these cells have entered 43 44 the blood ; and (2) upregulation of the pre-apoptotic molecule Fas .

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Figure 2. Immunocytochemical staining of human T lymphocytes in SCID mice lymph nodes (LNs). Frozen sections of LNs were obtained 4 days after i.v. injection of resting human PBLs. (A) and (B) were stained with anti-human CD3 Ab (brown) and counterstained with hematoxylin (blue). (A) is a LN section from a mouse i.v. injected with mock-treated resting human PBLs, and (B) is from a mouse given HIV-exposed PBLs. (C–F) are immunocytochemical colocalization of DNA fragmentation and lymphocyte surface markers. DNA fragmentation (apoptotic cells) was labeled by the TUNEL method (dark blue/black). Human T lymphocytes were stained by anti-human CD8 (C,E) or CD4 (D,F) Abs (brown). Double-labeled cells are dark brown or with dark nuclei (arrows). (A,B) X400 magnification, (C,D) X650, (E,F) X1000.

Within one or two days, many of these cells will migrate back into the blood as 58 a result of the normal lymph–blood circulation process , but when they enter the 55 blood, they exhibit accelerated homing back to lymph nodes and marrow . As

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Figure 3. Whole body γ camera scintiphotos localizing 111In-labeled CD4 lymphocytes in an uninfected and HIV-infected subject. Freshly isolated blood CD4 lymphocytes were labeled with 111In (500 µCi) in vitro, rinsed, and then i.v. reinfused back into the original donor. The subject was then scanned by a γ camera at 1 (A,D), 3 (B,E), and 24 (C,F) hr post-infusion. The intensity of the signals in organs corresponds to the number of radioactive lymphocytes that migrated from the blood within the indicated time points. Computer digitization of the radioactivity presented as the following colors: red > yellow > green > blue (negative). The images presented are 600-s exposures.

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Figure 4. The numbers of CD4+ T cells in the blood, lymph nodes, and at various time points following infection with HIV, and following HAART treatment.

the cells home, they transmigrate the high endothelial venules into the lymph nodes, at which point they receive second signals through various homing receptors. This signaling induces approximately one-half of these cells to die by apop54 tosis . Because these homing cells are abortively infected or only signaled by gp120 contact and are not making viral mRNA, it appears that “bystander cells” are dying. It is likely that if a surviving cell encounters an antigen specific for its TCR, it will respond normally and clonally expand. The virus, then, completes 40 its replication cycle and produces virus progeny . This process, in turn, results in a gradual increase in productively infected cells, which causes more resting cells to home and die. In response to highly active anti-retroviral therapy (HAART), release of infectious virus from the few productively infected cells + abates, and consequently, less virus can bind to and signal resting CD4 T cells. One effect would be that some CD4 cells appear to return to the blood because enhanced homing is stopped and CD4 cells are not being killed during the hom59 ing process. A disproportionate increase in memory T cells would be observed because HIV induces these normally non-homing cells to home.

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Figure 5. A schematic illustration of what may occur to CD4 lymphocytes in HIV+ individuals based on the studies of Wang et al.43,54 and Chen et al.55 The relatively rare productively infected cells making significant amounts of HIV (approximately 1 in 100,000 cells), depicted as squares producing virions, come into contact with surrounding lymphocytes, 98% of which would be resting. Virions binding to the resting CD4 lymphocytes or physical contact of the resting lymphocytes with the productively infected cell induces signals through CD4 in the resting cells, which leads to upregulation of L-selectin for a few days and Fas. These cells, through the normal lymph system-toblood circulation, will be back in the blood within one to two days. At that point, the enhanced Lselectin and Fas expression are maximized. These cells will home back to lymph nodes at an enhanced rate, and during the homing process one-third of them will be induced into apoptosis. These cells never make HIV virions and would appear to be “bystander” cells that are dying. Some of the surviving cells may actually come in contact with antigen specific for their TCR and would then be activated and clonally expand. Those cells will finish the replication cycle of HIV and will become HIV producing cells. The cycle of HIV signaling of resting cells in the lymph nodes then continues.

4. CONCLUDING REMARKS +

The above scenario can explain most of the events observed in HIV patients: loss of CD4 lymphocytes from the blood at the same time they are not decreasing in lymphoid tissues; cells dying by apoptosis in lymph nodes, but not in blood; cells not making virus in the lymph nodes being the predominant cells dying; the reappearance of mostly memory CD4 lymphocytes in the blood after

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HAART; and the reason steroid treatments stop the loss of CD4 lymphocytes 60 61 from the blood in patients . Steroids are known to downregulate L-selectin and retard homing to peripheral lymph nodes. If this is the major way HIV causes depletion of CD4 lymphocytes, then HIV may not cause AIDS if it used a different receptor on CD4 lymphocytes. The SIV endogenous to African green 62 monkeys is not pathogenic in that species, and these primates lack typical CD4 . Recent studies have shown that signaling through CD4 in certain ways (mAb crosslinking in solution; gp120–anti-gp120 crosslinking) causes changes in resting CD4 lymphocytes that mimic those induced by HIV contact. A number of viruses (arenaviruses, hemorrhagic fevers) cause disease by indirect mechanisms, not just by direct killing of the cells in which they replicate. Often the anti-viral immune responses or induced inflammatory reactions are the me63 diators of pathogenesis in those cases . AIDS could likely be a disease in which the signals induced by the virus through its cellular receptor on non-permissive cells are detrimental, and this may represent a rather unique pathogenic mechanism for viruses.

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18 INTEGRATING TRADITIONAL AND POSTGENOMIC APPROACHES TO INVESTIGATE LYMPHOCYTE DEVELOPMENT AND FUNCTION Yina Hsing Huang, Rina Barouch-Bentov, Ann Herman, John Walker, and Karsten Sauer*

1. INTRODUCTION The sequencing of the human and mouse genomes to over 95% coverage revealed that more than three quarters of the ~30,000 genes have not yet been as1-5 signed biological functions . Thus, a strong need exists to functionally annotate over 20,000 genes. This challenge provides the opportunity to discover novel 6 genes essential for disease pathology. If their encoded proteins are druggable , such genes represent promising targets for the development of new and improved therapies for severe human disorders including autoimmunity (rheumatoid arthritis, systemic lupus erythematosus, diabetes, etc.), allergies (asthma, dermatitis, etc.), and other inflammatory diseases. Traditional approaches to investigate gene function use results from biochemical, cell biological, molecular biological, and genetic studies, often accumulated over many years of intense research, to formulate a hypothesis that is then tested via targeted gene disruption, or more recently siRNA-mediated targeted knockdown for increased invivo relevance. This approach has been impressively powerful and productive. For example, over 100 genes involved in development and function of the adap7,8 tive immune system have been identified . On the other hand, the traditional approach is clearly slow and highly labor intensive, as many thousands of graduate students, postdocs, and technicians can attest.

Genomics Institute of the Novartis Research Foundation (GNF), 10675 John J. Hopkins Drive, San Diego, CA 92121, USA. *To whom correspondence should be addressed.

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A complete functional annotation of the genome is thus very unlikely to be achieved within the lifetime of even the youngest current graduate student, unless novel, faster approaches are used. The nearly complete genome sequencing and parallel development of powerful new technologies has enabled approaches that will investigate the functions of most of the un-annotated ~20,000 genes as well as unveil novel functions for known genes well within a young graduate student's lifetime. Here we discuss selected case studies to illustrate how the combination of novel, integrated technologies with traditional, hypothesis-driven research provides an accelerated, highly productive approach to investigate lymphocyte development and function at the dawn of the 21st century. First, we review how gene expression profiling and novel analysis tools allow gaining a broad perspective of the immunological transcriptome and its mining for potentially important genes. We then discuss the current status of much-needed genome-wide approaches to functionally interrogate such large sets of genes through highthroughput screens of cDNA or siRNA libraries. Narrowing the perspective to the single gene level and at the same time maximizing in vivo relevance, we discuss how large-scale forward genetics programs have led to identification of mouse mutants with defects in various aspects of immune function, ranging from lymphocyte development to complex in vivo responses in disease models. We use one particularly interesting mutant out of our own program, Ms. T9,10 less , as a case study to demonstrate how a combination of forward genetics and traditional approaches allowed us to identify inositol(1,4,5)trisphoshate-3kinase B (ItpkB) as a novel key regulator of T cell development.

2. THE IMMUNOLOGICAL TRANSCRIPTOME One of the key developments in the last decade was the establishment of DNA microarray technology, which now allows one to monitor expression of essentially the entire transcriptome simultaneously. In recent years, many studies have used this technology to discover novel genes, pathways, and gene networks involved in multiple cellular processes, ranging from T cell development to disease pathology. Gene expression profiling has opened new avenues to identify novel molecular targets for therapeutic intervention, and improved markers for 11 diagnosis and clinical prognosis . 2.1. Technology The most widespread methods for monitoring gene expression on a genomewide scale employ microarrays to measure nucleic acid abundance. Fluorescent dye-labeled cRNA or cDNA derived from various samples is hybridized to oligonucleotides or double-stranded DNAs synthesized or spotted onto a solid surface. For example, Affymetrix GeneChips can monitor ~39,000 mouse or

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~47,000 human transcripts per array. Each gene is represented by a probeset comprising 11 to 16 pairs of short, 25-base oligonucleotides on the array. Each pair includes a perfect match probe complementary to the gene sequence and a mismatch control probe that is identical except for a single base mismatch in its center. The bound fluorescence signal per probe is detected using an optical scanning device and quantified by comparison of the perfect match/mismatch differences per probeset. Another form of microarray uses double-stranded DNA or long single-stranded 60–80 base oligonucleotide probes. In this method, two different fluorescent dyes are used to label mRNA of interest and a reference mRNA. The intensity ratio between both dyes then represents the relative abundance of the mRNA of interest. Microarray approaches are fast and robust but expensive and restricted to the complexity of the arrayed probeset. Other technologies do not require individual probesets and are thus able to monitor samples of unknown complexity. Serial analysis of gene expression (SAGE), rapid analysis of gene expression (RAGE), and tandem arrayed ligation of expressed sequence tags (TALEST) are 12-14 some popular examples . These techniques make use of common restriction sites within the cDNA pool to generate short cDNA fragments whose identities and abundances are then determined by sequencing. This type of approach can lead to identification of new classes of genes not represented on microarrays, including noncoding or micro-RNAs. Once expression data have been generated, identifying genes and pathways that warrant individual follow-up is the next challenge. New and improved data 15 analysis software packages include GeneSifter (www.genesifter.net/web), Ingenuity (www.ingenuity.com), InforSense:BioSience (www.inforsense.com), and Interaction Explorer (PathwayAssist, www.stratagene.com). Additionally, open-source software packages such as Bioconductor or TM4 offer free analysis tools. These advanced software packages enable the scientist to analyze microarray data in the context of biological processes, compile gene annotation from multiple sources, identify and visualize expression patterns and networks of potentially interacting genes, as well as cross-reference biological networks gener15 ated from their datasets with well-known pathways . With the recent widespread use of gene expression profiling in many labs around the world, a paramount issue is the integration of results from different 16-19 laboratories and across all platforms . Similar studies performed by different labs can give very different results. The use of very standardized protocols by experienced operators helps to obtain more robust and cross-comparable data. Not unexpectedly, relative gene expression data comparing specific biological processes, for example, Gene Ontology (GO) categories, are more robust and biologically meaningful than absolute expression values. Several studies suggest that using similar experimental protocols, data analysis approaches, and mi16-20 croarray platforms are required for good validation across platforms . Documentation of experimental details and results following the Minimal Information About Microarray Experiments (MIAME) standards developed by the Microar-

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ray Gene Expression Data Society (MGED ) helps to improve experimental 20 reproducibility . Even though cross-platform and cross-experiment comparability still pose major issues, several institutions have started to assemble gene expression and annotation data warehouses accessible through the internet (Table 1). They provide public access to huge data collections and allow even a small lab to mine gene expression databases for genes showing a specific expression pattern across selected tissues or cell types. More advanced resources are starting to provide meta-analyses beyond simply plotting expression data. For example, ImmGen allows one to specifically investigate relationships between cell populations and expressed genes in the immune system (Christophe Benoist, personal communication). Table 1: Publicly Accessible Web-Based Gene Expression Data Warehouses Organization Genomics Institute of the Novartis Research Foundation (GNF) Gene Expression Omnibus (GEO)

URL symatlas.gnf.org

References [23]

www.ncbi.nlm.nih.gov/geo

[24]

ImmGen, Joslin Diabetes Center, Harvard University

www.immgen.org

University of Toronto Oncogenomics Normal Tissue Database, CCR, NCI, NIH, DHHS TeraGenomics

mgpd.med.utoronto.ca

[25]

ntddb.abcc.ncifcrf.gov/cgi-bin/nltissue.pl

[26]

www.barlowlockhartbrainmapnimhgrant.org/public/login.asp

[27]

genome-www.stanford.edu/microarray

[28,29]

Stanford Microarray Database

(Christophe Benoist, pers. communication)

2.2. Using Gene Expression Profiling To Investigate Lymphocyte Development And Function Gene expression analyses have been used by multiple labs to identify novel genes differentially regulated and thus possibly functionally involved in key 30-32 aspects of the immune system, including lymphocyte development , 33-36 37 38,39 40-43 activation , homeostatic proliferation , anergy , memory , or autoimmu44-46 nity . Here, we discuss selected examples to highlight the potential and limitations of this approach.

