Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1318
The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells
BY KRISTINA HOLMQVIST
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1 This thesis is based on the following papers, which will be referred to by their Roman numerals:
I Lingge Lu, Kristina Holmqvist, Michael Cross and MichaelWelsh. Role of the Src Homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation. Cell Growth Differ 2002;13:141-148.
II Kristina Holmqvist, Michael Cross, Debbie Riley and Michael Welsh. The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells. Cell Signal 2003;15:171-179.
III Kristina Holmqvist, Michael Cross, Robert Hägerkvist, Nader Rahimi and Michael Welsh. The Shb adaptor protein binds to tyrosine 1175 in the VEGFR-2 and regulates FAK activity, stress fiber formation and cellular migration. Submitted.
Reprints were made with the permission of the publishers.
2 TABLE OF CONTENTS
ABSTRACT
LIST OF PUBLICATIONS 2
TABLE OF CONTENTS 3
ABBREVIATIONS 5
INTRODUCTION 7 Angiogenesis 7 Receptor tyrosine kinases 9 FGFR-1 signalling 10 VEGFR-2 signalling 11 Shb 13 FAK 16 Src 18 Cytoskeleton 19 RNA interference 21
AIMS 22
METHODOLOGY 23 Cells 23 Protein assays; Immunoprecipitation 23 Western blot analysis 24 Fusion proteins, peptides and pull-down experiments 24 Rac1 and Rap1 assay 25 Rho assay 25 DNA synthesis 25 Proliferation and Viability 26 siRNA 26 Physiological assays; Tube formation 27 Morphology assay 27 Actin staining 27 Migration 28 Statistical analysis 28
3 RESULTS AND DISCUSSION 30 Paper I; The role of Shb in endothelial cell proliferation and differentiation. 30 Shb is necessary for tubular morphogenesis. 31
Paper II; Shb causes Src-dependent cell spreading and activation of FAK. 32 Shb and the downstream signalling of FGFR-1. 33
Paper III; Shb interacts with tyrosine 1175 in the VEGFR-2. 34 Shb regulates FAK activity, stress fiber formation and migration in VEGFR-2. 36
CONCLUSIONS 39
FUTURE PERSPECTIVES 40
ACKNOWLEDGEMENTS 41
REFERENCES 44
ORIGINAL PAPER 52
4 ABBREVIATIONS bFGF basic fibroblast growth factor (FGF-2) BSA Bovine serum albumin CHO Chinese hamster ovary CADTK Calcium dependent protein tyrosine kinase (Pyk2) CAK Cell adhesion kinase (Pyk2) cDNA complementary deoxyribonucleic acid CSF Colony stimulating factor CSK c-Src tyrosine kinase dsRNA double stranded RNA ECL Enhanced chemiluminescence ECM Extra cellular matrix EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor FAK Focal adhesion kinase FAT Focal adhesion targeting FCS Fetal calf serum FGF-2 Fibroblast growth factor 2 (bFGF) FGFR Fibroblast growth factor receptor FRNK FAK related non kinase GAP GTPase activating proteins GDI Guanine nucleotide dissociation inhibitor GEF Guanine exchange factor GPCR G-protein coupled receptor GST Glutatione-S-transferase HIF Hypoxia inducible factor HRE Hypoxia response element HRP Horseradish peroxidase HSPG Heparan sulphate proteoglycans IB Immunoblotting IBE cell Immortomouse brain endothelial cell IFN-J Interferon J IL-2R Interleukin 2 receptor IP Immunoprecipitation IPTG Isopropyl-thiogalaktoside LAT Linker for activation of T cell LPA Lysophosphatidic acid MAPK Mitogen activating protein kinase MLCK Myosin light chain kinase MMP Matrix metalloprotease NGF Nerve growth factor PAE Porcine aortic endothelial
5 PBS Phosphate buffered saline PDGF Platelet-derived growth factor PlGF Placenta growth factor PRNK Pyk2 related non kinase PTB Phosphotyrosine binding RAFTK Related adhesion focal tyrosine kinase (Pyk2) Rho Ras homologous RISC RNA induced silencing complex RNAi RNA interference RTK Receptor tyrosine kinase SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SH2/3 Src homology 2/3 SI/II Site I/II siRNA short interfering RNA SMC Smooth muscle cell TCR T-cell receptor TBS Tris buffered saline TBS-T Tris buffered saline-tween 20 TIME Telomerase immortalised microvascular endothelial TNFD Tumour necrosis factor D ts temperature sensitive VEGF Vascular endothelial growth factor VPF Vascular permeability factor (VEGF) VRAP VEGFR-associated protein
6 INTRODUCTION
Angiogenesis
Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels (Folkman et al., 1996, Risau et al., 1997). Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. This leads to receptor activation, an increase in the intrinsic tyrosine kinase activity and phosphorylation of specific intracellular tyrosine residues. As a consequence, certain signalling molecules bind to the phosphorylated tyrosines, and this initiates activation of downstream signalling cascades, leading to transcriptional changes and ultimately biological responses.
