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*%* +!-).!%*%! /*&!)+! //*&* 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. Several signaling proteins can then associate with the activated receptor, including Shb, Shc, PLC-γ1, PI3K, Grb2, Src, Crk and SHP-2, initiating signaling pathways leading to cell division and cell migration (for reviews see [1, 2]).

1.3 Hematopoietic receptors The T cell receptor (TCR) is an example of a hematopoietic receptor, and it consists of several subunits organized into three complexes. The main receptor is made up of an α and a β subunit and functions as a docking site for antigens presented to the T cell by macrophages or other antigen presenting cells. The adjacent CD3 complex consists of the δ, γ and ε components. The third complex is either made up of two ζ-chains or one ζ-chain and one η- chain. In the mature T cell, accessory proteins are also expressed, and are vital for a proper T cell response. CD4 and CD8 stabilize the interaction between the TCR and the antigen- presenting MHC (Major Histocompatibility Complex) [3], and are also vital for the initiation

9 Background of the T cell response, since the kinase Lck associates with CD4 and CD8. CD28 is also important for proper T cell activation, possibly via PI3K, which has the ability to associate with tyrosine phosphorylated CD28. Activation of the TCR in the absence of CD28 stimulation, renders the T cell unresponsive to antigen, as a mechanism against auto- immunity (for review see [4]).

T cell maturation occurs in the thymus as follows: Pre-T cells do not express TCR, or at low levels, and are referred to as ”double negative”, which means that they no not express CD4 or CD8 (CD4-/CD8-). The double negative cells mature into TCR-expressing, double positive, cells (CD4+/CD8+). These cells then give rise to single positive CD4+ (T helper cells) or CD8+ (T killer cells), which migrate from the thymus to the peripheral blood system [5].

The T cell response is initiated by the presentation of an antigen to the T cell receptor. This is followed by rapid tyrosine phosphorylation, by the tyrosine kinases Lck and Fyn, located on CD4 or CD8, of special motifs in the TCR ζ-chain called ITAMs (immunoreceptor tyrosine- based activation motifs) [6]. The tyrosine kinase, ZAP70, then associates with these phosphorylated ITAMs and hence phosphorylates several downstream targets, including, LAT, SLP-76, PLC-γ1 and Vav. This initiates a signaling cascade involving the activation of the ERK MAP kinase pathway through Ras, activation of JNK through Vav and Rac and also a release of Ca2+ from intracellular stores. Taken together this promotes activation of the transcription factors NFAT, AP-1 and NF-κB leading to activation of the Interleukin-2 gene and IL-2 production (see figure 2) (for reviews see [7, 8]).

1.4 Cytokine receptors Proteins secreted by cells of the immune system are usually referred to as cytokines, for example Interleukins. Cytokines are often involved in the inflammatory response acting as growth factors for immune cells. T cells upon antigen presentation secrete IL-2 (Interleukin- 2). The secreted IL-2 has the ability to associate with IL-2 receptors on other T cells or NK

10 Background cells (natural killer cells) and cause proliferation of T-killer, T-helper and NK cells. The IL-2 receptor is composed of three subunits, α, β and γ. The β-subunit is shared by the IL-15 receptor and homologous to subunits of the IL-3, IL-4, IL-5, IL-6, GM-CSF, IL-7 and IL-9 receptors. The γ subunit is referred to as the common γ chain since it is also present in the IL- 4, IL-7 and IL-15 receptors. The α subunit in not crucial for receptor activity but increases the affinity for IL-2. The IL-2 receptor has no kinase activity, but the tyrosine kinase JAK1 is associated with the IL-2 receptor β-chain and JAK3 is associated with the γ-chain and to some extent with the β-chain. JAK1 and 3 activate STAT proteins, leading to T cell proliferation (for reviews see [9, 10]).

Other proteins involved in IL-2 receptor signaling include: Lck, Syk, SHP-2, Grb2, PI3K, Gab2 and Shc. The kinase Lck associates with the IL-2 receptor, but is not essential for IL-2 induced proliferation. The tyrosine kinase Syk associates constitutively with the IL-2Rβ chain and is activated by JAK1 or JAK3 upon IL-2 stimulation [11, 12]. Shc is reported to associate with the IL-2Rβ chain on tyrosine 338 [13, 14]. Shc recruits Grb2 and Gab2 to the receptor. Gab2 can then recruit SHP-2 and PI3K to the receptor complex, where Syk phosphorylates some of these proteins [15]. Grb2, PI-3K and Shc have then the ability to activate the ras/ERK pathway, inducing T cell proliferation (see figure 3) (reviewed in [16]).

1.5 Protein kinases There are several cytoplasmic protein tyrosine kinases (PTKs) or serine/threonine kinases, participating in propagating the signal from the receptor to the nucleus in a cell. A kinase has the ability to phosphorylate tyrosine, serine or threonine residues on neighboring proteins. Phosphotyrosines can function as docking sites for other signaling proteins (see Adapter proteins). In my studies I have worked with the kinases: ZAP70, Lck, ERK1, ERK2, JNK, JAK1 and JAK3.

11 Background

ZAP70 is expressed mainly in hematopoietic cells, associates with the phosphorylated ITAMs in the ζ−chain, of the T cell receptor, and phosphorylates several of the proteins known to be involved in TCR signaling (see Hematopoietic receptors). In a ZAP70 deficient mouse, thymocyte development is arrested at the CD4+/CD8+ stage. In humans, ZAP70 mutations result in severe immunodeficiency [17, 18].

Lck is associated with cysteine residues in the cytoplasmic tails of CD4 and CD8 [19]. Crosslinking of CD4 or CD8 induces autophosphorylation of Lck, which enhances its kinase activity [20]. Lck then phosphorylates the ITAMs on the CD3 and ζ-chain-complexes of the T cell receptor, thus initiating the T cell response. Lck is also known to associate with the IL-2 receptor β chain in T cells, however, the role of Lck in IL-2 receptor signaling remains to be elucidated. In Lck deficient mice the thymocytes exhibit an incomplete block in the double negative (CD4-/CD8-) stage [21], however, if both Lck and Fyn are knocked out, the block is complete, and these mice show a complete absence of CD4+/CD8+ thymocytes. Other studied, performed in Lck deficient Jurkat cells, show that Lck is vital for TCR signaling [22].

ERK1 and ERK2 are MAP kinases involved in the Ras/MAPK pathway. When Ras is activated in a cell, it will recruit Raf-1 to the cell membrane, where it will be phosphorylated by membrane-bound kinases. Raf-1 has the ability to phosphorylate MEK (MAPKK), which can phosphorylate ERK1 and 2. Phosphorylated ERK translocates to the nucleus, where it can phosphorylate several transcription factors, including Elk1 and c-Fos. These transcription factors form homodimers and heterodimers, like AP-1, whose activity leads to transcription of genes, i.e. IL-2 or genes involved in cell proliferation (reviewed in [16]).

C-jun N-terminal kinase (JNK), also known as stress-activated MAP kinase (SAPK). JNK is activated by Rac1 and Cdc42, and known to regulate both cell proliferation and apoptosis. However, JNK has also more specialized functions in some cell types. As the name implies, JNK phosphorylates c-jun, but also other members of the AP-1 complex, including JunB,

12 Background

JunD and ATF2. JNK is of importance for the maturation of T cells in vivo. Jnk knockout mice are morphologically normal, but immunodeficient due to severe defects in T cell function. As mentioned above, JNK plays a role in the apoptotic machinery. The exact mechanism of how JNK mediates apoptosis is unclear. One potential target for JNK is the tumor suppressor p53. JNK can bind to p53, which promotes degradation of p53. However, JNK has also the capacity to phosphorylate the p53 protein, which will protect it from degradation. Other targets for JNK include Bcl-2 and Bcl-xL. These proteins have the capacity to be phosphorylated by JNK, which inhibits the anti-apoptotic functions of Bcl-2 and Bcl-xL (for review see [23])

Janus kinases (JAKs) are a novel class of non-receptor protein kinases. Four mammalian Jaks have been identified: JAK1, JAK2, JAK3 and Tyk2. Among them, JAK1, JAK2 and Tyk2 are ubiquitously expressed, and JAK3 is mainly expressed in hematopoietic cells. JAK1 and JAK3 have the ability to associate with the IL-2 receptor and phosphorylate several proteins, including the receptor itself. STAT5a and b, can then associate with the phosphorylated tyrosines 392 and 510 of the IL-2Rβ, where they are phosphorylated by JAK1 or 3. Two phosphorylated STAT proteins then dimerize to form a functional transcription factor, with the ability to translocate to the nucleus and initiate transcription of genes involved in cell division (see figure 2). STAT1 and 3 are also phosphorylated upon IL-2 stimulation of T cells, but they bind to the acidic domain of IL-2Rβ, in a phospho-independent manner (reviewed in [9]).