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The molecular mechanisms governing T cell development are still incompletely understood. To identify novel genes involved in thymic selection, a process that warrants generation of a functional T cell repertoire tolerant to self 47,48 49,50 antigens , several labs profiled gene expression during negative or positive 30,31,51 32,52 selection . These and other studies identified multiple genes differentially regulated during thymocyte development. Many of the results were confirmed by real-time PCR, immunoblot, or flow cytometry. Additionally, by using gene ontology databases, many of these genes have been placed into categories according to their known or predicted function. Besides the identification of individual genes, microarrays can be used to identify relationships between different signaling pathways or networks. To explore the differential contributions of TCR versus Notch signaling during positive selection, the Robey group investigated gene expression in thymocytes from mouse mutants deficient in genes 30 involved in TCR or Notch signaling on TCR transgenic backgrounds . They identified unique and common downstream targets of the TCR and Notch. In addition, TCR and Notch appeared to cooperate to induce a subset of genes, suggesting that Notch may sustain or enhance the effects of TCR signaling. Like many other studies, this group used a specialized subgenomic microarray, which is enriched for a relatively small number of genes related to immune function. Since then, the recent availability of genome covering microarrays now allows 31 more comprehensive studies . These examples clearly demonstrate the value of gene expression profiling to identify novel genes likely involved in thymocyte development. However, the fact that Th-POK, a recently identified differentially 53 expressed master regulator of thymocyte lineage commitment , was not identified in various gene profiling experiments pinpoints a limitation of this approach. Microarrays are useful for surveying large groups of genes, rather than conclusively interrogating specific individual genes. + CD8 T cells are crucial for host defense against invading pathogens and malignancies. Relatively little is known about intracellular signaling events that + control their differentiation into memory CD8 T cells, which are critical for an 40,54 efficient recall response against invading pathogens . To better understand + memory CD8 T cell development and survival, several groups monitored gene + expression in CD8 T cells during various kinds of stimulation, including viral 40-43 infection . However, although gene expression patterns can be profoundly connected to gene function, measuring many thousands of genes in parallel can result in a high false discovery rate. Thus, differential expression of individual 15 genes, or even correlations or associations may happen randomly . More detailed follow-up studies are thus essential to translate gene expression data into 43 meaningful biological insight. For example, the Ashton-Rickardt group used gene expression analyses to test the theory that memory T cells are direct progeny of effectors that escaped from death by identifying protective survival genes. They identified eight protective genes that were upregulated in memory + versus naive CD8 cells. Follow-up with retroviral transduction and pharmacological inhibition experiments led to identification of serine protease inhibitor 2A

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as a survival factor necessary for CD8 effector cell differentiation into memory + CD8 T cells. Gene expression profiling has been widely used to explore molecular events accompanying abnormalities in immunological disorders, including breakdown of tolerance and augmentation of the self reactive repertoire in autoimmune diseases. Among others, studies of type-1 diabetes used microarrays to investigate the gene expression program mediating central or peripheral tolerance. Combined with biochemical approaches, these studies helped to identify several im45,46,55 portant candidate molecules involved in disease pathology .

3. FUNCTIONAL GENOMICS: MODULATION OF GENE FUNCTION IN TISSUE CULTURE CELLS The major advantage of gene expression profiling is that with the most recent gene arrays it allows one to assess the expression of nearly the entire transcriptome, thus providing maximal genome coverage. Its major disadvantage is that gene expression data are mostly correlative and thus at best suited to identify relatively large sets of candidate genes that still need to be investigated functionally. Functional investigation requires testing the effects of overexpression or disruption of the gene in a relevant cell type and physiological context, best in whole animal models. Cost, time, and logistic issues limit whole animal studies to investigating a low number of genes at a time, in most cases no more than one. Cell lines provide a system in which gene function can be investigated at an intermediate scale, justifying their use as a first pass to investigate large gene numbers, albeit with reduced in vivo relevance. In the past, genome-wide functional genomic screens in lymphoid cells employed x-ray or chemical mutagenesis of Jurkat or other T cells to isolate clones deficient in surface expression of TCR components or other molecules, in par2+ ticular stimulation-induced activation markers, or defective in TCR-induced Ca mobilization or reporter gene expression. Isolation of individual clones required 56-59 complicated selection schemes . This approach resulted in the identification of a remarkable but clearly incomplete number of key components of the TCR sig56 naling machinery through the efforts of several labs over a period of ~20 years . More recently, screens of Jurkat or B cells with retroviral cDNA, peptide, or 60-63 anti-sense nucleotide libraries have been reported . These approaches used pooled libraries, necessitating selection protocols based on survival or iterative rounds of enrichment of the desired cell populations. For example, the Rigel group screened cDNA libraries to isolate Jurkat cell clones with impaired TCR 60 + low induced CD69 upregulation . Here, single CD3 CD69 cells were finally plated onto 96 well plates for isolation of over 2800 individual clones. These approaches led to the identification of several known mediators of TCR or BCR signaling, but also revealed several new candidates for proteins involved in these processes. Follow-up work unveiled SLAP-2 as a novel inhibitor of TCR and

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61

BCR signaling , and TRAC-1 as a novel E3 ubiquitin ligase with a positive role 64 in TCR signaling . The drawbacks of these approaches are that retroviral integration, spontaneous mutations, or epigenetic changes occurring during the long selection procedures may result in false positives. The Rigel group filtered out such cells using doxycyclin repression of the transduced cDNA and found that 60 1,323 out of 2,828 clones had a Dox-regulatable phenotype . Moreover, cDNA overexpression itself may elicit nonphysiologic effects, because overexpressed proteins may act as dominant negatives or even neomorphs. Thus, cDNA screens appear more appropriate in settings where phenotypes resulting from deficiency in unknown proteins are rescued, thereby identifying the missing protein or functional analogs thereof. Clearly, loss-of-function screens employing specific reagents to interfere with the expression of one gene at a time appear most appropriate to identify novel essential genes involved in lymphocyte function. 3.1. RNA Interference (RNAi) Technology Recently, large advances have been made in designing effective knockdown strategies using small interfering RNA (siRNA) and small hairpin DNA (shDNA). siRNAs are short double-stranded RNAs that target approximately 21 nucleotides of a particular mRNA. When transfected into cells, the anti-sense strand of the siRNA can form complexes with cellular mRNA and target it for degradation by the RISC complex. In this manner, 70–95% knockdown of target 65-68 mRNA levels can be achieved . This approach is a fast, easy, and inexpensive way of determining gene function compared to generating mutant cell lines or animals. Additionally, siRNA libraries have been used successfully in screens 67-72 with non-lymphoid cells to identify genes important in cellular processes . 69 However, gaining effective knockdown is not always easy . The degree of functional knockdown depends heavily on the target of interest. Targets that turn over rapidly and are limiting in their respective pathways are better than those that are abundant, have redundant function, or with long half-lives. Once an appropriate target has been identified, designing an siRNA sequence that will actually knock down its expression poses the next challenge. Computer algorithms, based on the testing of multiple siRNAs against thousands of targets, have been developed to design potentially effective siRNAs with a 20–75% chance of suc73,74 cess . Each potential siRNA must then be assessed for potential off-target effects, or knockdown of unintended targets with sequence similarity. Algorithms such as Smith-Waterman can identify some putative off-targets. However, sequence similarity in as little as 11 or even 7 consecutive nucleotides, particularly 75,76 in the 3′ UTR of the target mRNA, can mediate off-target effects . Clearly, the parameters defining the algorithms for designing siRNAs and identifying offtargets are by no means absolute. Thus, testing 3 to 5 different siRNAs per target and validating knockdown of RNA and ultimately protein expression are required. Because each siRNA has unique sequence-specific off-target effects, a

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large degree of confidence can be gained by observing the same cellular phenotype with at least two independent siRNAs and/or in a separate complementary 67,69 assay . To increase the probability of identifying an effective siRNA, some investigators and companies use a mix of several siRNAs against each target in hopes that one or two actually target the cellular transcript. However, since each siRNA in the mix is at a lower dose, the one or two effective siRNAs may knock down expression less efficiently than a single siRNA at full dose. The chance of eliciting off-target effects may also increase with the use of siRNA pools. siRNAs cannot replicate or propagate themselves. Therefore, knockdown is transient and limits the types of functional screens to short-term assays. To establish long-term knockdown, a retroviral or lentiviral plasmid-based form of 68,70,71,77,78 siRNA has been employed . Here, a RNA polymerase III promoter such as the U6 promoter drives expression of a small hairpin RNA (shRNA) from a shDNA containing the same short target sequence as the siRNA, followed by a hairpin and then the antisense target sequence. Within the cell, the transcribed shRNA forms a hairpin structure that is then processed by Dicer, ultimately generating an siRNA, which proceeds to target transcripts as above. However, in addition to the targeted cellular mRNA transcript, the viral shRNA is itself susceptible to knockdown by its encoded siRNA. Generating 1 to 3 mismatches (or wobbles) in the sense strand of the shDNA can decrease targeting of the viral 77 construct and improve knockdown efficiency . Because shDNA is plasmid based, cells in which the shDNA plasmid has stably integrated within the genome can be isolated via drug selection or sorting for coexpression of a marker such as GFP. Establishing stable knockdown lines enables functional analyses in a defined clonal population, and assays that are lengthy or require large cell numbers. Additionally, engineering of shDNAs into retroviral and lentiviral plasmids allows their delivery into difficult-to-transfect cells via viral transduction and may enable genome-wide RNAi screens in lym72 phocytes using arrayed siRNA/shDNA libraries . However, a problem with retro- or lentivirus-based screens is the difficulty in producing and packaging high-titer libraries in a high-throughput manner. Typically, ultracentrifugation is required to obtain the high multiplicity of infection (MOI) required for efficient viral transduction. Additionally, unless each shDNA has been validated, the knockdown efficiency is unknown. Testing three or more shDNAs per target can increase the likelihood that at least one shDNA is effective. Ineffective siRNAs and shDNAs can increase the false-negative rate and result in missing key effectors. Other drawbacks to shDNA knockdown include possible viral inactivation 65, 79 or interferon responses by the host cells . Nevertheless, recent advances in the generation of arrayed cDNA, siRNA, 72 and retro- or lentiviral shDNA libraries combined with establishment of efficient high-throughput transfection or transduction and assay technologies has enabled the parallel functional profiling of large gene sets that will soon reach genome-wide coverage and allow screens in lymphoid cells. These approaches

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provide a much needed complement to gene expression profiling and other broad technologies. They will allow one to interrogate and unveil entire pathways involved in a biological process of interest, and to prioritize genes for further investigation in more physiological systems. Large consortia like The RNAi Consortium (TRC, www.broad.mit.edu/genome_bio/trc), the Alliance for Cellu80 68,72 lar Signaling , or other institutions will make libraries, screening tools, and data accessible to the academic community. The major disadvantages of these approaches lie in the limited in-vivo relevance of immortalized cell lines and invitro assays, and in the proneness of cDNA overexpression or RNAi to yield artefactual, off-target results. To fully understand gene function in a natural, physiological context and thereby identify valid targets for pharmacological intervention, genetic studies in whole animals are essential. 4. MAXIMIZING IN-VIVO RELEVANCE: REVERSE AND FORWARD GENETICS 81

Over the last decades, reverse genetics, or gene targeting , has revealed important immunological functions for over a hundred genes in T and B cell develop7,8 ment, activation, and survival . Despite the rich amount of knowledge gained by this approach, reverse genetics is a time-consuming process that does not always easily bear fruit. Knockout mice are typically generated on mixed genetic backgrounds and require extensive backcrossing to eliminate strainspecific modifiers that can muddy data interpretation. Additionally, many genes have redundant functions, are ubiquitously expressed, or function at multiple stages of cellular development. Targeting genes with closely related family members can prove uninformative due to genetic compensation or redundancy. Null mutations sometimes lead to embryonic lethality or early developmental blocks that preclude analysis of the cells or process of interest. For this reason, tissue- or stage-specific conditional knockouts can be generated using the CreloxP or other systems, but require generation of and additional breeding to tissue-specific Cre transgenics. Despite these challenges, much of what we know about immunological processes is based on or confirmed by findings from the 7, 8 study of knockout mice . With the sequencing of several mammalian genomes, it became clear that only a fraction of the ~30,000 genes have an ascribed function. Reverse genetics performed in individual laboratories is unlikely to quickly reveal the function of un-annotated genes because the decision to invest in the targeting of individual genes is largely hypothesis driven and limited by imagination and current understanding. To alleviate this limitation, various public and private research consortiums or companies have established mouse strain repositories and begun to generate and distribute mouse mutants or ES cell libraries of targeted mutations (Table 2). Their availability significantly reduces the lead time required for generating knockouts; however, desired individual mutants may not yet be available.