Tumour cells secrete angiogenic factors, such as FGF and VEGF, that stimulate angiogenesis, facilitating the expansion of the tumour. VEGF and FGF can act synergistically in inducing angiogenesis (Stavri et al., 1995). The endothelial cells will, as a result of receptor activation, secrete proteases such as matrix metalloproteases (MMP) and plasminogen activators, leading to digestion of the basement membrane. This allows the endothelial cells to invade the surrounding tissue, where they migrate and proliferate to create a sprout (Fig. 1). The sprout elongates and the endothelial cells differentiate to form a functional lumen. The endothelial cells, in the newly formed vessel, will eventually start to secrete platelet-derived growth factor (PDGF-BB), which recruits supporting mesenchymal cells, that will differentiate into pericytes. This is critical for the stability of the vessel (Hirschi et al., 1997). Development of abnormal capillaries in pericyte-deficient PDGF-B -/- embryos shows that pericytes regulate microvessel structure (Lindahl et al., 1997). Physiological angiogenesis is restricted to embryonic development, ovulation and wound healing and in these cases the process is characterised by strict regulation.
7 Fig.1 Tumour cells
ECM
Migrat ion/ Proliferat ion & different iat ion Prot eolysis Angiogenic factors e.g. FGF & VEGF
Blood vessel Pericyt e/ Endothelial Cell Smoot h muscle cell
Figure 1. The process of angiogenesis in a tumour environment.
However, in pathological conditions, such as arthritis, growth of tumours and in the formation of metastasis, vessel formation is exaggerated and the vessels may be non-functional and lacking supporting pericytes. This leads to leakage from the vessel, explaining the oedematous nature of most tumours. Already in 1971, Folkman suggested that a tumour requires blood supply in order to grow beyond a few cubic millimeters and thereby predicted, if pathological angiogenesis is inhibited, there will be a reduction of tumour growth and metastasis (Folkman et al., 1972). Today, a wealth of data supports this theory (O’Reilly et al., 1997, Boehm et al., 1997).
The angiogenic switch, is triggered as a result of a shift in the balance between angiogenesis stimulators, bFGF and VEGF, and endogenous inhibitors, e.g. angiostatin and endostatin (Hanahan et al., 1996). Angiogenesis will be initiated when there is a change in the balance between activators and inhibitors, e.g. a reduction of inhibitor concentrations, by loss of tumour suppressor genes, or an increase in the levels of stimulators, by induction of VEGF, caused by hypoxia. In the absence of inducers or when the inducers are present but suppressed by higher levels of angiogenesis inhibitors the switch is off. Weidner et al., 1991 were the first to reveal a direct relationship
8 between metastasis and angiogenesis, in which they showed that a higher degree of angiogenesis in a primary tumour correlates with poor prognosis.
Receptor tyrosine kinases
Receptor tyrosine kinases (RTK) consist of an extracellular ligand binding domain, a transmembrane region and an intracellular tyrosine kinase domain. FGFR-1 and VEGF-2 are examples of subgroups from the RTK family. Upon ligand binding, the receptor will dimerize and induce phosphorylation of several tyrosines in the intracellular part, which will create docking sites for different signalling molecules containing specific motifs e.g. Src homology 2 (SH2) or phosphotyrosine binding (PTB) domain. The amino acid sequence adjacent to the phosphorylated tyrosine regulates binding specificity. Only a limited number of signalling proteins will have the right three-dimensional structure to bind to each specific site. The binding of one of these molecules will lead to downstream signalling and physiological responses.