1.6 Adapter proteins Several of the proteins involved in signal transduction have no enzymatic activity. However, some of these proteins have several protein-interaction domains, and can thus function as linkers between other proteins and also aid in the formation of protein complexes. These proteins with multiple binding domains/sites are usually referred to as adapter proteins or scaffolds (for reviews see [24-26]). Some of the most common binding domain includes:

13 Background

SH2 domains: Can associate with phosphorylated tyrosines, followed by a short amino acid motif, which is unique for each SH2 domain. SH2 domains have a conserved amino acid sequence, and was first described in the Src protein, where it got its name: Src Homology 2 domain. SH3 domains: Can associate with proline-rich domains, with the consensus sequence ”PxxP”. SH3 domains have also been named after the PTK Src, where it was first described. WW-domains: Has also the ability to bind to proline/tyrosine or proline/leucine motifs and are named after the two conserved tryptophanes in the binding motif. PTB-domains: Phospho-Tyrosine Binding domains can associate with phosphorylated tyrosines, with a short amino acid motif upstream (N-terminal) of the tyrosine. This motif is unique for each PTB domain, and less conserved than SH2 domain motifs. PTB domains have a conserved 3D structure, with similarities to the PH domain.

PH-domains: Pleckstrin homology domains associate with phospholipids, like PIP3, or the plasma membrane of the cell. PDZ-domains: Binds short motifs containing carboxy-terminal hydrophobic residues.

In my studies I have investigated two adapter proteins, Shb and Shf and their role in T cell signaling and PDGF signaling, respectively. Although the signaling pathways are similar in many cell types, the hematopoietic cells exhibit several unique features. There are for example several lymphocyte-specific adapter proteins. The relationship of the adapters LAT, SLP-76 and Gads to Shb is investigated in this thesis.

LAT is a 36 kDa adapter protein, expressed in hematopoietic cells and strongly phosphorylated upon T cell receptor engagement. LAT contains nine conserved tyrosine phosphorylation sites, where Tyr-132, Tyr-171 and Tyr-191 have been proposed as possible binding sites for Grb2 and Gads, while Tyr-171 and Tyr 226 are of importance for SLP-76 and PLC-γ1 interactions with LAT [27]. Two conserved cysteines near the transmembrane

14 Background domain of LAT suggested that LAT is palmitoylated, which turned out to be the case. Like many other palmitoylated proteins, including Lck and Fyn, LAT was enriched in glycolipid microdomains or lipid rafts. LAT has been shown to associate with PLC-γ1, SLP-76, p85 PI3K, Cbl, Grb2 and Gads, upon TCR engagement, and can therefore aid in targeting these proteins to the lipid rafts (reviewed in [28]). In LAT-deficient mouse, thymocyte development fails to proceed past the pro-T3 (CD4-/CD8-, CD25+/CD44-) stage [29], and LAT-deficient Jurkat cells exhibit deficencies in calcium flux, Ras-ERK activation and IL-2 gene activation [29-31].

SLP-76 is an adapter protein, mainly expressed in T cells, NK cells, monocytes and basophilic granulocytes. A SLP-76 homologue, SLP-65, is expressed in B cells, and has a role in B cell receptor signaling. Following TCR engagement, SLP-76 is phosphorylated on Tyr-113, -128 and –145 [32]. The Vav SH2 domain associates with SLP-76 via Tyr-113 and –128. SLP-76 can also associate with Nck, Itk, Gads, PLC-γ1 and SLAP-130. SLP-76 and LAT associate with each other upon TCR stimulation, likely via the Gads adapter protein [33]. Mice deficient in SLP-76, exhibit similar phenotypes as LAT deficient mice, since their thymocytes do not mature past the pro-T3 stage. SLP-76 deficient Jurkat cells, respond similar as LAT- deficient Jurkat cells, and exhibit no activation of ERK, PLC-γ1 or NFAT upon TCR stimulation [34]. This suggests a functional relationship between LAT and SLP-76 in TCR signaling (for review see [31]).

Gads, is a small adapter protein of the Grb2/Grap family, with two SH3 domains and one SH2 domain. Gads is mainly expressed in hematopoietic cells, and has been shown to associate with Shc, Bcr-Abl, HPK1 (hemopoietic progenitor kinase), LAT and SLP-76 [33, 35]. Overexpression of an SH2 domain defective Gads in Jurkat T cells, results in inhibition of TCR-induced NFAT activation as well as reduced tyrosine phosphorylation of HPK1 upon CD3 stimulation [36, 37].

15 Background

1.7 Monomeric G proteins and Guanine nucleotide exchange factors. Except for the tyrosine kinases, the receptors and the adaptor proteins, there are several other signaling proteins involved in the propagation of the signal from the cell membrane to the nucleus, including monomeric G proteins, Guanine nucleotide exchange factors and Phospholipases.

The monomeric G-proteins, sometimes referred to as GTPases, includes for example: Ras, Rap1, Rac1, Cdc42 and Rho and have important roles in the cell machinery. A Ras mutant was one of the first discovered oncogenic proteins, with the capacity to induce uncontrolled cell growth, and thus causes cancer. Resting monomeric G-proteins have an associated GDP (guanosine-di-phosphate) molecule, which is removed by a guanine nucleotide exchange factor (GEF) when the monomeric G protein is activated. The empty pocket is then quickly filled with a GTP (guanosine-tri-phosphate) molecule and the monomeric G protein is activated. Ras, for example, is activated by Sos (a GEF). Ras recruits Raf to the plasma membrane, where Raf is phosphorylated and activated by membrane-bound kinases, which is one of the early events in the ERK MAP kinase pathway, leading to cell proliferation (for review see [38]).

Another monomeric G protein, Rac1, is activated by Vav. Rac1 belongs to the Rho family of GTPases that controls cytoskeletal rearrangements, where Rac1 is involved in the promotion of membrane ruffling (reviewed in [38]). Rac1 activates the c-Jun N-terminal kinase (JNK). Active JNK can induce changes of cell morphology, as well as promote apoptosis or proliferation [23].

Vav is a guanine nucleotide exchange factor (GEF) for the GTPases Rac1 and Cdc42. There are three isoforms of Vav, Vav-1, which is expressed in hematopoietic cells, Vav-2 and Vav-3 which are more widely expressed. Vav-1-/- mice develop normally, however they exhibit reduced number of T cells and lack some B cells. T cells from Vav-1-/- mice are unable to

16 Background produce IL-2 and fail to proliferate in vitro [39-41]. These data from knock-out mice are confirmed in Jurkat cells, where Vav-1 has been shown to be important for NFAT activation, and hence IL-2 gene transcription [42]. Upon TCR engagement, Vav-1 is reported to be phosphorylated by Lck on Tyr-174, and this process activates the exchange activity of Vav [43], [44]. However, other studies have suggested tyrosine-174 on Vav as a putative candidate for phosphorylation by Syk and Zap70 [45], and Vav has the ability to bind both ZAP70 and Syk via its SH2 domain. A mutation of tyrosine-174 to phenylalanine, resulted in hyperphosphorylation of Vav-1, but had no effect on the exchange activity of Vav-1 [46]. The Y174F mutation in Vav-1 also increased Rac1 activity and potentiated the activation of the NFAT transcription factor [47]. Following T cell receptor activation, the Vav SH2 domain also has the ability to associate with Tyr-113 and Tyr-128 of SLP-76. Vav2 is tyrosine phosphorylated in response to growth factors, such as EGF and PDGF. According to [48], the major phosphorylation site in Vav2 is Tyr-159, which has the motif: ”GEDIY”. This motif is quite similar to the ”GDEIY” motif of Tyr-174 in Vav1 and also to the ”DEDIY” and ”EEDLY” motifs of Tyr-141 and Tyr-160 in Vav3. Engagement of CD28, the costimulatory receptor for the T-cell receptor, can induce a more sustained phosphorylation of Vav, compared to TCR induced phosphorylation of Vav. CD28 has the ability to recruit PI3K, which catalyses the reaction PIP2 to PIP3. PIP3 associates with the Vav PH domain, and helps in localizing Vav to the plasma membrane (for reviews see [49, 50]).

1.8 Phospholipases Several phospholipases are present at the plasma membrane, for example: Phospholipase A (PLA), Phospholipase C (PLC) and Phospholipase D (PLD). These have all different isoforms and PLC-γ has two isoforms: PLC-γ1 and PLC-γ2, which are involved in cell signaling in hematopoietic cells. PLC-γ1 is expressed in most tissues, while PLC-γ2 is expressed only in hematopoietic cells. Both PLC-γ1 and 2 can function as adapters, as well as phospholipases.