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Institution International Mouse Strain Resource (IMSR) International Gene Trap Consortium The Jackson Labs Mutant Mouse Regional Resource Centers (MMRRC) European Mutant Mouse Archive (EMMA) Oak Ridge National Laboratory Bay Genomics Lexicon Genetics Deltagen

URL www.informatics.jax.org/ imsr/index.jsp www.igtc.ca www.jax.org www.mmrrc.org/index.html www.emma.rm.cnr.it bio.lsd.ornl.gov/mouse baygenomics.ucsf.edu www.lexicon-genetics.com/ index.php www.deltagen.com/index. html

Key references [82] [83] [84] [84]

Fly geneticists have long used the power of forward genetics, which involves first identifying a phenotype followed by its causative mutation, to identify novel genes involved in complex biological processes like embryonic devel85 opment or innate immunity . A particular advantage of chemical mutagenesis is that it can result in complete loss-of-function (null/amorphic allele), partial lossof-function (hypomorphic allele), opposing or dominant negative function (antimorphic allele), or gain-of-function (hypermorphic allele) phenotypes. Alleles eliciting incomplete phenotypes allow genetic interaction analyses through breeding of different mutants, or sensitized mutagenesis screens in a mutant background. In flies this approach has allowed elucidation of entire signaling 86 networks, exemplified by groundbreaking work on the Ras/Erk pathway . Until recently, mouse genetics has been restricted to the slow characterization of natural mutations, resulting in identification of key genes like 87 FoxP3/Scurfy, a regulator of regulatory T cell development and autoimmunity , 53 or Th-POK, a master regulator of thymocyte lineage commitment , through many years of efforts. However, the recent sequencing of the mouse genome and the development of powerful technologies to map mutant genomic regions through the use of single nucleotide polymorphism (SNP) or microsatellite markers have greatly facilitated the positional cloning and molecular identifica88,89 tion of mutations in mice . Thus, the major current limitation to using mouse genetics for identification of novel gene functions in vivo is the lack of large numbers of uncharacterized mouse mutants. To generate and characterize libraries of novel mouse mutants, various institutions conduct genome-wide chemical mutagenesis screens in mice (Table 3). N-ethyl-N-nitrosourea (ENU) treatment of mice introduces point mutations within the genome at the approximate rate of 90 one per million base pairs . At the protein level, ENU treatment results mainly

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Table 3: Immunological ENU Screens Institution Australian Cancer Research Foundation Genetics Laboratory and Medical Genome Centre, John Curtin School of Medical Research, and Australian Phenomics Centre, Australian National University (ANU), Australia. URL: immunogenomics.jcs.anu.edu.au/acrf.htm; www.apf.edu.au Celltech, USA. (terminated) Genomics Institute of the Novartis Research Foundation, USA. URL: web.gnf.org

The Jackson Laboratory, Program for Genomic Applications (PGA), HLBS Center, USA. URL: pga.jax.org GSF/Institute of Experimental Genetics (IEG) Genome Project and Ingenium Pharmaceuticals AG, Germany. URL: www0.gsf.de/ieg; www.ingenium-ag.com The Scripps Research Institute (TSRI), USA. URL: www.scripps.edu/ imm/beutler

Walter and Eliza Hall Institute of Medical Research,

Immunological screens 1) Hematology 2) Flow cytometric analysis of PBLs 3) Antibody isotypes 4) Autoantibodies 5) Immunization screens (TH1/TH2 responses, T cell dependent/independent, memory)

1) Hematology 2) Flow cytometric analysis of PBLs 1) Hematology 2) Flow cytometric analysis of PBLs 3) T and B cell activation 4) Ig isotypes 5) DNP-KLH antibody responses 6) Autoantibodies 7) Dermatitis 8) Arthritis Hematology

1) Hematology 2) Lymphocyte populations and lineage markers 3) Basal immunoglobulin levels 4) Anti-DNA antibodies 5) Allergy: IgE level 6) Paw inflammation 1) Macrophage response to bacterial products/TLR ligands 2) CMV infection 3) Listeria infection 4) Pseudomonas aeruginosa infection 5) Tumor challenge 6) Visible mutations with immunological defect Hematology

Published genes and references 11 genes7, including Carma-192, NFκB293, Ikaros94, Roquin95, SLP-767. See also 89, 96.

CD8397. See also98. ItpkB9, 10, c-Myb99, CD4, DNA-PK, Lyn (AH, RB, BW, MC & KS, unpublished data).

PLCγ2100. See also 100-103.

12 genes104, including CD36105, Trif/Ticam1(Lps2)106, CD14107, TLR9108, TNF109. See also109-112.

c-Myb113

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Institution Australia. URL: www.wehi.edu.au/ research/divisions/chd Centre for Modeling Human Disease (CMHD), Canada. URL: www.cmhd.ca Riken Genomic Sciences Center (GSC), Japan. URL: www.gsc.riken.go.jp/index E.html Baylor College of Medicine (BCM) Mouse Genome Project, USA. URL: www.mousegenome.bcm.tmc. edu/Home.asp Oak Ridge National Laboratory (ORNL), USA. URL: bio.lsd.ornl.gov/ mgd/programs.htm

Immunological screens

Published genes and references

1) Hematology 2) Immunoglobulin levels 3) Delayed Hypersensitivity Hematology

Hematology

Hematology

fit1114

in missense mutations and to lesser degrees in splicing defects or nonsense mu91 tations . In a typical G3-screen to identify dominant and recessive mutants, a single ENU-treated founder male is bred to untreated females to eventually generate families of third-generation (G3) progeny. Each G3 mouse harbors a pool of point mutations on an otherwise homogeneous genetic background such as C57BL/6. G3 mice within the same family have overlapping pools of mutations, thereby providing a means of independent confirmation. Statistically, one in eight G3 mice should be homozygous for a given mutation. Informative mutations can be identified through phenotypic screens that can range from the simple analysis of peripheral blood cell type numbers to responses to complex immunological challenges. As long as preceding analyses do not interfere with successive ones, multiple screens can be performed in series on the same animals to maximize efficiency and minimize cost and effort (for excellent, detailed 7,89,96,98,104,115,116 recent reviews of forward genetic screening strategies, see ). As with any screen, its design determines the number and nature of the mutations identified. Robust screens with high signal-to-noise ratios, for example, activation of peripheral T cells via strong CD3/CD28 costimulation, are most likely to reveal strong mutant alleles but may miss weak alleles. In contrast, sensitive screens under suboptimal challenge conditions may reveal weak alleles,

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but generate more false positives. Moreover, weak alleles may be difficult to map due to high phenotype variance. Thus, screens that are designed to balance these concerns prove most fruitful. Simple screens, for example, using a CellDyne/HemaVet or flow cytometer, can be used to detect mutants with decreased numbers of peripheral blood cells. In-vitro activation of peripheral blood lymphocytes can reveal genes involved in B or T cell activation. Detection of serum antibodies by flow cytometry or highthroughput microscopy can reveal defects in isotype switching or B cell differentiation, as well as mutations that lead to increased predisposition to allergy (high IgE levels) or autoimmunity (anti-nuclear antibodies, ANA). Mice can also be immunologically challenged with antigen, irritants, or even viruses, microorganisms, or other disease inducing agents. Once a phenodeviant has been identified, it is backcrossed to evaluate phenotype heritability, and out-crossed to a different genetic strain such as Balb/c to identify the causative mutation. Mapping the mutation is achieved by analyzing C57BL/6 x Balb/c F2 offspring, whose genome is a mix of C57BL/6 and Balb/c, which are distinguishable by different genetic polymorphisms. At GNF, a panel 88 of 10,990 SNPs across 48 inbred mouse strains is available , allowing us to choose the most appropriate mapping strain for each given phenotype. This is important, because polymorphic modifier loci may significantly alter phenotypes in a background specific manner. This is a particular problem for complex 89,96 phenotypes such as disease susceptibility, which involves many genes . By linking the phenotype to homozygous C57BL/6 derived genomic fragments, the mutation can be restricted to a small chromosomal interval. Final identification results from genomic sequencing of candidate genes. These are prioritized by combining available expression data, human conserved synteny data, gene annotation, predicted or known function and biochemical data. Data warehouses like those listed in Table 1 facilitate candidate gene prioritization by providing easy electronic access to such data. Currently, mapping mutations to reasonably small genetic intervals and positionally cloning the mutated gene are the rate-limiting steps. To bypass the breeding required for mapping mutations, screening can be performed on hybrid mice. The mutagenized C57BL/6 male is crossed to untreated Balb/c females to eventually produce C57BL/6 x Balb/c hybrid G3 mice for screening. However, immune cells from different genetic backgrounds often respond very differently to stimulation. Thus, hybrid strains may be too phenotypically diverse to allow isolation of mutant alleles, resulting in weak or intermediate phenotypes. Once a mutation has been identified, follow-up requires hypothesis-driven research to tease out the mechanisms by which the affected gene functions. In a flow-cytometric screen for mutants with reduced peripheral blood T cell numbers, we recently identified a novel regulator of T cell development, inositol 9,10 (1,4,5) trisphosphate-3 kinase B (ItpkB) . ItpkB phosphorylates the second messenger IP3 to IP4. Loss of ItpkB protein expression due to an ENU-induced

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N-terminal nonsense mutation in our mutant Ms. T-less causes a block in posi+ + tive selection of CD4 CD8 double positive thymocytes, resulting in an almost complete peripheral T cell deficiency. Negative selection appears unimpaired (Y.H. Huang and K. Sauer, unpublished observation). To determine the mechanism by which IP4 regulates T cell maturation, we evaluated the ability of ItpkBdeficient thymocytes to transduce T cell receptor (TCR) signals involved in 2+ positive selection. Surprisingly, TCR-induced Ca signals were normal, even 2+ though IP3 is a key mediator of Ca release from intracellular stores. Instead, ItpkB deficient cells were defective in TCR-induced ERK activation, a process known to be essential for positive selection. Thus, our ENU-induced mouse mutant revealed a novel mechanism of TCR signaling involving ItpkB and very likely its phosphorylation product, the soluble small molecule IP4 (Figure 1). Further studies aimed at identifying the role of IP4-binding proteins, including IP4BP 117-119 the negative Ras regulator GAP1 , in TCR signaling and thymocyte development remain areas of active research. ENU-induced point mutations can reveal novel functions of known genes that might not be readily identified in mice harboring straightforward gene disruptions. For example, hypomorphic alleles can unveil functions of genes whose null mutation results in embryonic lethality. Anti- or hypermorphic alleles may unveil functions for redundant genes, whose disruption has no phenotype. The study of point mutant alleles may also provide more relevant disease models, considering that about 50 genetic polymorphisms often not resulting in complete 120-124 gene disruption have been linked to disease susceptibility in humans . 125 92 126, 127 Moreover, point mutant alleles of ZAP-70 , Carma-1 , or LAT can cause autoimmunity or hypersensitivity in mice, whereas disruption of the entire locus results in immunodeficiency. The GNF hematology screen recently yielded 99 Mega, a mouse mutant carrying a hypomorphic c-Myb allele . While c-Myb128 deficient mice die in utero , Mega is viable and fertile. It harbors a point mutation in the c-Myb transactivation domain, resulting in reduced activity and association with the transcriptional coactivator p300. This mutation resulted in increased proliferation of hematopoietic stem cells (HSCs), increased megakaryocyte development at the expense of red blood cell production, and blocks in

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Figure 1. Proposed role for ItpkB as a negative regulator of Ras in thymocytes through production of IP4. TCR stimulation results in activation of the receptor proximal protein tyrosine kinases Lck and ZAP-70, tyrosine phosphorylation (P) of multiple modular proteins, and assembly of a TCR signaling complex containing phospholipase Cγ1 (PLCγ). PLCγ1 hydrolyzes the membrane lipid PIP2 into IP3 and diacylglycerol (DAG). DAG recruits the Ras activator RasGRP1 and PKCs (not shown) to the membrane. IP3 mediates Ca2+ release from intracellular stores and is converted into IP4 through ItpkB. We hypothesize that IP4 may negatively regulate GAP1IP4BP, allowing RasGRP1 to fully activate Ras and resulting in Erk activation. Impaired IP4 production in ItpkB-deficient Ms. Tless mice would result in constitutive Ras inactivation by GAP1IP4BP. Inset: A block of thymocyte development in Ms. T-less at the CD4+CD8+ double positive stage (top) is associated with introduction of an N-terminal STOP codon into ItpkB which abrogates protein expression (center). This results in impaired TCR induced Erk-activation in Ms. T-less thymocytes (bottom). Rescued Erk activation after exogenous supply of the cell permeable DAG analog PMA suggests that ItpkB acts upstream of Ras. Details in9, 10.

early B and T cell development. Thus, a single point mutation in c-Myb revealed function of c-Myb at multiple stages of hematopoiesis. 4.1. The Promise of Forward Genetics in Understanding Disease The ultimate promise of genetics and genomics in biomedical science is to understand pathogenesis in order to be able to treat diseases with rationally designed therapies focused on molecular targets. Genome-wide association studies