Signalling molecules can have different roles in the cell e.g. enzymes (kinases, phosphatases and phospholipases) or adaptors (proteins that bind to many signalling molecules and cause complex formation). Adaptor proteins lack enzymatic activity, instead they have several protein interaction domains and can therefore function as linkers between different proteins. There are a large number of different binding domains. In this thesis, I have mainly focused on SH2-, SH3- and PTB-domains, where SH stands for Src Homology and PTB for phosphotyrosine binding domains. SH2 domains have the ability to bind to phosphorylated tyrosines, which are followed by a short amino acid motif, unique for every SH2 domain (Songyang et al., 1993). The SH3 domains associate with proline-rich sequences including the consensus motif PxxP whereas the PTB domains interact with phosphorylated tyrosines that have a short unique amino acid motif upstream of the tyrosine.
9 FGFR-1 signalling
The first identified pro-angiogenic molecule was FGF-2 (bFGF) (Shing et al., 1984). Within the FGF family, there are at least 20 factors, which share 30-70% identity in their amino acid sequence. The ligands (FGFs) will bind with high affinity to heparan sulphate proteoglycans (HSPG), which act as co-receptors and are located on the surface of endothelial cells and in the extracellular matrix. The extracellular part of the FGFR-1 consists of three immunoglobulin- like domains and together with the HSPG, they provide a binding site for the ligands. Heparin or heparan sulphates help to stabilise a complex constituting of two FGFs and two FGFRs, resulting in receptor dimerization and autophosphorylation of specific tyrosines, located in the intracellular part of the receptor (Plotnikov et al., 1999). A number of autophosphorylation sites have been identified in the FGFR-1. Activation of FGFR-1 can lead to different cellular responses e.g. differentiation, migration, survival or proliferation depending on the specific set of signalling molecules that binds to the activated tyrosines in the receptor.
There are four different FGF receptors (FGFR-1, -2 , -3 and - 4), that share a high degree of similarity. Furthermore, receptor variants have been generated through alternative splicing of FGFR mRNA (Jaye et al 1992, Johnson et al., 1993). Gene inactivation of FGFR-1 and FGFR-2 leads to embryonal death before gastrulation (Deng et al., 1994, Xu et al., 1998). This demonstrates the importance of these receptors in the development of embryos. By use of adenovirus-mediated expression of dominant negative FGFR-1, Lee and co-workers showed that FGFR-1 is required for development and maintenance of the vasculature (Lee et al., 2000).
However, gene inactivation of FGF-2 leads to mice with morphologically normal vasculature with the exception of low blood pressure, suggesting redundancy among the ligands (Dono et al., 1998). FGFR-1 is widely expressed and has been shown to be important for the development of the central nervous system and the skeleton (Ford-Perriss et al., 2001).
10 VEGFR-2 signalling
VEGF was first identified as vascular permeability factor (VPF) because of its ability to cause vascular leakage (Senger et al., 1983). VEGF is one of the most potent angiogenic factors. The VEGF family includes; VEGF-A or VPF, VEGF-B, VEGF-C, VEGF-D, VEGF-E and also the placenta growth factor (PlGF). Alternative exon splicing of the human VEGF-A gene gives rise to several isoforms including VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206, of which VEGF165 is the predominant form. The isoforms differ in their ability to bind heparan sulphate and the extracellular matrix (ECM).
VEGF expression is upregulated in conditions such as hypoxia, where the stability of hypoxia inducible factor-1 (HIF-1) is increased and will bind specifically to the hypoxia response element (HRE) in the promotor region of the VEGF gene (reviewed in Harris et al., 2000). Factors that can elevate VEGF receptor expression include VEGF and FGF (Shen et al., 1998, Barleon et al., 1997). The VEGF family members can bind to three structurally related receptor tyrosine kinase (RTK); VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4) (Matsumoto et al., 2001). VEGFR-1 is expressed on haematopoietic stem cells, macrophages, monocytes and vascular endothelium. VEGFR-2 is expressed on both vascular endothelium and on lymphatic endothelium, whereas the expression of VEGFR-3 is restricted to the lymphatic endothelium.