PLC-γ hydrolyses phosphatidylinositol 4,5 bisphospate (PIP2) to produce inositol 1,4,5-

17 Background

2+ triphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca from internal stores of the endoplasmatic reticulum into the cytoplasm. Ca2+ is a potent second messenger and mediates several cellular responses in T cells, like the activation of the calcium responsive protein calmodulin. The activation of calmodulin mediates its binding to, and activation of, the phosphatase calcineurin. Calcineurin can then dephosphorylate, and thus activate the transcription factor NFAT, which translocates to the nucleus and the IL-2 promoter, in the T cell. DAG is important in the activation of protein kinase C (PKC), leading to activation of both the ras/raf/MAPK signaling cascade and the NF-κB transcription factor (see figure 2) (for review see [51]).

T cell receptor engagement induces tyrosine phosphorylation of the adapters LAT and SLP- 76, which then have the ability to recruit PLC-γ1 to the ZAP70 tyrosine kinase. Phosphorylation of PLC-γ1, by ZAP70, contributes to the activation of its enzymatic activity. A mouse deficient in PLC-γ1 dies at embryonic day 9.0, before the onset of the generation of T cells [52]. PLC-γ1 deficient Jurkat cells exhibit defects in NFAT activation, and thus also in IL-2 production [53]. PLC-γ2 deficient mice are viable, but exhibit defects in B cell development [54].

1.9 The Shb adapter protein The Shb protein was cloned from a β-cell library in 1994 [55] but it has since found to be ubiquitously expressed. Shb is expressed as two isoforms of 55 and a 67 kDa. Both have a C- terminal SH2 domain, a central PTB-domain and four central putative tyrosine phosphorylation sites. However the 55kDa Shb has two, and the 67 kDa Shb has five N- terminal proline-rich regions (see figure 5).

The human Shb gene maps to chromosome 9 [56]. Resent advances in the HUGO project have revealed that the Shb cDNA is composed of six exons. The first, and largest, exon contains the proline-rich motifs and the PTB domain. The second and third exons contain the

18 Background putative tyrosine phosphorylation sites, followed by a short linker region in exon 4. The fifth and sixth exons make up the SH2 domain. This composition of the Shb gene could explain the evolution of the Shb homologues Shd and Shf, which simply lack the first exon and where the linker region displays a high degree of divergence.

Shb has been shown to associate with several signaling proteins. The SH2 domain of Shb associates with some receptors, including the PDGF-receptor α and β, the FGF-receptor-1 and the ζ-chain of the T cell receptor. The PTB domain of Shb has been found to associate with the kinase FAK and p36/38 (LAT). The proline-rich regions of Shb interact with the adapters Grb2, Eps8, Grap and the tyrosine kinase Src [57, 58], manuscript.

Fibroblasts overexpressing Shb display increased rates of cell death upon serum withdrawal, compared to control cells [59]. Overexpression of Shb can also induce apoptosis in β-cells of islets of Langerhans, isolated from Shb-transgenic mice, compared with control islets. This increase in apoptosis was observed both under basal conditions and after incubation with IL-1 beta + IFN-gamma. Overexpression of Shb enhances β-cell death under certain conditions and may thus contribute to β-cell destruction in type 1 diabetes [60].

Shb has several putative tyrosine phosphorylation sites and phosphorylation of Shb has been observed in response to FGF [61, 62], endostatin [62], NGF [63] and PDGF [64]. The SH2 domain of CrkII has been suggested to interact with tyrosine phosphorylated Shb [65]. Other putative SH2 domains that could interact with the tyrosine motifs in Shb include the proteins Abl, Nck and RasGAP [25, 66].

It has been shown that Shb is of importance in PC12 cells (a neuronal cell line), where overexpression of Shb induces a more rapid NGF- and FGF-2-dependent neurite outgrowth, compared to wild type PC12 cells [63]. NGF stimulation increases Rap1 activation 5 fold in PC12 cells overexpressing Shb, compared to normal PC12, while FGF-2 had no such effect.

19 Background

The NGF-induced Rap1 activation was found to depend on association of Crk-C3G complex to tyrosine phosphorylated Shb via the Crk SH2 domain. These results suggest a role for Shb in NGF-dependent Rap1 signaling and NGF- and FGF-2-dependent differentiation of PC12 cells [65].

Shb is also involved in angiogenesis. Endothelial cells (IBE cells) form tubular structures (capillary blood vessels) in response to FGF-2. IBE cells overexpressing Shb were able to form these tubes faster than control cells. However, IBE cells overexpressing a mutant form of Shb, with a defect SH2 domain, formed disorganized tubes to a lesser extent than control cells. These findings suggest that a functional SH2 domain of Shb is required for proper tubular morphogenesis, when new blood vessels are formed [65]. Shb has also been implicated in the regulation of apoptosis in endothelial cells. Overexpression of Shb in IBE cells (murine brain endothelial cells), induces a significant increase in the basal level of apoptotic cells, compared to vector-transfected cells. Addition of FGF-2, to these Shb expressing IBE cells, caused a significant inhibition of apoptosis, which was not seen in control IBE cells [62].

Shb is also important for PDGF-induced cytoskeletal reorganization. Fibroblasts over- expressing Shb display reduced membrane ruffling upon PDGF-AA or –BB stimulation, whereas filopodia structures were accentuated, compared to normal fibroblasts [64]. Rac activation was also abolished in the Shb-expressing fibroblasts upon PDGF stimulation, which probably explains the perturbed edge ruffle formation.

1.10 The Shd and She adapter proteins Since 1994 several Shb-like adaptor proteins have been cloned, including Shd and She [67]. These proteins all share a high homology to Shb in the SH2 domain. Shb and Shd also share four conserved putative tyrosine phosphorylation sites (see figure 5). These proteins were found in a yeast two hybrid screen, using the kinase domain of Abl as bait [67]. This suggests

20 Background that the SH2 domains of Shd and She associate with phospho-tyrosines in the Abl kinase domain. The Shd SH2 domain was functional and associates with several phospho-proteins in K562 cells. The tyrosine phosphorylation sites in Shd have the consensus sequence YxxP, which is a preferred site for phosphorylation by the Abl kinase. Shd was tyrosine phosphorylated by Abl, but not by a kinase dead form of Abl, when these proteins were co- expressed in COS cells [67]. Also, the Abl SH2 domain has the ability to associate with proteins containing phospho-tyrosines within a YxxP motif [25].

1.11 Subcellular localization The plasma membrane of several cell types, including T cells, contains certain areas that are rich in glycosphingolipids, cholesterol, GPI-anchored proteins, palmitoylated proteins or proteins with hydrophobic modifications. These areas are thought to be involved in cell signaling by concentrating signaling proteins, and thus mediate a quick association upon receptor stimulation. These areas are called ”lipid rafts” or ”glycolipid-enriched membrane microdomains” (GEMs). Lipid raft formation has so far been reported upon stimulation of the following receptors: IgE receptor, TCR, BCR, EGFR, insulin receptor, EphrinB1 receptor, Hedgehog and integrins [68]. Some signaling proteins are constitutively located in lipid rafts. In resting T cells LAT, Lck, Fyn, Syk, Cbl and Ras are located in rafts, whereas some other proteins are recruited to the rafts upon T cell receptor stimulation. The latter group includes SLP-76, Gads, Vav, ZAP70, TCR ζ-chain, PLC-γ1, Shc and PKC (reviewed in [68, 69]).

1.12 Gene activation Cell signaling through transmembrane receptors ultimately leads to the activation or deactivation of genes in the cell nucleus. This is mediated by transcription factors (TF), which are activated by phosphorylation (c-jun, c-fos or STAT) or dephosphorylation (NFAT) and then permitted to enter the nucleus. Some TF never leave the nucleus, instead the kinase will translocate to the nucleus and there phosphorylate its targets. These transcription factors sometimes have to dimerize to form a functional transcriptional activator, for example c-fos

21 Background and c-jun can form AP-1 (other combinations are also possible) and two STAT proteins have to dimerize to be functional. The regulatory region of a gene, where the transcription factors bind, often have several binding sites, to mediate the binding of different transcription factors and sometimes it has several sites for one transcription factor. The IL-2 gene in T cells is activated by TCR engagement and when activated mediates production of IL-2, which causes proliferation of T and NK cells. The IL-2 gene promotor region has binding sites for the transcription factors, AP-1, NFAT, NF-κB and Oct-1 (see figure 2) (for reviews see [4, 70]).