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have begun to yield exciting information toward identifying molecular targets after years of intensive efforts by several groups working on polygenic dis120-124 eases . The promise of forward genetics in this arena is only just beginning to be explored. The non-hypothesis-driven aspect of this type of genetic study could yield previously undiscovered mechanisms that lead to pathological immune responses and provide novel avenues for therapy. Some models of aberrant immune responses that generate pathology have been found surreptitiously by screening ENU mutant mouse libraries for non-disease-related phenotypes 92 such as high surface IgM (the Carma-1 mutant "unmodulated" ) or low B cell numbers (our “weeB” mutant; Ann Herman, Rina Barouch-Bentov, Mike Cooke & Karsten Sauer, unpublished observation). These mutants with altered immune phenotypes end up developing pathology as they age, atopy in the case of unmodulated and anti-nuclear antibodies (ANA) in weeB. The challenge of this type of study in a bona-fide disease model is that for an ENU-mutagenesis program there must be a robust response and a high incidence of disease in order to successfully isolate mutants away from background noise. Our best autoimmune disease models (collagen-induced arthritis, EAE, NOD, etc.) tend to have incidence in the 60–80% range, making it difficult to assess whether a mutation preventing disease onset has been inherited, or whether that mouse is simply a reflection of the 20% that would not have succumbed to disease anyway. Several groups are beginning to crack these issues by simplifying a disease process into steps. For example, the ENU effort led by Chris Goodnow has used anti-nuclear antibody (ANA) production to screen and isolate mutants that aberrantly develop this prelude to lupus-like disease at an 96 early age . Here at GNF we have embarked on a screen using the KRN serum transfer model of effector-stage arthritis developed by Mathis, Benoist, and col129 leagues . In this model, arthritogenic autoantibodies specific for glucose-6phosphate isomerase found in the serum of KRN T cell receptor transgenic animals on an NOD background are transferred into a naive recipient, in this case the G3 progeny of an ENU-mutagenized mouse. This model has been used extensively in reverse genetic studies, and is known to depend on IL-1, TNF-α (as seen in the human disease), as well as various cell types such as mast cells, neu130-133 trophils and NKT cells . The transient onset of arthritis is rapid and very robust, reaching approximately 95% incidence over a large cohort of mice depending on the strain. The high incidence has been amenable to usage in a highthroughput ENU screen, allowing us to identify several potential mutants that are resistant to the onset of arthritis. Our first confirmed mutant, “Knuckles,” is currently at the mapping stage (Ann Herman, Lisa Deaton & Karsten Sauer, unpublished observation). Further complications arise in mapping complex diseases, but these can be surmounted by using appropriate strains that do not affect disease phenotype directly. Thus, the usefulness of forward genetics approaches in complex disease models may be teased out with very robust models, or those that simplify a disease into its component parts. An unbiased approach to identi-

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fying disease-modulating loci will likely yield important information to achieve the promise of directed therapeutics in human disease. 4.2. The Future of Mouse Mutagenesis While the value of ENU mutagenesis has become apparent, developing an ENU program in a small laboratory setting is challenging. The cost and logistics of initiating large-scale mouse mutagenesis, a phenotyping program, and in particular a viable strategy for mapping and positional cloning of the mutated allele are often prohibitive. One solution for this problem that is readily accessible to small labs is to obtain mutants of interest from one of the mouse strain repositories or ENU facilities listed in Tables 2 and 3. It can be expected that within a few years gene-targeted or gene-trapped mice and eventually even conditional disruptions of most genes will be available through the current efforts of the institutions listed above, the European Mouse Mutagenesis Consortium and the 84 European Conditional Mouse Mutagenesis Program (EUCOMM) , the Knock134 83 out Mouse Project , the International Gene Trap Consortium , and other efforts. According to a recent analysis, nearly 2/3 of all murine genes have already 83 been trapped . Novel, faster, or more efficient gene disruption methods like 135-137 138 BAC-based targeting and recombineering , RNAi in ES cells or somatic 139 137 cells , or insertional mutagenesis via gene traps or MICER will significantly accelerate this process. However, all these technologies except RNAi induce larger genetic alterations in the targeted locus than point mutations and thus may elicit off-target effects. RNAi has the disadvantages of incomplete knockdown and high likelihood of unspecific off-target effects. Null alleles may not reveal all functions of the encoded genes due to lethality, developmental defects, or compensation by redundant genes. They may also not represent appropriate models for human disorders resulting from small genetic polymorphisms as discussed above. Thus, the need for point mutants carrying hypo- or hypermorphic alleles will continue to exist. Over time, some of the mutagenesis centers listed in Table 3 will provide growing libraries of ENU mutants. Resequencing of G1 offspring of mutagenized males will allow one to identify the point mutations each mouse harbors, and thus freeze libraries of sperm carrying pre-selected ENU induced alleles, providing the opportunity to select specific point muta115,116,140 tions for later propagation and hypothesis driven research . Yet, given that a comprehensive coverage of all naturally occurring protein interactions and functions, thought to underlie cellular traits, through interfering point mutations 7 might require on the order of several hundred thousand alleles , these libraries are unlikely to replace the benefit of unbiased, phenotype-driven de novo screens of random mutants for a significant while. More cost-efficient alternatives to ENU mutagenesis may become available by once again transferring invertebrate approaches to mammals. Insertional mutagenesis via transposable elements has been in use in fruit flies, worms, plants and yeast for many years. It can allow the direct cloning of the affected

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gene via inverse PCR, thus avoiding the currently rate-limiting sequencing of multiple candidate genes. Insertional mutagenesis also enables the identification of functionally relevant noncoding regions that may elude candidate gene sequencing approaches. The recent development of transposon systems like Sleeping Beauty that are active in mouse ES cells, embryos, and somatic cells may 116,137 enable transposon mutagenesis once certain issues are resolved . These issues include low transposition efficiency, the need to limit transposase expression to the germline to achieve stable preservation of identical insertions in all somatic cells, and the preferred close linkage of new integrations to the donor locus, reminiscent of "local hopping" in flies. An attractive and so far unavailable possibility enabled by transposon mutagenesis could be tissue-specific and conditional transposase expression. This would allow conditional mutagenesis in specific cells or tissues. Moreover, the "local hopping" phenomenon could be exploited to conduct screens within specific genomic regions. This approach has 141 recently been used successfully to mutate all genes in a 4-Mb region . Taken together, reverse genetic approaches employing classic or RNAi mediated gene targeting and forward genetics provide useful complements to functionally annotate the ~30,000 genes of the mammalian genome with maximal in 7 vivo and human disease relevance (Table 4) . Table 4: Comparison of in vivo mutagenesis approaches Gene targeting Requires prior knowledge about gene (hypothesis, structure, sequence, expression pattern)

RNAi Requires prior knowledge about gene (hypothesis, structure, sequence, expression pattern) Intermediate throughput

Transposons No prior knowledge required — potential for unanticipated, groundbreaking discoveries Intermediate throughput

~ 1 year to mutant

Intermediate time requirement

Simple logistics (KO facility)

Extensive logistics (many injections)

Phenotype first, intermediate time requirement Intermediate logistics

Low throughput: One to few genes at a time

ENU No prior knowledge required — potential for unanticipated, ground- breaking discoveries

Intermediate to high throughput: Many mutations at a time, but few may have effect. G1/F1 resequencing allows generation of mutant sperm collections which ultimately warrant high genome coverage and throughput Phenotype first, ~1 year to gene Extensive logistics (breeding, mapping, phenotyping)

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Table 4: Comparison of in vivo mutagenesis approaches Gene targeting Eliminates entire gene and its function, may be done conditionally/in tissue specific manner. Strong phenotypes unrelated to hypothesis, or redundancy may obscure hypothesized phenotype

RNAi Eliminates most gene functions, may possibly be done conditionally/in tissue specific manner Strong phenotypes unrelated to hypothesis, or redundancy may obscure hypothesized phenotype

Major disruption of genomic target region (many nucleotides, residual exogenous DNA) can cause artifacts Low probability of second site hits/effects on other genes

Minor disruption of genomic target region. Viral integration may cause off-target effects

Disease relevance: In most cases not representative of naturally occuring polymorphisms

Intermediate to high probability of off-target effects on other genes Disease relevance: In many cases not representative of naturally occuring polymorphisms

Transposons Eliminates most gene functions, may possibly be done conditionally/in tissue specific manner Strong phenotypes unrelated to hypothesis, or redundancy may obscure hypothesized phenotype, unless weak alleles are generated Intermediate disruption of genomic target region (transposon insertion)

ENU Phenotype selection allows selective modulation of a specific gene function, but mutation present throughout development.

Low ambiguity, since phenotype driven and low mutation rate

Low ambiguity, since phenotype driven and low mutation rate

Disease relevance: In many cases not representative of naturally occuring polymorphisms

Disease relevance: More representative of naturally occuring polymorphisms

Hypomorphic alleles may unveil phenotypes that are masked or absent in mice harboring disruptions or other null alleles. Anti- or hypermorphic alleles may unveil functions for redundant genes Very low disruption of genomic target region

5. CONCLUSIONS AND OUTLOOK Fast advances in gene profiling technology and the ability to perform functional genomics screens using arrayed cDNA and siRNA/shDNA libraries have drastically accelerated the pace of in-vitro and tissue culture-based functional gene annotation. High-throughput protein purification and crystallization methods allow unprecedented scale and speed of structure resolution, enabling fast elucidation of molecular mechanisms in vitro. Interactome maps will accelerate the description of protein interactions. Much needed are more efficient mutagenesis methods to accelerate high-resolution functional studies of proteins and their interactions. Here, small molecule modulator approaches may possibly provide useful complements to molecular biological and genetic approaches. The most

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severe bottleneck for target discovery is validation in animal models. A combination of large-scale forward and reverse genetics with population genetics and 88,142,143 QTL analyses in inbred mouse strains can alleviate this problem and will lead to accelerated discovery of targets with high disease relevance. While highthroughput small molecule screens are no longer rate limiting, the current bottleneck for the development of novel therapeutics are drug development and clinical studies. It is here where the highest need for the next "quantum leap" in biomedical technology may reside.

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19 PHOSPHOPROTEOMIC ANALYSIS OF LYMPHOCYTE SIGNALING Lulu Cao, Kebing Yu, and Arthur R. Salomon

1. INTRODUCTION The successful sequencing of the human genome is a monumental accomplishment. The application of this mountain of information to gain a better fundamental understanding of the molecular basis of disease represents a formidable challenge in the post-genomic era. For the first time, analysis of the whole complex system may be considered in parallel to traditional focused approaches. New global approaches must be developed to exploit this resource. One technique, the use of mRNA expression arrays, has gained new popularity in visualizing global patterns of transcription. Unfortunately, there is a relatively weak correlation between transcript abundance and the abundance of individual pro1 teins . Also, critical signaling pathway components such as posttranslational modifications and protein–protein interactions are overlooked by the expression arrays. The emerging field of proteomics seeks to address some of these challenges by focusing on which proteins are expressed, their abundance, how they interact, and how they are modified in a global, unbiased fashion. Modern mass spectral proteomic tools leverage the availability of genomic sequence databases to match experimental spectra to genome-derived peptide and protein sequence. This report will focus on some recent developments in bioinformatics and proteomics methods based on mass spectrometry that provide new capabilities to examine the structure of signaling cascades through global phosphorylation site analysis from complex lysates.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Box G-E335, Providence, RI 02912

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2. METHODS FOR PHOSPHOPROTEOMICS Phosphorylation sites from cell-derived signaling molecules are extremely difficult to detect due to their low abundance and low stoichiometry of phosphorylation in a cellular milieu of abundant unphosphorylated proteins. Although many approaches have been adopted to overcome this limitation, the limited specificity and efficiency of phosphorylation site isolation from highly multidimen2 sional chromatographic separations such as MudPIT or low pH, strong cation 3 exchange renders a unique technical challenge in revealing the phosphoproteome. Fundamentally, the poor quality of phosphopeptide tandem mass spectra arising from their low abundance, poor ionization, and abundant neutral loss of phosphate from phosphoserine and phosphothreonine residues necessitates manual validation of every candidate MS/MS spectra. This time-consuming burden can be overcome by increasing the quality of the data through the use of accu4 5 rate mass , better fragmentation methods such as ETD , more highly selective 6,7 8 chromatography , and development of new statistical algorithms . The limited dynamic range of mass spectrometers necessitates selective enrichment of phosphopeptides to obtain the highest numbers of phosphorylation sites from a complex mixture in the shortest amount of time. Furthermore, optimal MS/MS spectra are obtained in the positive ion mode, where the additional negative charge of the phosphate group reduces the ionization efficiency of phosphopeptides relative to unphosphorylated peptides. Unfortunately, chemical removal of the phosphorylation site to improve the ionization of phosphopeptides such as via beta-elimination leads to ambiguity of the presence of the original phosphoryla9 tion site due to possible nonspecific reactions . If we want definitive proof of the modification from the mass spectrum, it is possible to overcome the dynamic range problem and poor ionization of phosphopeptides by enriching the sample as much as possible. By far the most established method for phosphopeptide enrichment has 10 been immobilized metal affinity chromatography (IMAC) . A range of variations has been published, using different chromatographic materials, metal ions 10-13 and manual or automated setups . Often it is much easier to isolate phosphopeptides from complex mixtures than to identify “the” phosphopeptide(s) from individual proteins. Background binding of peptides containing a high percentage of acidic amino acids to the IMAC column leads to significant contamination of the enriched phosphopeptide samples and difficulty applying IMAC 6 alone to complex cellular lysates . Increased IMAC selectivity may be obtained through methyl ester derivatization of acidic amino acids, resulting in higher 6,7,11,14 yields of phosphopeptides .