PlGF, VEGF-A and VEGF-B can bind to VEGFR-1, while VEGF-A, VEGF-C, VEGF-D and VEGF-E can bind to VEGFR-2. Only VEGF-C and VEGF-D have the ability to bind to VEGFR-3 (Fig. 2.). VEGF-A has a higher affinity for VEGFR-1 than VEGFR-2. However, the in vitro kinase activity of VEGFR-1 is much lower than that of VEGFR-2, possibly explaining the few cellular responses found after VEGFR-1 activation in endothelial cells. VEGF-A exerts its physiological effects i.e. proliferation, survival, migration and permeability through VEGFR-2 in endothelial cells (Cross et al., 2001).
11 S S S S
soluble VEGFR-1
VEGFR-1 VEGFR-1 VEGFR-2 VEGFR-2 VEGFR-3
Endothelial cell
Basement membrane
HEMATOPOIETIC Pericyte STEM CELL MACROPHAGE Erythrocyte MONOCYTE VASCULAR ENDOTHELIUM LYMPHATIC ENDOTHELIUM
Figure 2. Overview of VEGF ligands, receptors and their expression. Figure adapted from Cross et al., 2003.
The VEGFR-1 gene codes for two products, a full length VEGFR-1 and a soluble form lacking the transmembrane and intracellular parts of the receptor. Since VEGF-A can bind both forms of the VEGFR-1, it has been suggested that the soluble receptor acts as decoy receptor preventing the VEGF-A from binding and activating VEGFR-2 (Shibuya et al., 2001). Also VEGFR-1 has been suggested to act as a decoy receptor by sequestering the ligand (Waltenberger et al., 1994).
Gene inactivation experiments have demonstrated the importance of both VEGFR-1 and VEGFR-2 in the development of the vasculature, since they lead to embryonal death at day 8.5 to 9.5 (Fong et al., 1995, Shalaby et al., 1995). In mice lacking VEGFR-1 there is an increase of haemangioblasts, the endothelial cell progenitors, leading to non-functional vessels. However, in the VEGFR-2 knock-out embryos the haemangioblasts fail to differentiate into endothelial cells, which could not assemble into blood vessels. Targeted inactivation of the VEGFR-3 gene leads to embryonal death at day 9.5, due to deficient blood and lymphatic vessel remodelling of 12 the embryo (Dumont et al., 1998). In the adult, the expression pattern of VEGFR-3 is mainly restricted to the lymphatic vasculature. However, some quiescent blood capillaries and angiogenic blood endothelial cells can express VEGFR-3 (Witmer et al., 2002). The main role of VEGFR-3 is in the development and function of the lymphatic system (Karkkainen et al., 2002).
All three of the VEGF receptors consist of an extracellular domain containing seven immunoglobulin-like structures, a transmembrane domain, a kinase domain with a kinase insert and a cytoplasmatic tail. VEGF-A binds to the second and third IgG loop of the VEGFR-2 initiating tyrosine phosphorylation of the known autophosphorylation sites in the intracellular part of the receptor; Tyr 951, 996, 1054, 1059, 1175 and 1214. VEGFR-associated protein (VRAP) has been shown to bind to Tyr 951 (Wu et al., 2000), while the adaptor protein Sck and the enzyme PLC-J can bind Tyr 1175 (Warner et al., 2000, Takahashi et al., 2001).
Shb
The adaptor protein Shb, was first identified in the insulin producing ETC-1 cell line by G1/G0 substraction screening. The ubiquitously expressed Shb protein exists in two isoforms, 56 and 67 kDa, probably because the Shb cDNA contains two methionine codons in the N-terminal part and can therefore code for two proteins of different sizes. (Welsh et al., 1994). This adaptor protein contains several domains, with different properties (Fig. 3);
* an SH2 domain, located in the C-terminus, that interacts with phospho-specific tyrosine residues in different receptors, such as FGFR-1, PDGFR-D and -E, TcR]-chain and IL-2R E- and J chains (Karlsson et al., 1995, Welsh et al., 1998, Lindholm et al., 1999, Lindholm, 2002).
13 * four central putative phosphorylation sites, that can interact with Crk II (Lu et al., 2000), SLP-76 (Lindholm et al., 2002) and RasGAP (Annerén et al., 2003).
* a PTB domain, that interacts with phospho-tyrosine residues in proteins like Linker for Activation of T cells (LAT) (Lindholm et al., 1999) and Vav-1 (Lindholm, 2002).