1.13 Cell survival and cell death Programmed cell death (apoptosis) was first identified in developing embryos, since apoptosis, is a requirement for development of a multicellular organism. Some cells are destined to die without a gross inflammatory response, to mediate morphological changes and organogenesis in the developing embryo. Apoptosis is an active and tightly regulated process, which can be triggered by various stimuli, including growth factor withdrawal, viral infections, death receptor ligands and DNA damage. Necrosis is often induced by trauma and in this process the cells die in an unordered fashion. The criteria for determining whether a cell is undergoing apoptosis versus necrosis includes morphological changes like nuclear condensation, DNA fragmentation and cytoplasmic swelling but also biochemical markers, like activation of the ICE-related proteases (caspases) and upregulation of p53 expression.

Death signals: As mentioned above, a family of effector proteins involved in the apoptotic pathway is the caspases, a family of proteases, which target proteins of the cytoskeleton and nuclear lamina. In the absence of survival signals, a protein called Bad associates with Bcl-2 at the mitochondrial membrane. Binding of Bad to Bcl-2 induces formation of pores in the mitochondrial membrane and a release of cytochrome C from the mitochondria. Cytochrome C associates with Apaf1, which activates caspase 9, which in turn activates caspase 3, and the apoptotic machinery is then set into motion. Activation of JNK and p38 can also activate caspase 9 and caspase 3, mediating apoptosis, in the absence of survival signals. Activation of the ERK pathway normally mediates cell survival, and caspase 3 has the ability to inhibit ERK activation, by inactivating Raf-1, which is upstream of ERK. Caspase 3 also activates

22 Background

PKCθ and PKCδ, leading to the nuclear condensation that is associated with apoptosis. JNK also has the ability to upregulate Fas ligand (FasL) expression. Fas is a receptor on the cell surface that can cause apoptosis when engaged by its ligand (FasL). Fas is the most widely expressed example of a ”death receptor”.

Survival signals: ERK activation has been shown to rescue cells from apoptosis, induced by

H2O2, hypoxia and chemotherapeutic agents. This is due to several signaling events in the cell. One target for ERK is the Bad protein, which is essential for the release of cytochrome C from the mitochondrial membrane. When Bad is phosphorylated by ERK or Akt/PKB (substrate for PI3K), it can associate with the 14-3-3 protein, which targets this Bad/14-3-3 complex to the cytosol, away from the mitochondria and the Bcl proteins, thus inhibiting the apoptotic pathway. In the absence of Bad, the Bcl proteins are anti-apoptotic, and inhibit the pore formation and the release of cytochrome C from the mitochondria (for reviews see [71- 73]).

23 Methodology

2 METHODOLOGY 2.1 DNA Subcloning PCR (Polymerase Chain Reaction) was used to amplify DNA. Added to the PCR reaction mix, except buffer, was, two primers, annealing to the different strands of the fragment to be amplified, dNTP (a mix of dCTP, dTTP, dATP and dGTP) and Taq polymerase. The reaction was subjected to 25-35 temperature cycles as follows: denaturation for 1-2 minutes in 96°C, annealing for 1-2 minutes in 50-60°C and extension for 1-4 minutes in 72°C. The PCR product was then either cut with appropriate restriction enzymes and ligated into an appropriate vector, or ligated directly into a vector.

DNA constructs Several plasmids were constructed, to express wild-type protein or proteins altered in some way, in mammalian cells. In this thesis the following expression plasmids were used: WT Shb, R522K Shb (with a point mutation in the SH2 domain, rendering it non-functional) and ∆PTB-Tyr Shb (with the PTB domain and the four putative tyrosine phosphorylation sites deleted) in pcDNA1, WT Shf in pBABE.HA, WT Vav in pEF116, WT SLP-76 (aa1-534), SLP-76-Tyr (aa1-155) and SLP-76-SH2 (aa415-534) in pSG5A, WT-IL-2Rβ and IL-2Rβ- Y5,6F in pCMV4neo, WT JAK1 and JAK3 in Prk5. Plasmids to produce fusion proteins in bacteria were also used, were the protein of interest were fused to GST or His-tag (pET), to mediate purification of the protein. These plasmids include: The Shb-SH2-GST (containing the Shb SH2 domain), Shb-PTB-Pro-GST or p55 Shb∆SH2 (containing the Shb PTB domain and two proline-rich regions), Shb-PTB-GST (containing the Shb PTB domain), p55 Shb-pET (containing full-length p55 Shb), SLP-76- Pro-GST (containing the SLP-76 proline-rich regions), SLP-76-SH2-GST (containing the SLP-76 SH2 domain) and Shf-SH2-GST (containing the Shf SH2 domain).

24 Methodology

Sequencing: The DNA sequence was verified using a Perkin Elmer sequencer. Plasmid DNA was mixed with ”Ready Reaction Mix” (Perkin Elmer) and sequencing primer. This mix was temperature cycled 25 times as follows: 10 seconds in 95°C, 5 seconds in 50°C and 4 minutes in 60°C. The product was precipitated with ethanol, reconstituted in ”Template suppression reagent” and sequenced.

2.2 RNA RT-PCR mRNA was prepared from the Ba/F3 clones, to assess expression of Shb (III), using the Oligotex Direct mRNA Kit (Qiagen, GmbH, Germany). cDNA synthesis and RT-PCR was then performed using the OneStep RT-PCR Kit (Qiagen, GmbH, Germany). Primers for GAPDH were used as a control of mRNA quality and quantity. RT-PCR was then performed using primers for human Shb, and the products were separated on a 1.8 % agarose gel.

Northern Blot: Northern blotting was used to assess the tissue distribution of Shf mRNA (IV). Human Shf cDNA was labeled with [32P] dATP and hybridized to a multiple human tissue mRNA blot. The blot was washed and the radioactivity was detected using a Phosphor Imager.

2.3 Proteins SDS-PAGE and Western Blotting: SDS-Poly Acrylamide Gel Electrophoresis is used to separate proteins according to their molecular weights (in kDaltons). The proteins are denatured by boiling and addition of SDS, and then separated on the SDS polyacrylamide gel. The separated proteins are transferred onto a filter which is blocked in 5% BSA, 0,5%Tween-20, incubated with primary antibody (protein-specific) and then horseradish-peroxidase coupled secondary antibody (specific to the

25 Methodology primary antibody). The proteins are detected using ECL (Enhanced Chemiluminescence) detection system.

2.4 Protein-protein interactions Studies of protein interactions using fusion proteins: Fusion proteins are composed of two proteins that are fused to each other. One protein, GST (Glutathione-S-Transferase) or a stretch of Histidines (His-tag) has the ability to bind well to a compound (glutathione or Nickel) that can be coupled to beads, and thus easily purified. This protein can then be fused to the protein, or part of protein, that one would like to study. In my studies I have utilized fusion proteins comprising different parts of Shb, full-length Shb, the Shf-SH2 domain and also different parts of SLP-76 coupled to GST. These GST- fusion proteins are then incubated with cell extracts from cells treated with growth factors, antibodies or cytokines. The sepharose coupled fusion proteins can then be used to ”fish out” interaction partners that associate with Shb, Shf or SLP-76 upon cell stimulation. The whole protein complex can then be resolved on SDS-PAGE and Western blot, and interaction partners can then be identified using antibodies against different proteins.

Peptide inhibition When Shb/Shf interacting proteins have been found, using fusion proteins of different domains of Shb or Shf, the more exact binding site can be identified using short peptides. If the fusion protein contains an SH2- or PTB-domain the tyrosine phosphorylation sites in the interacting protein are possible targets. Short phospho-peptides can be synthesized, of 14-16 amino acids, containing the peptide sequence flanking the possible tyrosine, which is phosphorylated. These peptides are then included in excess when cell extracts are incubated with GST-fusion protein. If the peptide has the ability to bind the fusion protein SH2- or PTB- domain, the peptide will compete with the protein from the cell extract in binding to the fusion protein. In my studies I have used phospho-peptides with sequence from tyrosine-sites in LAT, PDGF receptor α, Vav, SLP-76, IL-2 receptor β and the ITAMs of the TCR ζ-chain.

26 Methodology

Co-immunoprecipitations Co-immunoprecipitations can be used to study interactions between proteins, using antibodies against one protein and protein A sepharose, to pull down the protein complex formed around that particular protein, as a result of cell signaling. After washing, this protein complex can then be resolved using SDS-PAGE and Western blotting, and the blot can be probed with different antibodies against candidate interaction partners, or with phosphotyrosine, to assess tyrosine phosphorylation.

Immunpreciptation is also used for in vitro kinase (IVK) assays where the protein complex, coupled to sepharose, is incubated with [γ-32P] labeled ATP and exogenous substrate. For the Lck IVK assay, enolase was used as substrate (Fig 1C). The samples are then washed, resolved on SDS-PAGE and the [32P]-incorporation was assessed by autoradiography.