3. PHOSPHOPROTEOMIC PLATFORM Here I will describe an efficient phosphoproteomic method capable of discovery of hundreds of phosphorylation sites within a single day based on methyl esters

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Figure 1. Proteomic platform for the analysis of phosphorylation sites from complex cell lysates (adapted with permission from L.M. Brill et al., Anal. Chem. 76(10), 2763–1772 (2004), Copyright American Cancer Society). (a) After addition of a standard phosphopeptide used for external relative quantitation, proteins are optionally purified by phosphotyrosine immunoprecipitation prior to tryptic digestion of peptides. The resulting collection of peptides is methylated in methanolic HCl to increase the selectivity of subsequent separations and residual acid removed on a reversed-phase C18 column. Phosphorylated peptides are enriched on an immobilized metal affinity chromatography (IMAC) column and then eluted to a second reversed-phase C18 column. Peptides are then slowly eluted with a gradient of acetonitrile into the mass spectrometer through (b) a custom-made electrospray emitter tip at 20 nl/min. (c) All columns are hand prepared on a pressure bomb through the application of 600 psi of pressure to an emulsion of chromatographic resin through a 360-µm o.d. glass capillary fitted with a porous frit.

6

6

IMAC . This method has been improved significantly from its original imple11,14 mentation (Figure 1a). First, complex mixtures of proteins are obtained either from cells or tissue. Standard protein purification techniques such as immunoaffinity purification of phosphotyrosine-containing proteins are useful in focusing attention on smaller subpopulations of proteins such as tyrosine-phosphorylated proteins. After addition of a standard phosphopeptide(s), the cell or tissue derived proteins are most typically digested with trypsin and the resulting peptides are converted to methyl esters with acidic methanol. Although global internal labeling such as with D3/D0 methyl esters or iTRAQ reagents provides the best 15 relative quantitation , we have found addition of synthetic, non-genomic phosphopeptides to be a simple yet powerful method for relative quantitation of 14 phosphorylation . The addition of a reversed-phase C18 “desalting” column after peptide methylation and before the IMAC column efficiently removes the residual acidity from the methylation reaction and increases phosphopeptide

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retention on a high-capacity IMAC column. Peptides are then slowly introduced to the high-capacity IMAC column through gradient elution from the desalting column. Phosphorylated peptides are retained on the IMAC column while unphosphorylated proteins are removed with wash buffer. Finally, peptides are slowly introduced to a 75-µm i.d. C18 “pre-column” with phosphate buffer and eluted into the mass spec through a 5-µm home-pulled electrospray emitter tip pulled on a 75-µm i.d. capillary and packed with C18 resin (analytical column; Figure 1b). To maximize sensitivity, all columns in the system are prepared in glass capillaries with a “pressure bomb” (Figure 1c).

4. AUTOMATION OF THE PHOSPHOPROTEOMIC PLATFORM Automation of proteomic methods is essential to increase both the reproducibility and throughput of the analysis. The rapid and reproducible identification of phosphorylation sites from complex cell-derived mixtures is a crucial tool to accelerate signaling pathway discovery. It is critical to define not only the location of phosphorylation on proteins but to define each phosphorylation site’s biological significance within the pathway. The analysis of site-directed mutants and protein disruptions of novel phosphorylation sites will provide insights into the placement of sites within pathways. As a complement to traditional approaches, the ability to define the perturbations in global patterns of phosphorylation resulting from disruption of a protein or novel phosphorylation site will provide vital clues about whether a new phosphorylation site is relevant to a given signaling pathway and its location within the pathway. A single timecourse phosphoproteomic experiment typically generates information about hundreds of sites of phosphorylation. A high-throughput system is therefore essential to perform both the initial time-course phosphoproteomic experiments but also the time-course data-inspired phosphoproteomic mutant analyses. Here I describe a fully automated phosphoproteomic system for highthroughput phosphorylation site analysis controlled by custom-made automation 7 software adapted from Ficarro et al. (Figure 2). This system not only includes critical automation of the necessary phosphopeptide separations and introduction 7 of peptides into the mass spectrometer as originally described by Ficarro et al , but also the bioinformatic analysis of the resulting data. A typical time-course experiment generates tens of thousands of MS/MS spectra in a single day that need to be compared to theoretical spectra derived from genomic sequence databases with algorithms such as SEQUEST. Also, software must be written to provide the storage, organization, visualization, and statistical analysis of the data. We have directly coupled our automated mass spectrometer to a 16-cpu sequest cluster and a custom-made phosphoproteomic relational database. After completion of data acquisition, the automation software controls the transfer of data to the SEQUEST cluster, searching of the data, and deposition of the searched data into our custom-made proteomic relational database on a 3 terra-

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Figure 2. Fully automated system for analysis of phosphorylated peptides. (Adapted from S.B. Ficarro et al., Rapid Commun. Mass. Spectrom. 19(1), 57–71 (2005), Copyright John Wiley and Sons Ltd.; reproduced with permission.)

byte data server. After loading an autosampler with methylated peptides from complex mixtures, the system provides fully automated, unattended multidimensional chromatography with IMAC, nanospray with peak parking, SEQUEST searching on a cluster of computers, and bioinformatic analysis of the SEQUEST results in our database. To maintain 24/7 operational status, the automation software alerts the operator if any problems arise via email through its diagnostic capabilities.

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Table 1. Phosphopeptides Detected in Pervanadate-Treated Jurkat Cells (Adapted with permission from L.M. Brill et al., Anal. Chem. 76(10), 2763–2772 (2004))

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Currently, there are no commercially available relational databases for the analysis of large proteomic data sets. We have designed a proteomic relational database that contains functionality to accelerate both the manual validation of spectra and the bioinformatic analysis of proteomic data. The database has the ability to display proteomics comparative analyses in a user-friendly, intuitive interface allowing the investigator to quickly discern temporal and spatial patterns of phosphorylation sites. One key feature of the database is an automated match of newly acquired spectra to previously manually validated spectra (2732 spectra so far), obviating redundant manual validation. Another critical feature of the database is an interactive MS/MS spectral editor allowing the electronic storage of manual annotations added directly to spectra during manual spectral validation. We have integrated other newly developed databases, such as the 16 Human Reference Protein Database (HPRD) , directly within our database to provide automated lookup of phosphorylation sites and protein–protein interactions from the literature and hyperlinking of the relevant journal articles directly to the proteomic data. This feature allows the user to quickly investigate literature connections to the novel phosphorylation sites revealed in phosphoproteomic experiments, minimizing manual PubMed searching. Ambiguous protein naming in the literature is clarified through querying HPRD by peptide sequence (not currently available through the HPRD website). While the data are loaded into the database with the automation software, every peptide is BLAST searched against the genomic database. These archived BLAST searches allow the user to rapidly analyze the many names associated with a peptide assignment and permanently reassign the correct name to each peptide. Together, this bioinformatic infrastructure provides for rapid analysis of phosphoproteomic data.

5. PHOSPHOPROTEOMICS APPLIED TO LYMPHOCYTE SIGNALING To determine whether this IMAC technology platform is sensitive enough to view phosphorylation sites from human T cells, pervanadate-treated Jurkat T 6 cells were analyzed as described in Brill et al . This analysis of phosphotyrosine immunoprecipitated and IMAC-enriched phosphopeptides from 20-minute pervanadate stimulated Jurkat cells revealed 182 unique phosphorylation sites residing on 151 different peptides. A total of 80 tyrosine, 83 serine, and 19 6 threonine phosphorylation sites were observed (Table 1) . Most importantly, this experiment established that all of these phosphorylation sites are associated with a single cell line from a single experiment and that our methodology is sensitive enough to see them. In contrast with traditional approaches, these phosphorylation sites were not inferred from site-directed mutagenesis or in vitro kinase reactions but were obtained directly from MS/MS sequencing of

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L. CAO ET AL. Table 2: Phosphopeptides from Anti-CD3/CD4 Stimulated Jurkat Cells

Protein Jurkat CAS-L

NCBI GI # 5453680

Peptide sequence

PO4 site

TGHGYVpYEYPSR

Y166

CBL

115855

IKPSSpSANAIpYSLAAR

S669, Y674

CD3δ

4502669

NDQVpYQPLR

Y149

CD3δ

4502669

DRDDAQpYSHLGGNWAR

Y160

Known site

Y674

4502671

DLpYSGLNQR

Y199

Y199

CD3ζ

115997

NPQEGLpYNELQK

Y110

Y110

19

CD3ζ

115997

RKNPQEGLpYNELQK

Y110

Y110

19

CD3ζ

115997

MAEApYSEIGMK

Y122

Y122

19

CD3ζ

115997

GHDGLpYQGLSTATK

Y141

Y141

19

115997

REEpYDVLDKR

7656965

pSHAENPTASHVDNEpYSQP

Y83

Y83

Y453

PRNpSR

S439, Y453, S460

5 min

x

x

x

x

x

x

x

x

x

x

x

x

x

RHpTDPVQLQAAGR

T262

HS1

4885405

GFGGQpYGIQK

Y198

LIM lipoma 5031887

YYEGYpYAAGPGYGGR

Y301

PYK2

YIEDEDpYpYKASVTR

Y579, Y580

Y579, 21 Y580

RIDTLNSDGpYTPEPAR

Y292

Y292

22

PMPMDTSVpYESPpYSDPEEL

Y315, Y319 Y492, Y493

Y315, 23 Y319 Y492, 22 Y493

x

x x

x

x

x

x

x

x

x

20

4758476

x

x

x

x

x

x x

ZAP-70

1177033

ZAP-70

1177044

ZAP-70

1177044

ALGADDSpYpYTAR

ZAP-70

1177044

ALGADDSYpYTAR

Y493

Y493

YIEDEDpYpYKASVTR

Y579, Y580

Y579, 21 Y580

KDK

2 min

x

19

GADS

4758976

1 min

x

CD3ε

CD3ζ

0.5 min

17

18

CD5

0 min

x x

x

x

x

x

x

22

x

x

x

Jurkat Lck depleted (J.CaM1.6) PYK2

4758976

x

Times are after anti-mouse IgG treatment (TCR crosslinking). Shaded areas indicate time points not analyzed in the experiments. Copied with permission from A.R. Salomon et al, Proc. Natl. Acad. Sci. USA, 100(2), 443-448 (2003).

peptides derived from whole cell lysates. The fact that many of these phosphorylation sites are known to play critical roles in T cell signaling suggests that pervanadate stimulation is a useful technique that provides many biologically relevant phosphorylation sites. 17-23

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In an initial approach to understand the signaling pathways involved in T cell activation, phosphorylation sites were comprehensively determined after T 14 cell receptor stimulation as described in Salomon et al . This experiment utilized an earlier version of the phosphoproteomic technology platform lacking the desalting column and using the less sensitive LCQ mass spectrometer. Phosphorylated peptides were isolated and sites of phosphorylation characterized by MS/MS sequencing after receptor activation (0-, 0.5-, 1-, 2-, and 5-minute time 14 points) . Upregulation of ZAP-70 catalytic activity results from phosphorylation 493 319 of Tyr by Lck and autophosphorylation of Tyr following tandem interactions of the ZAP-70 SH2 domains with immunoreceptor tyrosine-based activation 23,24 motifs (ITAMs) on CD3ζ . Although we found fewer phosphorylation sites due to the older methodology used in this experiment, this time-course experiment was able to show the sequential phosphorylation of CD3ζ ITAM phosphorylation sites prior to ZAP-70 phosphorylation with careful relative quantita14 tion (as illustrated in Salomon et al ). All of the known in vivo tyrosine 292 315 319 492 phosphorylation sites of ZAP-70 were detected (Tyr , Tyr , Tyr , Tyr , and 493 22,23 Tyr ) . Furthermore, tyrosine phosphorylation was effectively absent in the Lck-depleted mutant cell line J.CaM1.6 on CD3/CD4 stimulation (Table 2). Interestingly, despite the large volume of published work on T cell signaling, these experiments also identified unreported sites of tyrosine phosphorylation 166 such as Tyr of Cas-L, which contains a strong consensus site for binding to the Lck SH2 domain. Cas-L is known to bind to Lck kinase in CD3-stimulated T 25 cells, but the site of interaction has not been defined ; phosphorylation of an Lck SH2 site on Cas-L suggests that Lck could bind this site. Another novel 198 phosphorylation site was observed at Tyr of HS1. HS1 is known to be tyrosine phosphorylated as a consequence of anti-CD3 stimulation, possibly through the 26,27 action of Syk , but again the site of phosphorylation was not previously identified. Induced phosphorylation of CD3ζ, CD3δ, and CD3ε was consistent with 19 the expected phosphorylation of these ITAM regions .

6. CONCLUDING REMARKS Through fusion of innovations in high-throughput chromatographic separations of phosphopeptides, detection by mass spectrometry, and bioinformatic analysis, we have assembled a formidable tool to complement the traditional methodologies typically used to study signaling pathways. Although discovery of a large and dynamic set of cell-derived phosphorylation sites in a single proteomics experiment is an impressive accomplishment, understanding which of these sites participate in a given signaling pathway and the nature of this participation is the fundamental challenge confronting the proteomics researcher. It is simply not feasible to make hundreds of site-directed mutants and the associated knockout mice with current methodologies within a single lab in a reasonable amount of time. Therefore, complimentary high-throughput follow-up strategies must be

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developed to ascertain whether newly discovered phosphorylation sites participate in pathways and the nature of this participation. Our new approach streamlines the usual signaling pathway analysis paradigm by allowing for the production of mutants of phosphorylation sites and signaling proteins shown to exist within cells as opposed to the usual motif-driven site-directed mutagenesis approach. Also, phosphoproteomic characterization of perturbations of global phosphorylation patterns in mutant cells will likely provide a useful perspective on signaling pathways and ultimately a more rapid understanding of the molecular basis of a wide array of diseases.

