! "# $ # # % & '( )!&!* &!+",& *%* +!-).!%*%! /*&!)+! //*&* 00 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medical Cell Biology presented at Uppsala University in 2002. ABSTRACT Lindholm, C. K. 2002. Shb and Its Homologues: Signaling in T Lymphocytes and Fibroblasts. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1129. 53 pp. Uppsala. ISBN 91-554-5260-4. Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis. Key Words: Shb, T cell receptor, IL-2 receptor, PDGF receptor, cell signaling, adapter proteins, LAT, Vav, PLC- γ1, SLP-76, JAK1, JAK3, Jurkat cells, T cells, NK cells, Apoptosis. Cecilia K. Lindholm, Department of Medical Cell Biology, Biomedicum, Uppsala University, Box 571, S-75123 Uppsala, Sweden. Cecilia K Lindholm 2002 ISSN 0282-7476 ISBN 91-554-5260-4 Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2002 2 To my family De spår jag kommer att avsätta blir inte som en elefants. De kanske blir som en liten nalles. Okej. Klaus Rifbjerg, Vaxdukshjärtat 3 REPORTS CONSTITUTING THE THESIS (Referred to in the text by their Roman numerals) I Cecilia K Lindholm, Erik Gylfe, Weiguo Zhang, Lawrence E Samelson and Michael Welsh (1999) Requirement of the Src Homology 2 Domain Protein Shb for T Cell Receptor-dependent Activation of the Interleukin-2 Gene Nuclear Factor for Activation of T cells Element in Jurkat T Cells. J. Biol. Chem. 274(39) 28050-28057. II Cecilia K. Lindholm, Maria L. Henriksson, Bengt Hallberg and Michael Welsh (2002) Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells. Submitted III Cecilia K. Lindholm (2002) IL-2 receptor signaling through the Shb adapter protein in T and NK cells. Manuscript IV Cecilia K. Lindholm, J. Daniel Frantz, Steven E. Shoelson and Michael Welsh (2000) Shf, a Shb-like Adapter Protein, Is Involved in PDGF-α-receptor Regulation of Apoptosis. Biochem. Biophys. Res. Comm. 278, 537-543. 4 TABLE OF CONTENTS: ABSTRACT 2 REPORTS CONSTITUTING THE THESIS 4 ABBREVIATIONS 6 INTRODUCTION AND AIMS 7 1. BACKGROUND 9 1.1. Receptors and their ligands 9 1.2. Tyrosine kinase receptors 9 1.3. Hematopoietic receptors 9 1.4. Cytokine receptors 10 1.5. Protein kinases 11 1.6. Adapter proteins 13 1.7. Monomeric G proteins and Guanine nucleotide exchange factors 16 1.8. Phospholipases 17 1.9. The Shb adapter protein 18 1.10. The Shd and She adapter proteins 20 1.11. Subcellular localization 21 1.12. Gene activation 21 1.13. Cell survival and cell death 22 2. METHODOLOGY 24 2.1. DNA 24 2.2. RNA 25 2.3. Proteins 25 2.4. Protein-protein interactions 26 2.5. Cellular processes 27 3. RESULTS AND DISCUSSION 31 3.1. A role for Shb in the early events of TCR signaling. 31 3.2. Effects of overexpression of Shb, with a defect SH2 domain, in Jurkat T cells. 35 3.3. Requirement of the Shb SH2 domain for NFAT activation and IL-2 gene transcription 37 3.4. Subcellular localization of Shb upon T cell receptor engagement. 38 3.5. Association of Shb with the IL-2 receptor signaling complex. 38 3.6. A physiological role for Shb in IL-2 receptor signaling and regulation of apoptosis. 41 3.7. A role for the Shf adapter protein in PDGF-receptor signaling and cell survival. 43 4. CONCLUSIONS 45 ACKNOWLEDGEMENTS 46 5. REFERENCES 48 5 Abbreviations used: TCR T cell receptor BCR B cell receptor LAT linker for activation of T cells PLC-γ1 phospholipase C-γ1 ZAP70 zeta-associated protein-70 PI3K Phosphatidyl inositol-3 kinase PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor FGF fibroblast growth factor NGF Nerve growth factor SH2 Src homology 2 SH3 Src homology 3 PTB phosphotyrosine binding PH Pleckstrin homology MAPK mitogen-activated protein kinase NFAT nuclear factor for activation of T cells AP-1 activating protein-1 NF-κB Nuclear factor κB PTK protein tyrosine kinase ITAM immunoreceptor tyrosine–based activation motif ERK extra-cellular signal-regulated protein kinase IL-2 Interleukin-2 IL-2R Interleukin-2 receptor GM-CSF