Subcellular localization (GEM) To test if Shb localizes to lipid rafts upon TCR engagement (II), Jurkat cells were CD3 stimulated or not, before lysis in Triton lysis buffer. The lysate was mixed with an equal volume of 80% Sucrose, to obtain 40% Sucrose, which was overlaid with 30% sucrose and 5% sucrose. This gradient was subjected to ultracentrifugation at 45 000 rpm for 24 hours. Aliquots were collected and the proteins were precipitated using TCA. The proteins were reconstituted in a smaller volume and the proteins were separated using SDS-PAGE and Western blotting. The blot could then be probed with different proteins, and the lipid rafts were typically present in fraction 3-5.

2.5 Cellular processes Cells and transfections Several cell lines have been used in this study, including the T-helper cell line Jurkat (I, II), NIH-3T3 fibroblasts (IV), PAE aortic endothelial cells (IV), COS-7 kidney cells (II, III), Ba/F3 IL-3 dependent pre-B cells (III) and NK-92 natural-killer cells (III). I have also used

27 Methodology primary human T cells (III), isolated from human blood as follows: BC (Buffy coat) was collected from the blood bank and purified on a ficoll-pague gradient. The lymphocyte-layer was washed and resuspended in normal media supplemented with PHA (Phytohemaglutinin). IL-2 (Interleukin-2) was added to the cells on day 3 after PHA addition and the cells were then expanded for 10 to14 days with IL-2. To obtain stable clones in Jurkat cells (I, II), these cells were electroporated with Shb R522K expression vector and vector carrying a neomycin resistance gene. Geneticin resistant clones were isolated, and assayed for overexpression of Shb. To obtain stable clones overexpressing Shf (IV), NIH-3T3 cells were transfected with Shf cDNA in expression vector, using Lipofectamine. Puromycin was then added to the media, and growing clones were tested for Shf overexpression. To obtain clones expressing WT-IL-2Rβ+R522K Shb, IL-2Rβ- Y5,6F+WT-Shb or WT-IL-2Rβ+WT-Shb in Ba/F3 cells (III), the cells were electroporated with the above mentioned plasmids. Cells overexpressing IL-2Rβ+Shb (or mutations of Shb and IL-2Rβ) were selected using the neomycin gene in the IL-2Rβ vector. Several independent clones, from each transfection, were established.

Transient transfections were also performed, using electroporation of Jurkat cells (I) and Lipofectamine for NIH3T3 (IV) and COS (II, III) cells.

Cell proliferation To assess the proliferation of NIH3T3 cells overexpressing Shf (IV) compared to normal NIH3T3 cells, the cells were counted using a Bürker chamber. 3 x 104 cells were seeded into media containing 0,1% serum, with or without PDGF-AA, and counted after 24, 48, 72 and 96 hours.

28 Methodology

Cell viability When cells are not viable, they undergo programmed cell death, usually referred to as apoptosis. This can be measured using Annexin V, which is a protein with the ability to recognize phosphatidyl-serine on the cell surface, which is a marker for apoptosis. To assess if overexpression of Shf had an effect on cell viability (IV), NIH3T3-Shf and NIH3T3-control cells were cultured in a low serum content (0.1% serum) with or without the addition of PDGF-AA for four consecutive days. Cell viability was then measured by double staining of the cells with FITC labeled Annexin V and Propidium iodide, and cell sorting using a flow cytometer. Flow cytometry revealed two populations with altered characteristics compared with the main population. The apoptotic population showed strong staining for annexin (FL-1) and slightly elevated levels of propidium iodide staining (FL-3). The second population was strongly positive for propidium iodide with no or little annexin-positivity, and this was considered to consist of necrotic cells.

To determine a possible role for Shb in IL-2 mediated regulation of apoptosis (III), cells from two independent clones each of the Ba/F3 cells overexpressing WT-IL-2Rβ+R522K-Shb, IL- 2Rβ-Y5,6F+WT-Shb and WT-IL-2Rβ+WT-Shb were cultured in the absence of IL-3, but in the presence of IL-2. The cells were cultured for 9 days, and new media containing IL-2 was added daily, to keep the cells at constant cell density. On day 5 and 9 after IL-2 addition, the cells were washed and labeled with Annexin V-FITC and propidium iodide, and sorted on a flow cytometer, as described above.

Measurements of cytoplasmic Ca2+ One effect of TCR engagement involves the release of Ca2+ from intracellular stores, contributing to the activation of the NFAT transcription factor, in the IL-2 gene. To measure release of Ca2+, fura-2 acetoxymethylester was added to the Jurkat cells or Jurkat cells expressing Shb R522K (I). After washing, the cells were placed in a cuvette in a time-sharing

29 Methodology multichannel spectrophotofluorometer [74]. The cytoplasmic Ca2+ concentration was then measured after the addition of anti-CD3.

T Cell activation Engagement of the T cell receptor initiates a signaling cascade that will ultimately lead to activation of the IL-2 gene, and consequently IL-2 production. The promotor region of the IL- 2 gene contains binding sites for several transcription factors, the most vital one being the NFAT transcription factor. Activation of this transcription factor can be measured using a reporter gene construct composed of a triplet of the NFAT binding site from the IL-2 promoter region coupled to a CAT (chloramphenicol acetyltransferase) reporter. When the NFAT element is activated, CAT will incorporate radioactive carbon into the cells, which can be measured using a scintillation counter. Activation of the NFAT transcription factor was measured after CD3 stimulation of normal Jurkat cells and Jurkat cells overexpressing Shb R522K (I). IL-2 production can also be measured directly using PE (phycoerytrin) conjugated anti-IL-2 antibody. Jurkat cells, CD3 stimulated or not, were fixed in 4% paraformaldehyde and permeabilized. The cells were then stained with the PE-rat anti-human IL-2 antibody and analyzed by flow cytometry (FACS). The fraction of cells producing IL-2 upon T cell engagement can then be determined, and compared between R522K and control Jurkat T cells (I).

30 Results and Discussion

3 RESULTS AND DISCUSSION 3.1 A role for Shb in the early events of TCR signaling (I, II). The T cell response is initiated by the presentation of an antigen to the T cell receptor. This is followed by rapid phosphorylation of three tyrosine-motifs on the TCR ζ-chain called ITAMs. The tyrosine kinase ZAP70 associates with these ITAMs upon T cell engagement, and has then the ability to phosphorylate several downstream targets. It has been shown that Shb also has the ability to associate with the TCR ζ-chain, upon TCR stimulation in Jurkat cells [58] and primary human T cells (Fig 1A), isolated from human blood. Shb associates mainly with the first ITAM (ζ1) but also with the second ITAM (ζ2) of the ζ-chain (Fig 1B), since the association could be inhibited using phospho-peptides corresponding to these sites.

In paper I we decided to evaluate the possibility of TCR-dependent phosphorylation of Shb, since Shb previously has been shown to be phosphorylated in response to NGF, FGF and PDGF stimulation [62-64]. Shb displayed increased tyrosine phosphorylation upon CD3 stimulation in Jurkat T cells. Four putative tyrosine phosphorylation sites in Shb have since been identified (IV), and implied to be targets for the CrkII SH2 domain [65].

Several proteins have been found to associate with Shb upon CD3 stimulation of Jurkat T cells. LAT, PLC-γ1, SLP-76, ZAP70 and Vav all co-immunoprecipitate with Shb. LAT and Vav were shown to associate with the PTB domain of Shb (I, II). The Shb binding sites in LAT and Vav have been identified using phosphopeptides corresponding to different motifs of LAT and Vav proteins. Peptides corresponding to the putative phosphorylation sites Tyr-127 and Tyr-171 of LAT, could efficiently block out the binding between LAT and Shb (I). Other proteins interacting with Tyr-127 in LAT have not yet been reported. Tyr-132, Tyr-171 and Tyr-191 in LAT are binding sites for Grb2 and Gads, while Tyr-171 and Tyr 226 are of importance for SLP-76 and PLC-γ1 interactions with LAT [27]. Tyr-174 in Vav was shown to mediate the binding between Shb and Vav (II). Vav is reported to be phosphorylated by Lck on Tyr-174

31 Results and Discussion

Figure 1. (A) Primary T cells were stimulated (+), or not (-), with CD3 antibody for 2 minutes in 37°C and lysed. Cell extracts were immunoprecipitated using Shb antisera and rabbit serum (NRS). The precipitates were resolved on SDS-PAGE and immuno-blotted with anti-phosphotyrosine (4G10). (B) Jurkat T cells were stimulated (+), or not (-), with CD3 antibody for 2 minutes in 37°C, and lysed. Cell extracts were incubated with the Shb-SH2-GST fusion protein, in the absence or presence of phospho-peptides. The peptides correspond to the ITAMs in the ζ-chain (ζ1, ζ2 and ζ3). The ζ1(p3) and ζ1(p14) are phosphorylated on one tyrosine each. The protein complexes were resolved on SDS-PAGE and blotted with 4G10 anti-phosphotyrosine antibody. (C) Jurkat-R522K and Jurkat-neo cells were CD4 stimulated for 2 minutes in 37°C, and lysed. Cell extracts were immunoprecipitated using anti-Lck antibody, and subjected to an in-vitro kinase (IVK) assay using [γ-32P]ATP and enolase as substrate. Immunoprecipitates and IVK samples were resolved on SDS-PAGE and subjected to Western blotting using 4G10 or autoradiography using hyperfilm MP. The positions of the TCR ζ-chain and Lck are indicated in the figure.