7. REFERENCES 1.

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12. D. C. Neville, C. R. Rozanas, E. M. Price, D. B. Gruis, A. S. Verkman, and R. R. Townsend, Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry, Protein Sci 6(11), 2436–2445 (1997). 13. A. Stensballe, S. Andersen, and O. N. Jensen, Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis, Proteomics 1(2), 207–222 (2001). 14. A. R. Salomon, S. B. Ficarro, L. M. Brill, A. Brinker, Q. T. Phung, C. Ericson, K. Sauer, A. Brock, D. M. Horn, P. G. Schultz, and E. C. Peters, Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry, Proc Natl Acad Sci U S A 100(2), 443–448 (2003). 15. Y. Zhang, A. Wolf-Yadlin, P. L. Ross, D. J. Pappin, J. Rush, D. A. Lauffenburger, and F. M. White, Time-resolved Mass Spectrometry of Tyrosine Phosphorylation Sites in the Epidermal Growth Factor Receptor Signaling Network Reveals Dynamic Modules, Mol Cell Proteomics 4(9), 1240–1250 (2005). 16. S. Peri, J. D. Navarro, R. Amanchy, T. Z. Kristiansen, C. K. Jonnalagadda, V. Surendranath, V. Niranjan, B. Muthusamy, T. K. Gandhi, M. Gronborg, N. Ibarrola, N. Deshpande, K. Shanker, H. N. Shivashankar, B. P. Rashmi, M. A. Ramya, Z. Zhao, K. N. Chandrika, N. Padma, H. C. Harsha, A. J. Yatish, M. P. Kavitha, M. Menezes, D. R. Choudhury, S. Suresh, N. Ghosh, R. Saravana, S. Chandran, S. Krishna, M. Joy, S. K. Anand, V. Madavan, A. Joseph, G. W. Wong, W. P. Schiemann, S. N. Constantinescu, L. Huang, R. Khosravi-Far, H. Steen, M. Tewari, S. Ghaffari, G. C. Blobe, C. V. Dang, J. G. Garcia, J. Pevsner, O. N. Jensen, P. Roepstorff, K. S. Deshpande, A. M. Chinnaiyan, A. Hamosh, A. Chakravarti, and A. Pandey, Development of human protein reference database as an initial platform for approaching systems biology in humans, Genome Res 13(10), 2363–2371 (2003). 17. H. Steen, B. Kuster, M. Fernandez, A. Pandey, and M. Mann, Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway, J Biol Chem 277(2), 1031–1039 (2002). 18. I. de Aos, M. H. Metzger, M. Exley, C. E. Dahl, S. Misra, D. Zheng, L. Varticovski, C. Terhorst, and J. Sancho, Tyrosine phosphorylation of the CD3-epsilon subunit of the T cell antigen receptor mediates enhanced association with phosphatidylinositol 3-kinase in Jurkat T cells, J Biol Chem 272(40), 25310–25318 (1997). 19. E. N. Kersh, A. S. Shaw, and P. M. Allen, Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation, Science 281(5376), 572–575 (1998). 20. J. M. Vila, I. Gimferrer, O. Padilla, M. Arman, L. Places, M. Simarro, J. Vives, and F. Lozano, Residues Y429 and Y463 of the human CD5 are targeted by protein tyrosine kinases, Eur J Immunol 31(4), 1191–1198 (2001). 21. D. D. Schlaepfer, C. R. Hauck, and D. J. Sieg, Signaling through focal adhesion kinase, Prog Biophys Mol Biol 71(3-4), 435–478 (1999). 22. J. D. Watts, M. Affolter, D. L. Krebs, R. L. Wange, L. E. Samelson, and R. Aebersold, Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70, J Biol Chem 269(47), 29520–29529 (1994). 23. V. Di Bartolo, D. Mege, V. Germain, M. Pelosi, E. Dufour, F. Michel, G. Magistrelli, A. Isacchi, and O. Acuto, Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling, J Biol Chem 274(10), 6285–6294 (1999).

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24. A. C. Chan, M. Dalton, R. Johnson, G. H. Kong, T. Wang, R. Thoma, and T. Kurosaki, Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function, Embo J 14(11), 2499–2508 (1995). 25. H. Kanda, T. Mimura, K. Hamasaki, K. Yamamoto, Y. Yazaki, H. Hirai, and Y. Nojima, Fyn and Lck tyrosine kinases regulate tyrosine phosphorylation of p105CasL, a member of the p130Cas docking protein family, in T-cell receptormediated signalling, Immunology 97(1), 56–61 (1999). 26. J. E. Hutchcroft, J. M. Slavik, H. Lin, T. Watanabe, and B. E. Bierer, Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28, J Immunol 161(9), 4506–4512 (1998). 27. M. Ruzzene, A. M. Brunati, O. Marin, A. Donella-Deana, and L. A. Pinna, SH2 domains mediate the sequential phosphorylation of HS1 protein by p72syk and Srcrelated protein tyrosine kinases, Biochemistry 35(16), 5327–5332 (1996).

Author Index

Acuto, Oreste, 189 Altman, Amnon, 1 Bandaranayake, Ashok D., 89 Barouch-Bentov, Rina, 1, 245 Blanchet, Fabien, 189 Braciale, Vivian, 229 Browne, Cecille D., 29 Cao, Lulu, 277 Carmo, Alexandre M., 127 Castro, Monica A.A., 127 Ching, Keith A., 29 Cloyd, Miles W., 229 da Rocha Dias, Silvy, 115 Deckert, Marcel, 107 Demydenko, Dmytro, 207 Douziech, Nadine, 157 Dupuis, Gilles, 157 Fortin, Carl, 157 Fukamachi, Toshihiko, 219 Fülöp, Tamàs, 137, 157 Garçon, Fabien, 15 Giacconi, Roberta, 137 Grasis, Juris A., 29 Harada, Yohsuke, 207 Herman, Ann, 245 Huang, Yina Hsing, 245 Jeon, Myung-shin, 207 Ji, Jiaxiang, 229 King, Philip D., 73 Kobayashi, Hiroshi, 219 Lao, Qizong, 219 Lapinski, Philip E., 73

Larbi, Anis, 129, 157 Liu, Yun-Cai, 207 MacGregor, Jennifer N., 73 Marti, Francesc, 73 Miyamoto, Suzanne, 171 Mocchegiani, Eugenio, 137 Moreno-García, Miguel E., 89 Mustelin, Tomas, 53 Muti, Elisa, 137 Nunès, Jacques A.,15 Nunes, Raquel J., 127 Okamura, Shinya, 219 Rawlings, David J., 89 Rottapel, Robert, 107 Rudd, Christopher E., 115 Saito, Hiromi, 219 Salomon, Arthur R., 277 Sauer, Karsten, 245 Schneider, Helga, 115 Schurter, Brando T., 189 Shao, Yuan, 207 Smith, Melissa, 229 Sommer, Karen M., 89 Tsoukas, Constantine D., 29 Turner, M., 43 Valk, Elke, 115 Venuprasad, K., 207 Vigorito, E., 43 Walker, John, 245 Wei, Bin, 115 Yang, Chun, 207 Yu, Kebing, 277

289

Index APCs, 117 interacting with cholesterol enrichment of T cells, 161–162 Apoptosis, 137–149, 230 during aging, 146–148 and lipid rafts, 142–143 Apoptotic resistance, 147 Arg 415, 111 Arginine methylation of NIP45, 197–198 regulation by peptidylarginine deiminases, 200–201 and signal transduction, 190–202 Arginine methyltransferases, 190–195 Arthritis, 60 and forward genetics, 260 Asp-based phosphatases, 54 Atypical dual-specificity phosphatases (aDSPs), 54 Autism, 59–60 Autoimmunity and E3 ligases, 210 and PTPs, 60–61 and TSAd, 75–76 Avidin, 47 Avidity, 117–118

A A20 as de-ubiquiting enzyme, 210–211 Abl, 109 Acidification and CTIB, 219–225 Acidosis and CTIB, 221–222 on signal pathways, 220–221 ACP1, 61 Actin cytoskeleton and regulation by itk, 32, 34–38 Actin polymerization and Itk, 29, 32, 34–38 Actin remodeling, 37 Activation-induced cell death (AICD). See AICD ADAP, 117 Adapter proteins, 45, 92, 107–112 intracellular, 73 and Itk, 33 transmembrane, 73 Adhesion CTLA-4 mediated, 118–124 and integrins, 116–118 Adhesion and degranulation-promoting adaptor protein. See ADAP Aging of the immune system, 144–148 apoptosis, 146–148 T cell functions, 145–146 AICD, 137–140, 144, 147, 148, 149 AIDS, 229 AIP4, 212 Akt/PKB serine/threonine kinase, 23 Alzheimer's disease, 59, 160 ANA, 260 Anti-CD3, 118–123 stimulation, 161–163 Anti-CD3/CD28 antibodies, 5 Anti-CD28 stimulation, 161–163 Antigen-presenting cells. See APCs Anti-nuclear antibody. See ANA AP-1, 83, 111 activation and 3BP2, 110 activation by protein kinase C-theta (PKCθ), 5–6

B Bad and lipid rafts, 142, 143 Bannayan-Zonana syndrome, 58 BAP37, 180 BAP29 and TSAd, 81 B cell antigen receptor. See BCR B cells development and ERK activation, 44–46 and PI3K subunits, 43–44 and PTPs expression, 56 Bcl-2 protein, 7 Bcl-10, 3–4, 99, 101 in NF-κB activation, 95–97 Bcl-xL protein, 7

291

292 BCR, 44 and activation of NK-κB, 89–101 and 3BP2, 110 coligation with CD19, 46–49 and ERK activation, 44–46 signaling and PKC isoforms, 93–94 BimEL, 7 β2 integrins, 116–117 Bioconductor, 247 Bladder cancer, 107, 112 BLNK, 44, 92–93 Bmx, 15 3BP2 and cherubism, 111–112 in leukocyte cell signaling, 110–111 and pathology, 111–112 structure and expression of, 107–110 as tumor suppressor gene, 112 Bright transcription factor, 20 Bruton's tyrosine kinase. See Btk Btk, 15, 19–20, 92, 93 PH domain, 16 visualization of, 22 Bubonic plague, 61 Bystander cells, 230

C C1858T, 60 C3G, 212 c-Abl, 107, 111 Calcineurin, 5 Calcium flux and 3BP2, 110 mobility in T cell signaling, 141 mobilization, 160 and protein kinase C-theta (PKCθ), 5–6 cAMP, 221 Cancer, 62 bladder, 107, 112 and mRNA translation, 181 and PTPs, 56–58 Canonical NF-κB1 pathway, 3 Cap-binding protein, 172 Cap formation, 34 Cardiac hypertrophy, 59 Cardiovascular diseases and PTPs, 59–60 CARM1, 195, 196 CARMA1, 3–4, 258 in IKK activation, 99–100 in NF-κB activation, 95–101

INDEX Cas-L, 285 Caspase-3, 140, 143, 146 Caspase-8, 100, 140, 143, 146 Catalytic cleft, 64 Caveolae, 19 Cbl, 22, 212 Cbl-b, 209–210, 212 CBP/p300, 196 c-Cbl, 111 CCK receptor, 160 CC-PDZ linker region, 96 CCR, 212 CD2, 131 mobility of, 129 CD3δ, 285 CD3ε, 285 CD3ζ, 285 CD4 lymphocytes depletion of in HIV infection, 230–238 CD4+ T cells, 56 CD5, 131 CD8, 56, 132 and gene expression analysis, 249–250 in HIV infection, 233 and protein kinase C-theta (PKCθ), 6–8 CD8αα, 132 CD8αβ, 132 CD9, 131 CD11/CD18, 117 CD19 coligation with BCR, 46–49 CD28, 115–116, 131 and cholesterol, 158, 164–167 and Itk, 30 and lipid rafts, 141 mediated costimulatory signaling, 198–200 in T cell signaling, 142 CD28 co-stimulatory receptor, 18 CD28 co-stimulatory receptor antibodies, 75 CD43 during aging, 145 CD45, 91 in autoimmunity, 61 CD62L, 234 CD79a, 44 CD79b, 44 CD80/86, 115–116 CD95, 142 CD244 and 3BP2, 111 Cdc14A phosphatase, 58 Cdc14B phosphatase, 58

INDEX CDC25, 62 CDC25 phosphatases, 55 Cdc25A phosphatase, 58 Cdc25B phosphatase, 58 Cdc25C phosphatase, 58 CDC48, 82 Cdc42 and Itk, 35 Cell–cell junctions, 59 Cell cycle progression, 58 Cell-mediated autoimmunity and E3 ligases, 210 Cell signaling. See Signaling Central nervous system and PTPs, 60 Central T cell tolerance, 78–81 Ceramide, 139, 142 Charcoat-Marie-Tooth syndrome type 4B, 58 Chemokine receptors, ubiquitination of, 212 Cherubism, 107, 111–112 Cholesterol extraction from T cell membranes, 158–160 functional effects on Jurkat cells, 162–165 increase in T cell membranes, 160 in lipid rafts, 143 modulation by cyclodextrin, 158–160 and size of T cells, 161–162 and T cell response in aging, 157–167 Cholesterol pool, 159 Citrulline, 200 Class I PI3Ks, 43–44 subclass IA, 43–44 subclass IB, 44 CLIC-4 and TSAd, 81 c-Myb, 258–259 c-Myc and TSAd, 81 Coactivator associated arginine methyltransferase I. See CARM1 Costimulatory signaling, 198–200 Cowden disease, 58 c-Rel, 89 CrkL, 212 Csk, 91 Csk kinase, 60 c-Src, 111 C-terminus protein of IκB-β. See CTIB CTIB, 219–225 in acidic conditions, 221–222 in immune cells, 223 regulating NF-κB functions, 222–223 as splicing variant, 223–224