Granulocyte-Macrophage colony stimulating factor ECL enhanced chemiluminescence system CAT chloramphenicol acetyltransferase NRS normal rabbit serum PBS phosphate-buffered saline PAGE polyacrylamide gel electrophoresis BSA bovine serum albumin JNK c-Jun N-terminal kinase JAK Janus kinase STAT signal transducer and activator of transcription GST glutathione-S-transferase P-Y phosphotyrosine GEMs Glycolipid-enriched membrane microdomain GDP Guanosine-di-phosphate GTP Guanosine-tri-phosphate PHA phytohemagglutinin NK Natural Killer DMEM Dulbecco’s Modified Eagle Media WT Wild type MHC major histocompatibility complex 6 Introduction INTRODUCTION A multicellular organism is composed of a variety of specialized cells, each with its own place and function in the organism. Both development and homeostasis of an organism requires strict regulation, that controls cell growth, differentiation, survival and death, generally referred to as cell signaling. This includes cell-cell contacts, activation of receptors and intracellular signal transducers, like kinases, phosphatases and transcription factors. The cells in an organism are constantly exposed to signals, activating different pathways, leading to the transcription of several genes. The repertoire of expressed proteins, and their levels of expression in the cell, then determine the characteristics of the cell. The immune system is composed of several specialized cells, with the aim to protect the organism from infections caused by bacteria, virus or fungi. There are two types of immune responses, innate and acquired immunity. The acquired immune response is mediated by B and T cells, and as the name suggests, has the ability to adapt to the environment. In humans, diseases like SCID (Severe combined immunodeficiency) and AIDS (Acquired immunodeficiency syndrome) derive from T cell defects. SCID is caused by a genetic defect, while AIDS is acquired through a retrovirus. However, both these diseases are lethal, and implicate the significance of a functional immune system for survival of the organism. In my studies I have tried to elucidate a role for the Shb adapter protein in T cells. I have studied the importance of Shb for proper T cell receptor signaling and established a role for Shb in IL-2 receptor signaling. I have also studied the importance of the Shb-relative, Shf, in fibroblast function and regulation of apoptosis. 7 Introduction The aims of this thesis were: • To determine the role for Shb in T cell receptor signaling, and find possible Shb interaction partners in T cells. • To study the effects of the Shb SH2 domain mutant on T cell receptor signaling in T cells. • To identify a possible role for Shb in Interleukin-2 receptor signaling in T and NK cells. • To find Shb homologues and define a role for the Shb family of adapter proteins in cell signaling. 8 Background 1 BACKGROUND 1.1 Receptors and their ligands Factors outside the cell, such as growth factors, hormones or proteins on other cells, can effect intracellular events, such as cell growth, cell death (apoptosis), production of new proteins or changes in cell morphology. These factors are collectively referred to as ligands, and they have the ability to associate with their cognate receptors. My focus here will be on tyrosine kinase receptors, hematopoietic receptors and cytokine receptors. 1.2 Tyrosine kinase receptors The PDGF receptors are examples of tyrosine kinase receptors. The tyrosine kinase receptors are transmembrane proteins with the ability to dimerize upon binding to their ligands which brings the two subunits in close contact with each other. These receptors have usually kinase domains with the ability to autophosphorylate tyrosines on the receptor itself and tyrosines on proteins that associate to the receptor. The PDGF receptor has two homologous isoforms, α and β which can form homo- or heterodimers (αα, ββ or αβ receptors), upon PDGF binding.