32 Results and Discussion which activates the exchange activity of Vav [43], [44]. However, other reports claim that Tyr- 174 is a possible site for negative regulation of Vav [75], since a Y174F point mutation in Vav, increased both basal Rac activity and NFAT activation [47].

The LAT-peptides could not inhibit the Shb-Vav or Shb-SLP-76 interactions, just as the Vav peptides could not inhibit LAT-Shb or Shb-SLP-76 associations. Three peptides corresponding to tyrosine phosphorylation sites in SLP-76, were also tested. Two of these peptides (Tyr-113 and Tyr 128) correspond to the Vav binding site in SLP-76, but none of these peptides had any effect on SLP-76 or Vav binding to Shb. This leads us to conclude that although some of these proteins have the ability to associate with each other, LAT, Vav and SLP-76 all associate independently to Shb (II).

PLC-γ1, Gads, SLP-76 and ZAP70 also have the ability to associate with Shb in T cells. PLC-γ1 and Gads associate constitutively with the proline-rich domains of Shb, independent of TCR engagement, and this association can not be inhibited using phosphotyrosine (I, II).

The SH2 domain of SLP-76 was found to associate with tyrosine phosphorylated Shb, since a fusion protein consisting of GST coupled to the SLP-76 SH2 domain associated with Shb after CD3-stimulation. The SH2 domain of SLP-76 and Shb co-immunoprecipitated, when overexpressed in COS cells, upon pervanadate treatment. The exact tyrosine in Shb, responsible for the recruitment of SLP-76, has not yet been determined.

ZAP70 was found to associate with both the Shb Pro-PTB fusion protein (containing the PTB domain and two proline rich regions) and the Shb SH2 fusion protein, and these associations were slightly increased after CD3 stimulation. The interaction between the SH2 domain and ZAP70 was completely blocked with phosphotyrosine, whereas binding of ZAP70 to the Pro- PTB fusion protein was only partially inhibited under these conditions. We believe that both

33 Results and Discussion

Figure 2. A model of T cell receptor interactions with Shb. TCR engagement results in recruitment of ZAP70 tyrosine kinase to the TCR, where Shb is also associated. Shb can then associate with LAT, PLC-γ1, Grb2, SLP- 76, Gads and Vav, which are brought into the vicinity of ZAP70 and are thus phosphorylated. This signaling complex can then recruit a number of effectors, leading to activation of the AP-1, NFAT and NF-κB transcription factors, and IL-2 gene transcription. interactions of ZAP70 with the Shb-SH2 domain and the Shb PTB-Proline-rich domain are indirect, the former mediated by the TCR ζ -chain and the latter with components of the Shb signaling complex.

34 Results and Discussion

We have suggested a model for the interactions involving Shb in T cells (Fig. 2). ZAP70 is bound to the ITAMs of the ζ-chain, to which Shb with its associated proteins also is attached, and thus brought in proximity to the tyrosine kinase ZAP70. Shb can thus function as a linker between the LAT-Vav-SLP-76-Gads-PLC-γ1 complex and the TCR ζ chain and ZAP70, and according to this model Shb is vital for the propagation of the signal from the TCR to the nucleus.

3.2 Effects of overexpression of Shb, with a defect SH2 domain, in Jurkat T cells (I, II). Since the SH2 domain of Shb was found to associate with the TCR ζ chain, a Shb mutant, with a defective SH2 domain, was constructed. This mutant Shb, where Arg-522 was replaced with Lysine, was then overexpressed in Jurkat T cells. Cell extracts from mutant clones (Jurkat R522K-2 and –3), showed weaker or no tyrosine phosphorylation in response to TCR stimulation by CD3 crosslinking compared to control cells (I). Several proteins were affected, especially the proteins migrating as 160 kDa, 100 kDa, 66 kDa and 36/38 kDa. These proteins were later identified as PLC-γ1, Vav, SLP-76 and LAT (I, II). As mentioned above, these proteins also associate with Shb in different ways.

To further study the effects of the R522K mutation on PLC-γ1, Vav, SLP-76 and LAT phosphorylation, we performed immunoprecipitations of these proteins in Jurkat-R522K and control cells (I, II). Western blot analyses of these immunoprecipitates revealed in the Shb SH2 defective clone (Jurkat-R522K), a failure of CD3 stimulation to increase the phosphorylation of either PLC-γ1, Vav, SLP-76 or LAT. The same experiment was performed on ZAP70 in these cells, with no detectable difference in stimulation between mutant and control. This is consistent with the view that ZAP70 operates upstream of Shb, whereas PLC-γ1, Vav, SLP-76 and LAT are effectors downstream of Shb in the signaling pathway following TCR engagement. It could also be argued that the negative effects on protein phosphorylation in the Jurkat R522K cells, might be due to perturbed Lck activity. However, CD4-induced Lck activity was not affected in the Jurkat R522K cells, compared to control Jurkat cells (Fig 1C).

35 Results and Discussion

Active PLC-γ1 is known to regulate intracellular Ca2+ levels through inositol phospholipids, and as anticipated, the Jurkat R522K cells displayed an abolished Ca2+ response upon TCR activation (I). Activation of calcineurin and the transcription factor NFAT, in T cells, is dependent on proper Ca2+ signaling.

To assess the effects of the R522K mutation in the Shb SH2 domain, on the activation of the MAP kinases ERK-1, ERK-2 and JNK, upon TCR stimulation, blots from cell extracts of CD3 stimulated and unstimulated Jurkat R522K and control cells, were probed with antibodies that recognize phosphorylated ERK and JNK. The degree of phosphorylation reflects the degree of activation. Jurkat R522K cells displayed a decreased phosphorylation of both ERK (p42 and p44) and JNK, after CD3 stimulation compared to the control cells (I, II). SLP-76 and LAT have both been shown to be involved in ERK activation. It is therefore not surprising that the Jurkat R522K cells, with abolished TCR dependent SLP-76 and LAT phosphorylation, exhibit defects in ERK activation. The observed lack of JNK activation in the Jurkat R522K clone is probably due to the abolished activation of Vav and Rac1 in these cells, since several reports have shown that activated Vav is required for Rac1 mediated JNK activation in lymphoid cells. [76, 77]. The Ras family member Rap1 has also been implicated in TCR signaling but in contrast to Ras, Rap1 signaling promotes an anergic state. When the cells are in this state, even Ras activation fails to induce proliferation. TCR-induced activation of Rap1 was also decreased in the Jurkat R522K cells compared to control cells (unpublished data).

Another mutant of Shb, with a deletion of the PTB domain and the tyrosine phosphorylation- sites, was overexpressed in Jurkat T cells (II). Cell extracts from Jurkat cells transiently transfected with wild-type Shb and the ∆PTB-Tyr mutant of Shb, showed that tyrosine phosphorylation of a number of proteins was decreased in the ∆PTB-Tyr Shb, upon CD3 stimulation, compared to WT Shb. Particularly, decreased tyrosine phosphorylation of proteins corresponding to 160 kDa (PLC-γ1), 75 kDa and 36 kDa (LAT) was observed. The 75 kDa

36 Results and Discussion protein co-migrated exactly with SLP-76. These results indicate that both the SH2 domain and the PTB-Tyr region of Shb, are vital for the phosphorylation of several proteins upon TCR engagement, including SLP-76, PLC-γ1 and LAT.

3.3 Requirement of the Shb SH2 domain for NFAT activation and IL-2 gene transcription (I). It is known that the Ras/MAPK pathway, the Vav/JNK pathway and the Ca2+ pathway cooperate in T cells to activate the IL-2 promoter through the transcription factors NFAT and AP-1. The Ras/MAPK pathway can activate AP-1 and also synergize with the calcium pathway to activate the T cell specific transcription factor NFAT. The Vav/JNK pathway is also important for AP-1 and NFAT activation in TCR activated T cells. To assess if NFAT activation was perturbed in the Jurkat R522K cells, we used a reporter gene construct composed of a triplet of the NFAT binding site from the IL-2 promoter region coupled to a CAT (chloramphenicol acetyltransferase) reporter. This reporter construct was transfected into Jurkat cells together with empty vector or Shb R522K plasmid. The TCR mediated NFAT activation could then be determined, using the ability of CAT to incorporate radioactive carbon into the cell, when the NFAT elements were activated. Jurkat R522K transfected cells display a poor NFAT-mediated induction of IL-2 transcription in response to CD3 crosslinking+TPA, compared to control cells (I). We also examined the endogenous IL-2 levels in Jurkat cells transiently transfected with vector, wild-type Shb or mutant R522K Shb. These cells were labeled with a PE-conjugated anti-IL-2 antibody and sorted by flow cytometry (FACS). We then observed a fourfold increase in IL-2 expression in the vector and wild-type Shb-transfected cells after stimulation with CD3 and TPA, whereas there was no increase in IL-2 production in the R522K Shb transfected cells after CD3+TPA stimulation (I). These results indicate that Shb is involved in the propagation of the signal from the TCR to the IL-2 gene in T cells.