293 CTLA-4, 115–116 upregulating LFA-1 adhesion, 118–123 CTL differentiation and protein kinase C-theta (PKCθ), 6–8 CXCR, 212 CXCR4, 212 Cyclodextrin modulating cholesterol, 158–160 Cysteine in PTPs, 54–55 Cytoplasm and TSAd functions, 83–84 Cytoskeleton and lipid rafts, 130, 143

D DAG, 46, 93, 176, 259 and protein kinase C-theta (PKCθ), 1 DAP12, 111 Death effector domain. See DED Death-inducing signaling complex. See DISC Death receptors, 138, 139–140 DED, 140 Demethylimination, 200 DEP1, 57, 59 Detergent-insoluble glycolipid domains. See DIGs De-ubiquiting enzyme, 210–211 Diabetes, type 1, 60 Diabetes, type 2, 59, 62 Diaylglycerol. See DAG Diffusion rate in lipid rafts, 129, 131 DIGs, 127 DISC, 137, 140, 143–144 Drugs inhibiting PTPs, 62

E E1, 207 E1B-AP5, 194 E2s, 207 E3s, 207, 208 E3 ligases in cell-mediated autoimmunity, 210 in T cell anergy, 209–210 in T cell differentiation, 208–209 EEA1, 19 EGF receptor, 22 Egr 2, 210 Egr 3, 210 eIF2, 172–173, 174–175

294 eIF3, 172–175 eIF4B, 173 eIF4E, 172, 181 eIF4F, 172–173 eIF3j subunit, 175 Elemental rafts, 128 ENU mutagenesis, 254–256, 262–263 ERK, 5, 7, 44 activation by PI3K subunits, 44–46 phosphorylation of, 47–49 ERK1, 44 ERK2, 44 Experimental autoimmune encephalitis (EAE), 8 Extracellular signal regulated protein kinases. See ERK

F 4E-BP1/2, 172 FADD, 139–140, 143, 146 Fas, 137, 146, 147, 232 in lipid rafts, 143–144 trimerization, 139, 142 Fas-associated death domain. See FADD Fas/Fas-L death receptor, 139–140, 143–144 Fas-L, 137, 139, 140, 147 Fast extractable cholesterol pool, 159 FK506, 175, 178–179 Fluorescence recovery after photobleaching. See FRAP Fluorescence resonance energy transfer. See FRET Fluorescent proteins, 21 Forward genetics, 254–263 in understanding disease, 259–261 FoxP3/Scurfy, 254 Frame-shift mutations, 58 FRAP, 22–23, 129 FRET, 22, 129 FSC and T cell-bead contact formation, 161 Functional genomics, 250–253 Fyn, 129–130 Fyn-binding protein (FYB), 73

G G2A, 221 Gads, 141 Gain-of-function mutations, 58 GALT, 233

INDEX GAP1, 259 GA repeats, 76 G-CSFR, 109 GEMs, 127 Gene clusters, 182 Gene expression analysis in lymphocyte development, 248–250 Gene expression profiling, 247 GeneSifter, 247 Gene targeting, 253, 262–263 Gene transcription and 3BP2, 110 Genome, functional annotation of, 245–246 Germline mutations, 58 Gleevec, 63 GLEPP1, 57, 59 Gly 420, 111 Glycolipid-enriched membranes. See GEMs Glycosylphosphatidylinositol. See GPI gp33, 6 gp120, 230 GPCR family, 221 GPI, 127, 129 GPR4, 221 Graves' disease, 60 Grb2, 22, 45, 73 Grb-2-related adapter protein-2 (GRAP-2), 73 Grb7 adapter proteins, 108 GRIP1, 195 Growth receptor-bound protein-2. See Grb2 GTPases, 211

H H-2Kb, 78 HAART treatment, 234–235 Haloacid dehalogenase (HAD)-related enzymes, 54 Hayflick's theory of cellular sensescence, 145 HDL, 158, 159 Hematopoietic cells, 56 Hematopoietic tyrosine phosphatase (HePTP), 57 Highly active anti-retroviral therapy. See HAART treatment Histones, arginine methylation of, 190, 191–192 HIV, pathogenesis of, 229–230 Hmt1, 191 HPRD, 283 Hrs, 212

INDEX HS1, 285 HSP60 and TSAd, 81 Human disease and PTPs, 56–61 Human Reference Protein Database. See HPRD HuR, 196 Hydroxypropyl-β-cyclodextrin, 160 H-Y TCR model, 78

I ICAM-1 in CTLA-4 mediated adhesion, 117, 120–123 ICOS, 115–116, 210 IDO, 116 IFN-α/β, 196 IFN-γ, 197 and tec kinase localization, 19–20 and TSAd, 81 Igα/Igβ heterodimer, 90–91 IgE, 61 IgM, 47–49 IκB, 210 IκBα, 89 IκBβ, 89, 222–223 IκBε, 89 IκB kinase (IKK) complex, 3 IκBs, 89–90 IKK, 210–211 activation by CARMA1, 99–100 IKKα, 4, 89 IKKβ, 3, 89, 99 IKK complex, 89–90, 94, 96 IKKγ, 89 IL-2 and apoptosis, 139 and 3BP2, 111 expression and protein kinase C-theta (PKCθ), 1 and lipid rafts, 141 and TSAd, 75, 81, 84 IL-4, 197 IMAC, 278 ImmGen, 248 Immobilized metal affinity chromatography. See IMAC Immune cells and CTIB, 223 Immune regulation by ubiquitin conjugation, 207–214 Immune response to acidification, 219–225 Immune system and protein kinase C-theta (PKCθ), 6–9

295 Immunodeficiency and PTPs, 60–61 Immunological synpase, 34, 118 Immunoreceptor tyrosine-based activation motif. See ITAM Immunosenescence, 144–145 Immunosuppressants and mRNA translation, 178–179 Indoleamine 2,3-dioxygenase. See IDO Inducible costimulatory molecule. See ICOS Inducible T cell tyrosine. See Itk Infection in HIV, 232 InforSense:BioSience, 247 Ingenuity, 247 Inhibitory κB (IκB) proteins, 2–3 Inositol-1,4,5-P3. See IP3 Inositol (1,4,5) triphosphate-3 kinase. See ItpkB Inside–out signaling, 211 Insulin resistance to, 58–59 and signal pathways, 62, 175–176 Integrins and adhesion, 116–118 signaling, 211–212 Interaction Explorer, 247 Intercellular adhesion molecule-1. See ICAM-1 Interleukin-2 inducible kinase. See ITK Internal pH (pHi), 220, 221 Internal ribosome entry element. See IRES Ionomycin, 178 IP3, 93, 176, 221 IP4, 257–259 IRES, 181 ITAM, 44 and BCR induced signaling, 90–92 Itch, 208–209 Itk, 15, 29–38, 117 and actin polymerization, 29 and adapter proteins, 33 enzymatic activity of, 30–32, 35 nuclear localization of, 20 PH domain of, 30–31 and regulation of actin cytoskeleton, 32, 34–36 SH2 domain of, 32–33 SH3 domain of, 31–32 structure of, 30 TH domain of, 31–32 and TSAd, 84 Itk kinase, 18 ItpkB, 257–259

296 J Jak2, 195 JBP1, 195 JNK, 5, 7 JNK1, 209 JunB degradation, 209 Jurkat cells, effects of cholesterol modulation on, 162–165 Juvenile arthritis, 60

K K48 linkage, 208 K63 linkage, 208 Knockdown of gene targets, 251–252 Knockout mice, 253 KRN, 260

L Lafora's epilepsy, 58 Laforin, 58 LAR, 57 LAT, 18, 33, 36, 73, 258 and 3BP2, 110 and lipid rafts, 130, 132 in T cell signaling, 141–142 and TSAd, 83 Lck, 21–22, 61, 128, 129–130, 285 and lipid rafts, 132 and TSAd, 83–84 Lck-interacting transmembrane protein (LIME), 73 LCMV, 6 LDL, 158 Leukemia, 175 and PTPs, 57 Leukocyte cell signaling, 110–112 Leukocyte-specific protein of 76 (SLP-76), 33 LFA-1, 117–118, 211–212 adhesion mediated by CTLA-4, 118–123 Lhermitte-Duclos disease, 58 Linker for activated T cells. See LAT Lipid microdomains, 128–129 Lipid rafts, 3, 91, 164 in activation-induced cell death, 137 during aging, 146 and apoptosis, 142–143

INDEX and cholesterol modulation, 157, 158 definition of, 127–128 and Fas, 143–144, 147–149 protein interactions in, 131–132 in T cell receptor signaling, 129–131 in T lymphocyte signaling, 141–142 visualizing, 128–129 LMPTP, 59, 61 Lovastatin, 163–164, 165, 166 Low MR PTP (LMPTP), 55 LPS, 196 L-selectin, 232, 234 Lung inflammation and protein kinase C-theta (PKCθ), 8–9 Lymph nodes and CD4 lymphocytes content, 231–232 migration of CD4 lymphocytes in HIV infection, 234–235 Lymphocytes activation and tec family kinases, 15–23 development and gene expression analysis, 248–250 and PTP expression, 56 and translation of individual mRNAs, 177–182 Lymphocyte signaling and mRNA translation, 171–182 and phosphoproteomics, 283–285 Lymphoid tyrosine phosphatase (LYP), 60 Lymphomas, 95 Lyn, 91, 92

M MAGUK, 96, 101 MALT1, 3–4, 99 in NF-κB activation, 95–96 MAP kinase phosphatases (MKPs), 54, 55, 57 MAP kinases, 57 MAPKs, 5, 44, 47 in acidosis, 220 pathway, 175 Matrix metalloproteinse-9. See MMP-9 MBCD, 142, 146, 147, 149 extracting cholesterol, 158–160, 161, 163, 164 MEFs, 96 Mega, 258 MEKK1, 209

INDEX Mekk2 kinase, 84 Membrane-associated guanylate kinase homologue. See MAGUK Membrane cholesterol. See Cholesterol Metabolic syndromes and PTPs, 58–59 Methylation, 190 Methyl-β-cyclodextrin. See MBCD Methylosome, 195 MHC-peptide, 78 Microarray analysis, 177–178, 246–247 Microdomains, 22 Minimal Information About Microarray Experiments (MIAME) standards, 247–248 Mitogen-activated protein kinases. See MAPKs MKP-1, 57, 59, 62 MMP-9, 220 Monogenic inherited syndromes, 58 Mono-methylarginine, 190 Mono-ubiquitination, 207 Mouse mutagenesis, 261–263 Mouse mutants, 254–256 mRNA expression arrays, 277 translation of, 172–182 and ubiquitin, 210 MTA, 197 MTMR2, 58 MTMR13, 58 mTOR pathway, 175 Multiple mono-ubiquitination, 207 Multiple sclerosis, 61 Murine embryonic fibroblasts. See MEFs Muscular dystrophy, x-linked, 58 Mutagenesis ENU, 254–256, 262–263 mouse, 261–263 Mutations and inherited syndromes, 58 in mice, 254–256 mapping, 257–258 Mycobacteria, 61 Myelin oligodendrocyte glycoprotein (MOG), 8 Myotubularin, 58 Myotubularin-related protein 2, 58 Myotubularin-related protein 13, 58

297 N Necrosis, 138 NEMO, 3, 99 Neoplastic diseases and PTPs, 56–58 N-ethyl-N-nitrosourea. See ENU Neurological diseases and PTPs, 60 NF-AT, 5–6, 83, 110, 111, 176 NF-AT activation, 197–198 NF-ATc1, 220 NF-AT-interacting protein 45. See NIP45 NF-κB, 210–211 activation and AP-1, 5 and BCR, 89–101 and PKCβ, 94–99 and PKC isoforms, 93–94 and protein kinase C-theta (PKCθ), 2–4 and CTIB, 222–223 dimers, 89–90 NF-κB1, 89 NF-κB2, 4, 89 NIK, 4 NIP45, arginine methylation of, 197–198 Non-canonical NF-κB1 pathway, 3 Noonan syndrome, 58 Notch signaling and gene expression, 249 Nuclear entry by TSAd, 82 Nuclear Factor-κβ. See NK-κB Nuclear localization site (NLS), 19 Nuclear proteins, 192 Nucleic acid, measure of, 246 Nucleus localization by tec kinase, 19–21 O Obesity, 62 OGR1, 221 Oseoblasts and 3BP2, 111–112 OT-I cells, 6–7 Ovalbumin peptide, 6, 7 OxLDL, 163–164 P p50α, 43, 45 p55α, 43, 45 p56lck, 141 p85α, 43 activating ERK, 44–46 and PI3K activity, 37–39