* two to five proline-rich domains, that can interact with the SH3 domains of Grb2, Eps8, v-src and p85D PI3-kinase, PLC-J (Karlsson et al., 1997, Welsh et al., 1998 and Lindholm et al., 1999).
64 kDa 56 kDa Y333 Y384
N P1 P2 P3 P4 P5 PTB SH2 C
Y355 Y423
Grb2 P 36/38 (LAT)Crk II PDGFR p85 PI3-K Vav SLP-76 FGFR-1 PLC-γ RasGAP TCR Src IL-2R Eps 8 JAK 1/2
Figure 3. Structure of the Shb protein.
The human Shb gene is located in chromosome 9 (Yulug et al., 1994). The Shb cDNA consists of six exons. The first exon corresponds to the proline-rich motifs and the PTB domain, while the second and third exons contain the putative phosphorylation sites. The fourth exon is a short linker region, while the fifth and sixth exons correspond to the SH2 domain.
14 Several factors including; PDGF, NGF, FGF, CD3 and IL-2, can induce phosphorylation (activation) of Shb (Hooshmand-Rad et al., 2000, Karlsson et al., 1998, Lindholm et al., 1999, Lindholm 2002). The endogenous angiogenesis inhibitors; Angiostatin and Endostatin have opposing effects; Angiostatin inhibits, while Endostatin potentiates Shb phosphorylation (Claesson-Welsh et al., 1998, Dixelius et al., 2000). The Src-like kinase FRK/RAK, also known as GTK, has the ability to induce phosphorylation of Shb (Annerén et al., 2000).
Shb is also known to be involved in T-cell receptor signalling by generating multi-protein signalling complexes that associate with the TCR (Welsh et al., 1998, Lindholm et al., 1999). Overexpression of Shb induces c-myc independent apoptosis of cells maintained in low serum (Karlsson et al., 1996). This adaptor protein affects differentiation of neural cells through transmitting bFGF- and NGF- dependent differentiation signals in PC12 cells (Karlsson et al., 1998, Lu et al., 2000). In transgenic mice expressing Shb, controlled by rat insulin promotor, the E cell formation at late embryonal stages is increased, as is also the E cell death, under stressful conditions, which may contribute to E cell destruction in type 1 diabetes (Welsh et al., 1999).
Furthermore, Shb plays a role in growth factor induced cytoskeletal rearrangements. In Patch mouse fibroblasts, PDGF- stimulation leads to decreased membrane ruffle formation and generation of filopodia in Shb overexpressing cells but not in SH2- domain mutant cells compared with control cells (Hooshmand-Rad et al., 2000). Shb is also involved in angiogenesis. The anti-angiogenic factors angiostatin and endostatin as well as the angiogenic growth factors FGF and PDGF signal through Shb. Another study revealed that Shb binds to tyrosine 766 in FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation (Cross et al., 2002).
15 FAK
Focal adhesion kinase (FAK) is a 125 kDa non-receptor tyrosine kinase, which localises in focal adhesions upon agonist and integrin-mediated stimulation (Schaller et al., 1992). Integrins are a family of D and E heterodimeric transmembrane glycoproteins, which are responsible for binding to components of the extracellular matrix (ECM). Upon ECM binding to their extracellular domain, integrins associate in clusters at the cell membrane ultimately leading to the formation of focal adhesions (Burridge et al., 1988). FAK is associated with focal adhesions and is phosphorylated by both integrin-mediated cell adhesion and by a number of ligands binding to their cognate receptor tyrosine kinases or via G-protein coupled receptors (GPCR). Hence multiple stimuli converge at the level of FAK phosphorylation.
FAK is expressed in most cells and tissues. Studies have shown that FAK is also very conserved between different species (Hanks et al., 1992). FAK has several tyrosine phosphorylation sites that upon activation, can interact with a number of molecules leading to downstream signalling and cellular responses (Fig.4). Tyrosine 397 is the major autophosphorylation site, while tyrosine 576 and 577 are present in the catalytical part of FAK and necessary for its maximal activity (Calalb et al., 1995). Phosphorylation of tyrosine 397 creates a high affinity binding site for the SH2 domain of Src family kinases leading to their recruitment and activation (Cobb et al., 1994). This site is also important for the recruitment of other SH2 containing proteins including the p85 subunit of phosphoinositide 3-kinase (PI3 kinase), PLC-J and Grb7.