37 Results and Discussion

3.4 Subcellular localization of Shb upon T cell receptor engagement (II). Recent attention has focused on the concentration of effector molecules into subdomains, so called GEMs or lipid rafts, upon receptor stimulation. These membrane subdomains are characterized by their detergent insolubility. It has recently been shown that several proteins involved in TCR signal transduction, including SLP-76, Vav, ZAP-70 and Gads, localize to these lipid rafts, upon TCR stimulation, whereas others are constitutively enriched in these domains, including LAT, Cbl, Lck, Fyn and Ras. To assess the localization of Shb to lipid rafts, Jurkat T cells were left unstimulated or stimulated with anti-CD3 antibody and then lysed in a Triton X-100-based buffer. Lysates were subjected to sucrose density gradient ultracentrifugation to separate the detergent resistant GEMs from the Triton-soluble fractions. Shb was found to localize to the GEM fraction, (fraction 3 and 4) after CD3 stimulation (II). SLP-76, LAT, Vav and ZAP70 were also seen to localize to fraction 3 and 4 upon CD3 stimulation, which is in agreement with what previously has been shown by others [78-81].

We decided to also investigate the localization of R522K Shb, into lipid rafts, upon CD3 stimulation. We then saw that the mutant Shb (R522K) did localize to the lipid rafts, in a CD3- independent manner, and the relative amount of Shb present in the lipid rafts was less than in normal Jurkat cells. This leads us to conclude that Shb is most likely recruited to the lipid rafts by LAT, which exhibits a weak basal degree of phosphorylation, also in the absence of TCR engagement.

3.5 Association of Shb with the IL-2 receptor signaling complex (III). This study describes the interactions between Shb and components of the IL-2 receptor signaling complex. Using fusion proteins, I observed that the Shb SH2 domain had the ability to associate with phospho-proteins reminiscent of the IL-2 receptor β and γ chain upon IL-2 stimulation, in primary T cells and the NK cell line NK-92 (III). To test if these actually were the IL-2Rβ chain, IL-2Rβ was immunoprecipitated followed by dissociation of the complex in SDS and subsequent fusion protein pull-down with the Shb SH2 domain. This showed us that

38 Results and Discussion

Shb does associate directly to the IL-2Rβ chain, since IL-2Rβ bound to Shb, subsequent to lysate denaturation. The results using COS cells, where IL-2Rβ and Shb co-immunoprecipitated upon pervanadate treatment, in the absence of lymphocyte specific proteins, reinforce the view that Shb and the IL-2Rβ chain associate directly.

The cytoplasmic tail of the IL-2 receptor β chain contains six tyrosine residues, four in the acidic region (Y338, Y355, Y358 and Y361), and one in each of the distal segments (Y392 and Y510). To investigate the binding site for Shb in the IL-2 receptor β subunit, I used phospho- peptides corresponding to two tyrosine-motifs in the IL-2Rβ chain, which matched the consensus binding sequence for the Shb SH2 domain (pY-T/V/I-T-L/F, where Leucine in position +3 is the main determinant for Shb SH2 binding). The Shb SH2 domain was found to associate with tyrosine 510, and to some extent with tyrosine 392, in the IL-2R β chain, upon IL-2 stimulation. Tyr-392 and Tyr-510 are also known as the STAT5 binding sites in the IL- 2Rβ chain [82, 83].

Shb and IL-2 receptor β were co-transfected in COS cells, to assess their intracellular association. Shb was also co-transfected with the IL-2Rβ-Y5,6F mutant, with Tyr-392 and Tyr- 510 mutated to Phe. The cells were stimulated, or not, with pervanadate to increase tyrosine phosphorylation of Shb and IL-2Rβ. Shb was then immunoprecipitated and Shb and IL-2Rβ do co-immunoprecipitate, and this association was inhibited by approximately 60%, by the Tyr to Phe mutations in the IL-2R. These results indicate that, even though Tyr-510 and possibly Tyr- 392, in the IL-2Rβ chain, are main docking sites for the Shb SH2 domain, other sites of the receptor might also contribute to Shb-binding.

39 Results and Discussion

Figure 3. A model for the interactions between Shb and components of the IL-2 receptor signaling complex.

JAK1 and JAK3 were also found to associate with Shb, but in contrast to the IL-2 receptor, JAK1 and 3 appear to associate with the proline-rich regions of Shb in T and NK cells. To test if Shb could associate with JAK1 and 3 in the absence of other T or NK cell specific proteins, I co-transfected Shb together with either JAK1 or JAK3 in COS cells. We pervanadate- stimulated these cells and performed immuno-precipitations using anti-Shb sera. Shb was found to co-immunoprecipitate both JAK1 and JAK3, especially upon pervanadate stimulation. Both the co-immunoprecipitation of JAK1 and JAK3 with Shb in the absence of IL-2 receptor and the association of JAK1 and 3 to the proline-rich domains of Shb, indicates that the interaction between JAK1/JAK3 and Shb occurs independently of the IL-2 receptor.

This provides us with a model, where Shb can both aid in binding JAK1 and 3 to the IL-2 receptor, but also aid in recruiting additional signaling proteins to the activated IL-2 receptor complex, possibly Grb2 and PI3K (Fig 3). 3.6 A physiological role for Shb in IL-2 receptor signaling and regulation of apoptosis (III). To elucidate a physiological role for Shb in IL-2 receptor signaling, clones overexpressing Shb and IL-2Rβ in the IL-3 dependent murine pre-B cell line Ba/F3, which expresses the common γ chain endogenously, were established. When these cells are transfected with IL-

40 Results and Discussion

2Rβ, they gain the capacity to proliferate upon IL-2 stimulation, in the absence of IL-3 [84]. Clones expressing wild-type IL-2Rβ together with the mutant R522K Shb (with a non- functional SH2 domain) and clones expressing the mutant IL-2R β Y5,6F (with tyrosines 392 and 510 mutated to phenylalanine) together with wild-type Shb were also established, and these are anticipated to exhibit diminished IL-2Rβ-Shb interactions.

We have previously observed tyrosine phosphorylation of Shb in response to FGF, NGF, PDGF [Claesson-Welsh, 1998 #30; Karlsson, 1998 #13; Dixelius, 2000 #76; Hooshmand- Rad, 2000 #72] and TCR stimulation (I). I therefore decided to evaluate the possibility of IL-2 dependent phosphorylation of Shb in these pre-B cells, which revealed an increased tyrosine phosphorylation of p55 and p67 Shb upon IL-2 stimulation only in the clone expressing WT Shb and WT IL-2Rβ. No stimulatory effect of IL-2 could be detected in the clone expressing WT-Shb together with IL-2Rβ with Tyr 392 and 510 mutated to Phe (Y5,6F), or in the clone expressing WT-IL-2Rβ together with R522K-Shb.

Since Shb and its homologues previously have been implicated in the regulation of apoptosis, we decided to investigate if Shb could be involved in IL-2 receptor mediated survival/apoptosis. Two independent clones each of the Ba/F3 cells expressing R522K- Shb+IL-2Rβ, WT-Shb+IL-2Rβ-Y5,6F and WT-Shb+IL-2Rβ, were grown in the absence of IL-3, but in the presence of IL-2. The cells were harvested on day 5 and day 9 after IL-2 addition, and stained with annexin (as an indication of apoptosis) and propidium iodide. Flow cytometry revealed that the frequency of apoptosis is significantly lower in the cells

41 Results and Discussion

Figure 4. A model for the dual role of Shb in T cell signaling. Shb is involved in T cell receptor signaling as well as IL-2 signaling in T lymphocytes. overexpressing WT-Shb+WT-IL-2Rβ compared to the cells overexpressing the mutant Shb+WT-IL-2Rβ or the mutant IL-2Rβ+WT-Shb, after 5 and 9 days of culture in IL-2 containing media. These results indicate that Shb-IL-2 receptor interactions participate in the regulation of apoptosis subsequent to IL-2 stimulation.