298 p101, 44 p110α, 45 p110β, 45 p110δ activating ERK, 44–46 and PI3K activity, 47–49 p110γ, 44 PAD4, 200 PADs, 200–201 PAmRNAs, 180 PD98059, 178–179 PDK1, 4 PDZ-SH3-GUK domain, 96 Peptidylarginine deiminases. See PADs Peripheral T cell tolerance, 76–78 Phalloidin, 34 PH domain and activation of Tec family kinases, 16–19 of 3BP2, 108 of Itk, 30–32 mutations of, 34–35 and movement of Btk, 20 Phenodeviant, 257 Phogrin, 60 Phorbol myristate acetate. See PMA Phosphatidylinositol-(3,4,5) triphosphate. See PIP3 Phosphatidylinositol-3,4-5-triphosphate. See PtdIns-3,4,5-P3 Phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], 16, 18 visualization of, 22 Phosphoinositide 3-kinase. See PI3K Phospholipase C. See PLC Phosphopeptides, 279–280, 284–285 Phosphoproteomics and lymphocyte signaling, 283–285 methods for, 278 Phosphoproteomics platform, 278–280 automation of, 280–283 Phosphorylated peptides, 279–280, 281–283 Phosphorylation, 189 automation of, in phosphoproteomics, 280–285 of 3BP2, 109–110 of ERK, 44, 45, 47–49 of ITAM, 90–92 and Itk, 30, 32–33, 35–36 of serine, 53 site isolation, 278 of tyrosine, 53–54, 116, 131, 164, 166, 285

INDEX PH-TH domain of Itk, 35–36 PIAS1, 213 PIAS as SUMO ligase, 213 PI3K, 16, 92, 116, 175 in CD19 signaling, 47 subunits of, 43–44 and ERK activation, 44–46 PIP3, 43 PIP5K, 93 PKB, 47 PKCβ in NF-κB activation, 93–99 PKCδ, 94 PKC isoforms involved in B cell signaling, 93–94 PKCθ activating AP-1, 5–6 activating NF-κB, 2–4 in CTL differentiation and survival, 6–8 in immune system, 6–9 membrane localization and activation, 1–2 in T cells, 1–2 in Th2 cell responses, 8–9 PKC-regulated domain. See PRD PKCζ, 94 Plasma membrane localization by tec family kinases, 16–19 PLC, 176 PLCγ, 46 and 3BP2, 110–111 PLCγ1, 18, 83 PLCγ2, 93 Plectstrin Homology domain. See PH domain PMA, 176, 178–179 Point mutant alleles, 258 Point mutations, 58 Polymorphism in PTPs, 60 Poly-ubiquitination, 207 PRD, 98–99 PR domain of 3BP2, 108, 109 Pristane, 76 PRL357 PRMT1, 191, 194, 196, 199 PRMT2, 194 PRMT3, 191, 194–185 PRMT4, 195, 196 PRMT5, 195 PRMT6, 195 PRMT7, 195 PRMT8, 195

INDEX PRMTs, 20, 190–195 type I, 190, 191 type II, 190, 191 Pro 418, 111 Pro-apoptotic genes, 81 Programmed cell death. See Apoptosis Proinflammation, 211 Proline-rich domain. See PR domain Protein arginine methyltransferases. See PRMTs Protein B cell linker protein. See BLNK Protein interactions in lipid rafts, 131–132 Protein kinase C-theta. See PKCθ Protein synthesis, 175–176 Protein tyrosine kinases. See PTKs Protein tyrosine phosphatases. See PTPs Protein ubiquitination, 207–214 Proteomics, 177–178, 277 PtdIns-3,4,5-P3, 92 PtdIns-4,5-P2, 93 PTEN, 18 and cancers, 57 mutations of, 58 PTKs, 15–16, 53 in T cell signaling, 141 PTP, inhibitors of, 62 PTP1B, 58–59, 62 PTPα, 57 PTP-BAS, 57 PTPD2, 57 PTPH1, 57 PTPκ, 59 PTPµ, 59 PTP-MEG2, 60 PTPN6, 60 PTPN22, 60 PTPRN, 60 PTPs abundance of, 55–56 in cardiovascular and neurological diseases, 59–60 class I family, 54 class II family, 55 class III family, 55 as drug targets, 62 expression in immune cells, 56 in human disease, 56–61 in immunodeficiency and autoimmunity, 60–61 in metabolic syndromes, 58–59 in monogenic inherited syndromes, 58

299 in neoplastic disease, 56–58 overview of, 53–55 as tools for immune evasion, 61

R Rac1, 110 RAGE, 247 Rap-1, 212 in CTLA-4 mediated adhesion, 120– 124 Rap-1-N17, 122–124 Rap-1-V12, 123–124 Rapamycin, 175, 178 Rapid analysis of gene expression. See RAGE Ras, 83 and ERK activation, 45–46 RasGAP1, 83 RasGEFs, 83 RasGRP1, 83 RasGRP3, 46 Ras guanine nucleotide exchange factors, 83 Rch1α, 20–21 RelA, 89 RelB, 89 Reverse cholesterol transport, 158, 159 Reverse genetics, 253–254 Rheumatoid arthritis, 60 RhoA ubiquitination, 212 Ribonomic profiling, 181–182 Ribonomics, 181–182 Ribosomes 40S ribosome, 174–175 48S ribosome, 172, 174 60S ribosome, 173 80S ribosome, 172 Ribosomal loading, 182 RING domain, 210 Rlk, 15, 19–21 RNA interference (RNAi) technology, 251–253, 261–263 Ro3290430, 178–179 Roquin, 210 RPTPγ, 57 RPTPρ, 57

S S564, 98 S649, 98

300 S657, 98 S-adenosyl-L-homocysteine. See SAH S-adenosyl-L-homocysteine hydrolase. See SAHase S-adenosyl-L-methionine. See SAM SAGE, 247 SAH, 196 SAHase, 196 Salmonella, 61 SAM binding domain, 191, 192 Scr-PTKs, 92 SEQUEST, 280–281 Serial analysis of gene expression. See SAGE Serine phosphorylation of 3BP2, 109–110 Serum response element (SRE), 111 SH3BP2, 107 Shc, 45 SH2-containing leukocyte protein of 76 kD (SLP-76), 73 SH2 domain, 17, 18, 92 of 3BP2, 108, 109 of Itk, 32–33 mutations of, 34–36 of TSAd, 74, 82 SH3 domain, 17, 18, 21, 107 and 3BP2, 109 of Itk, 31–32, 37 and movement of Btk, 20 shDNA, 251–253 SHIP, 18 SHP1, 57, 60 SHP2, 58, 59 Signaling and 3BP2, 110–112 CD28-mediated costimulatory, 198–200 induced by BCR, 90–92 and Itk, 29, 33, 37–38 and lipid rafts, 129–131, 141–142 lymphocyte and mRNA translation, 171–182 Signaling pathways, 44 controlling translation in lymphocytes, 175–177 leading to PKC activation, 92–93 through CD4, 239 Signaling proteins, 44 and protein kinase C-theta (PKCθ), 2 Signal memory, 201 Signalosome, 44, 141 Signal pathways and effect of external acidosis, 220–221

INDEX Signal transduction and arginine methylation, 190–202 receptor-mediated, 189 regulation by TSAd, 75 and role of TSAd, 82–84 in T cells, 73 Simvastatin, 164 Single-particle tracking. See SPT siRNA, 251–252 SLAP-2, 250 SLIM, 213 Slow extractable cholesterol pool, 159 SLP-76, 18, 36, 110 SMAC, 118 and protein kinase C-theta (PKCθ), 1 Small hairpin DNA (shDNA). See shDNA Small interfering RNA. See siRNA Small ubiquitin-like modifier. See SUMO S-methionine, 178–179 Smurf1, 212 Sos, 45 SOS, 83 SPAK, 5 S-palmitoyl fatty acids, 128 SPT, 129 SptP, 61 Src kinases, 17, 18 and movement of Btk, 20 Src-PTKs, 90–92, 93 STAT, ubiquitination of, 213 STAT1, 196, 213 STAT4, 213 STAT5, 20 STAT6, 196 Statins, 164 STAT-interacting LIM protein. See SLIM Steroids in HIV treatment, 239 SUMO, 208 Superantigen-induced cell death, 76–78 Supramolecular activation cluster. See SMAC Syk, 92, 93, 107, 111 Syk-family kinases, 109 Systemic lupus erythematosus, 60, 61

T 3'UTR, 181 TAK1, 99, 211 TALEST, 247 Tandem arrayed ligation of expressed sequence tags. See TALEST

INDEX TARPP, 196–197 Tat, 230 T cell adapters, 73–84 T cell receptor. See TCR T cells activation by protein kinase C-theta (PKCθ), 1–9 activation from cell surface receptors, 115–116 activation-induced cell death in, 137, 138–139 activation regulation by TSAd, 75 during aging, 145–149 anergy, 209–210 bead contact formation, 161, 163 death of, 76–78 development and Itk, 29 differentiation and ubiquitination, 208–209 in immunosenescence, 144–145 and integrins, 117 modulation of cholesterol by cyclodextrin, 158–160 and protein kinase C-theta (PKCθ), 1–2, 5–6 signaling and cholesterol extraction, 159–160 and lipid rafts, 141–142 signal transduction, 73 and role of TSAd, 82–84 size of cholesterol-enriched cells, 161–162 tolerance and TSAd, 76–80 central, 78–81 peripheral, 76–78 translation of, 174–175, 177–182 T cell-specific adapter protein. See TSAd TCPTP, 59 TCR, 18 and CD28-mediated costimulatory signaling, 198–200 and cholesterol, 158 and Itk, 29 and lipid rafts, 143 signaling, 176 and cholesterol, 164–167 and functional genomics, 250–251 and gene expression, 249 and lipid rafts, 129–131 and TSAd, 83–84 and TSAd entry into nucleus, 82 and TSAd regulation, 75

301 TCR/CD3 complex, 3, 120–121, 129 and Itk, 30–31 TCR/CD28, 137 signaling and protein kinase C-theta (PKCθ), 3–4 stimulation and protein kinase C-theta (PKCθ), 1–2 TCR/CD29 signaling, 147 TCR complexes and Itk, 30–33, 34 TCR-Vβ8 T cells, 76 TCRζ/CD3, 115–116 TDAG8, 221 Tec family kinases, 5–6, 15–23, 29, 92 localization at nucleus, 19–21 at plasma membrane, 16–19 visualization of, 21–23 FRAP, 22–23 FRET, 22 TIRF, 21–22 Tec Homology domain. See TH domain Telomere shortening, 145 TFII-I, 20 Th1 cells, 208 and protein kinase C-theta (PKCθ), 8–9 Th2 cells, 208, 209 and protein kinase C-theta (PKCθ), 8–9 Th2 cytokines, 36–37 TH domain, 17 of Itk, 30, 31–32 T-helper memory T cells, 232 Th-POK, 254 Threonine-histidine/tryptophan loop. See THW loop THW loop, 191 Thymic involution, 145–146, 149 Thymic selection, 76–80 Thymocyte cyclic-AMP regulated phosphoprotein. See TARPP Thymocyte development and gene expression analysis, 248–250 Thymocytes and TSAd, 75, 78–80 TIRF microscopy, 21–22, 129 TLR signaling, 211 TM4, 247 TNFR1, 139, 142 TNF-related apoptosis-inducing ligand. See TRAIL Total internal reflection fluorescence. See TIRF microscopy

302 TRAC-1, 251 TRAF2, 142 TRAF6, 99, 211 TRAIL, 139 Transcription factors, 208 Transcriptome, 246–250 Translation control by signal pathways, 175–177 of individual mRNAs, 177–182 inhibition of, 181 initiation of, 172–173 in T lymphocytes, 174–175, 177–182 Translation initiation factor, 172 Transphosphorylation and Itk, 30, 32–33, 35–36 Tregs, 80 T regulatory cells. See Tregs Triton X-100, 128 TSAd and autoimmune disease, 75–76 cytoplasmic functions of, 83–84 nuclear function of, 82 regulation of T cell activation, 75 structure and expression of, 74–75 in T cell signal transduction, 82–84 and T cell tolerance, 76–80 central, 78–81 peripheral, 76–78 Tumor necrosis factors (TNFs) as death receptors, 139 Txk/Rlk complex, 19 Type 1 diabetes, 60 Type 2 diabetes, 59, 62 Tyr 183, 109 Tyr 446, 109 Tyrosine 482, 47 Tyrosine 513, 47 Tyrosine 805, 82 Tyrosine kinase, 38 Tyrosine phosphorylation, 53–54, 116, 131, 164, 166, 285 of 3BP2, 109 Tyrosine residue, 2 U Ubc13, 99 Ubiquitiin-conjugating enzymes. See E2s Ubiquitin, 207–214 Ubiquitin activating enzyme. See E1 Ubiquitination of chemokine receptors, 212 of STAT, 213

INDEX Ubiquitin conjugation, 207–214 Ubiquitin ligases. See E3s Uev1A, 99 Upstream AUG (uAUGs), 181 Upstream open reading frames (uORFs), 181 3'UTR, 181

V Valosin-containing protein (VCP), 82 Vascular biology and PTPs, 59–60 Vav, 18 and 3BP2, 110–111 and Itk, 35–36 Vav1, 2, 20, 111, 117 and arginine methylation, 199–200 Vav1/2, 110 Vav3, 111 VEGF activity, 220 VH1-like phosphatase PTPs, 54

W WASP, 18, 37–38 WeeB, 260 Wolf-Hischorn syndrome, 107, 112

X X-linked agammaglobulinemia, 15

Y Y753, 93 Y759, 93 YEN motif, 109 Yersinia pestis, 61, 62 YopH, 61, 62

Z ZAP-70, 129, 141, 258, 259, 285 Zinc finger domain, 194

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