These findings indicate a dual role for Shb in T cells, where Shb is involved in both the T cell receptor and the IL-2 receptor signaling complexes (see figure 4). Other proteins reported to be involved downstream of both the TCR and the IL-2 receptor include: Grb2, Shc, PI3K and Lck. Grb2 and Shc form a complex with each other and are known to activate the Ras/ERK pathway. Shb has also been shown to associate with Grb2 and p85 PI3K [57, 58] and might be an additional component of this signaling complex.

42 Results and Discussion

Figure 5. (A) Schematic view of Shb and Shf with their binding domains. (B) Comparison of the putative tyrosine phosphorylation sites, and the SH2 domains of Shf, Shb and Shd.

3.7 A role for the Shf adapter protein in PDGF-receptor signaling and cell survival (IV).

A small adapter protein of 53 kDa, migrating as 64 kDa on SDS-PAGE, with sequence similarities to Shb, was found using database searches and named Shf (IV). Shf is mainly expressed in skeletal muscle, brain, liver, prostate, testis, ovary, small intestine and colon. Shf contains an SH2 domain and four putative tyrosine phosphorylation sites (Fig. 5). Both the SH2 domain and the tyrosine phosphorylation sites are conserved between Shf, Shb and Shd (another adapter protein of the Shb family). The consensus sequence for the tyrosine phosphorylation sites are: Y-X-(D/E/Q/T)-P-(Y/F/W)-(E/D). Proteins with SH2 domains that could bind these peptide sequences include: Abl, Crk, Nck and RasGAP [25, 66]. We have

43 Results and Discussion also seen that Shf is strongly tyrosine phosphorylated in NIH3T3 cells, if we inhibit dephosphorylation with pervanadate, but we see no basal phosphorylation and no detectable PDGF-AA induced phosphorylation of Shf, in these cells. The SH2 domain of Shf was found to bind to tyrosine-720 in the PDGF-α-receptor but not to the PDGF-β-receptor in PAE cells. Tyr-720 in the PDGF-α-receptor also has the ability to recruit Shb. It is therefore possible that Shb and Shf compete for this binding site in the PDGF receptor if simultaneously expressed.

Fibroblasts overexpressing Shf increased in cell number faster than control cells when grown in the presence of PDGF-AA. We speculated that this might be due to a decrease in apoptosis. Fibroblasts overexpressing Shf did indeed display significantly lower rates of apoptosis than control cells. We have speculated above that Shf might compete with the related adapter Shb. It has previously been shown that overexpression of Shb in NIH3T3 cells increases apoptosis and decreases cell proliferation upon serum-starvation. It is therefore possible that Shf and Shb can regulate downstream signaling events by competing for the same binding site on the PDGF-α-receptor. The PTB-domain and proline-rich domain of Shb may interact with downstream targets and since Shf can not bind these targets, but possibly others shared with Shb via its tyrosine phosphorylation sites, the cellular response will be different upon PDGF- receptor stimulation depending on the relative expression of the two proteins. For example, overexpression of Shf did not alter PDGF- or pervanadate-induced activation of ERK1, ERK2 or JNK (unpublished data). However, overexpression of Shb in PhB cells (patch mice fibroblasts), diminished PDGF-BB dependent Rac1 activation [64]. If this effect is due to binding of Shb to activated Vav-2, possibly targeting Vav-2 away from Rac1, is still unknown. However, it is not surprising that Shf does not display these effects, since it lacks the Shb PTB domain. Overexpression of Shb in NIH-3T3 cells, did not effect PDGF-induced activation of ERK1 and 2 [57]. In conclusion, our results suggests a role for the Shf adapter in PDGF-receptor signaling and regulation of apoptosis.

44 Conclusions

4 CONCLUSIONS

ƒ Shb is vital for the transduction of the signal from the T cell receptor to the IL-2 gene in T cells, by associating with LAT, PLC-γ1, Vav, SLP-76, ZAP70, Gads and the ζ-chain of the T cell receptor (I, II).

ƒ The Shb SH2 domain is vital for T cell receptor induced phosphorylation of LAT, PLC- γ1, Vav and SLP-76 and for release of Ca2+ from intracellular stores, activation of the MAP kinases ERK1, ERK2 and JNK and also for NFAT activation and IL-2 production in Jurkat T cells (I, II).

ƒ Shb was found to localize to the membrane subdomains referred to as ”lipid rafts” after T cell receptor engagement in Jurkat T cells (II).

ƒ The Shb SH2 domain has the ability to associate with both the IL-2 receptor β and γ chain upon IL-2 stimulation, in primary T cells and the NK cell line NK-92. JAK1 and JAK3 were also found to associate with Shb, in T and NK cells, however, the interaction between JAK1/JAK3 and Shb occurs independently of the Shb/IL-2 receptor interaction (III).

ƒ Overexpression of Shb and IL-2Rβ in the pre-B cell line Ba/F3 that expresses the γ chain constitutively, revealed an IL-2 induced tyrosine phosphorylation of Shb. These cells were also less apoptotic in the presence of IL-2 than cells overexpressing Shb or IL-2Rβ mutants, inhibiting the IL-2Rβ-Shb interaction (III).

ƒ The Shb homologue, Shf, has the ability to associate with the PDGF receptor, and regulate apoptosis when overexpressed in NIH3T3 cells (IV).

45 Acknowledgements

ACKNOWLEDGEMENTS My study was carried out at the Department of Medical Cell Biology, Uppsala University. I am very grateful for having had the opportunity to work in the stimulating environment of this department, with all these nice and knowledgeable people around me. I would especially like to thank the following people:

Professor Michael Welsh. The best supervisor anyone could ever wish for, and without whom this thesis would never have been written. Thank you for sharing some of your vast knowledge of signal transduction, both in theory and in the lab. Special thanks for never being impatient when I asked all those, sometimes silly, questions, and never giving up when things didn’t go our way, but always helping me to figure out a new strategy when everything seemed hopeless. And also, thank you, Michael and Lena, for taking such good care of me in Boston.

Professor Arne Andersson. Thank you for your continuous support, and for arranging all those nice pasta-lunches.

Professor Gottfried Romans. Thank you for your support, and for always taking the time to help me, even though I know that your schedule is tight.

My co-authors, J. Daniel Frantz, Steven E. Shoelson, Eric Gylfe, Weiguo Zhang, Lawrence E Samelson, Maria L Henriksson and Bengt Hallberg, for fruitful collaborations.

Dr Doreen Cantrell, for providing the ζ-chain phospho-peptides.

Håkan Borg, Carina Carlsson, P-O Carlsson, Ulf Eriksson, Claes Hellerström, Leif Jansson, Stellan Sandler and Nils Welsh. Thank you all for your pleasant company, interesting discussions, good advice and encouragement.

My room mates, Cecilia Anneren, Jonas Cederberg, Peppi, Rickard Olsson and Parri Wentzel. Thank you for good company and the nice conversations.

All the PhD students of the department, both former and present. Thanks for all the good times, your friendship, your support, and for creating such a good atmosphere at the department.

Ing-Marie Mörsare and Ing-Britt Hallgren. Thank you for excellent technical assistance.

Göran Ståhl. Thank you so very much for helping me with all those dreadful technical things, like fixing my pocket calculator or helping me put the chain back on my bicycle.

All the members of the Department of Medical Cell Biology, past and present. Thank you for being so nice and contributing to the nice atmosphere of the department.

Mamma och Pappa. Tack för att ni har stöttat mig hela vägen, både i skolan och hemma. Tack mamma, för att du väckte mitt sy-intresse med alla fina dockkläder som du sydde åt mig, och pappa, för mitt serie-intresse som vi grundmurade med Kalle Anka varje vecka.

46 Acknowledgements

Min ystra syster Mille, som är min bästa vän, såväl som min syster.

Min pojkvän Håkan, som vet att vetenskap är stort…och som jag tycker väldigt mycket om.

Mina goda vänner, Anne Kiuru, Leif Rob Eriksson, Katarina Sewerin, Nene Lundström, Åsa Karlström, Carina Gunnarsson och Natalja Fredriksson. Tack för att ni alltid ställer upp och är så bra kompisar.

Tolkiensällskapet. Tack för att ni finns och förgyller min tillvaro.

Alla mina andra goda vänner och bekanta, tack för allt roligt vi har haft och för att ni finns i mitt liv.

During my Ph.D. studies I have received financial support from the Liljewalchs fund, Rektors-fonden, Kungliga Vetenskapssamhällets forskningsstipendier, Uppsala Läkareförening and Sven Brolins fund.

My work has been supported by grants from the Juvenile Diabetes Foundation International, Swedish Medical Research Council, the Swedish Diabetes Association, the Novo-nordisk Foundation and the Family Ernfors Fund.

47 References

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