Tyrosine 861 and 925 are localised in the focal adhesion targeting (FAT) sequence, consisting of approximately 100 amino acids, in the C-terminus of FAK (Hildebrand et al., 1993). Paxillin, a focal adhesion containing protein, has been shown to bind to the FAT sequence (Hildebrand et al., 1995). The C-terminal part of FAK contains several protein-protein interaction sites. There are two proline rich regions, denoted Site I (SI), which provides a binding site for the SH3 domain of Cas, and Site II, which is the interaction site for the SH3-domains of two regulators of small GTPases, GRAF and ASAPI. FAK also contains four recently mapped serine 16 phosphorylation sites (S 722, 843, 846 and 910) of which three of them have an unknown function. Cdk5-induced FAK phosphorylation at Serine 732, in neural cells, is important for microtubule organisation, nuclear movement and neuronal migration (Xie et al., 2003). The cytoplasmatic domain of E-integrin has been reported to interact with the N-terminal region of FAK (Schaller et al., 1995).
SH3 Cas ASAP/GRAF SH3
SH2 SH3 Src Growth factor receptors Paxillin Integrins 51 511 PxxP718 PxxP881
Y 397 Y576 Y577 Y861 Y925
S845 FAT N S722 S843 S922 C
Cytoplasmatic Catalytic FRNK interactions domain
Figure 4. Overview of FAK. Adapted from Parsons 2003.
Another member of the FAK family is Pyk2 also known as cell adhesion kinase (CAK) beta and related adhesion focal tyrosine kinase (RAFTK) or calcium dependent protein tyrosine kinase (CADTK). Pyk2 shares a high degree of similarity with FAK, but has a more restricted expression, located to the central nervous system and hematopoetic cells (Avraham et al., 2000). The non-catalytic C- terminal part of FAK and Pyk2 is denoted FAK related non kinase (FRNK) and Pyk2 related non kinase (PRNK). It has been suggested that they act as negative regulators (Nolan et al., 1999, Xiong et al., 1998). FAK is essential for embryonic development since in mice, the homozygous FAK knockout is embryonically lethal (Ilic et al., 1995). Fibroblasts derived from these mice display poor spreading and migration, suggesting that FAK plays a role in these processes. Furthermore, overexpression of FRNK blocks cell spreading and haptotactic and chemotactic migration (Richardson et al., 1997). Overexpression of FAK in Chinese hamster ovary (CHO) cells results in increased cell migration (Cary et al., 1996). Retransfection of FAK 17 in FAK-deficient cells restored migration, while reintroducing the FAK Y397 mutant failed to restore haptotactic migration (Owen et al., 1999, Sieg et al., 1999, Cary et al., 1998). It is now becoming clear that FAK is upregulated in a number of epithelial cancers, where its expression correlates with the invasiveness of the tumour (McLean et al., 2003).
Focal adhesions are cellular structures that link the extracellular matrix on the outside of the cell, through integrin receptors, to the actin cytoskeleton inside the cell. A large number of proteins can associate in these complex structures, including FAK, paxillin, vinculin, Src, D-actinin, talin, VASP, ERK, PAK and many more, leading to downstream signalling (reviewed in Parsons 2003). Whilst there is a wealth of information detailing the signalling pathways downstream of FAK, the pathways responsible for direct activation of FAK are poorly understood.
Src
The first identified oncogene was viral Src (v-Src), which is encoded by Rous sarcoma virus (Hunter et al., 1980). The Src family of protein tyrosine kinases consists of several members including Src, Yes, Fyn, Lck, Blk, Lyn, Hck, Yrk and Fgr. Src, Yes and Fyn are ubiquitously expressed in mammalian cells, while the other Src family members have a more restricted expression pattern. The Src family members are 52-62 kDa proteins that contain an N-terminal consensus myristoylation motif, an SH3 domain, an SH2 domain and a kinase domain followed by a C-terminal tail with regulatory elements (reviewed in Schlessinger 2000). In order for Src to associate with the plasma membrane the amino terminal part has to be myristyolated. Src is localised in the cytoplasm in focal adhesions, in fibroblasts, and in cell-cell contacts in epithelial cells. The SH3 domain has the ability to bind to proline-rich sequences, whereas the SH2 domain interacts with specific phospho-tyrosines.
The SH2 domain of Src has been shown to bind directly to tyrosine 397 in FAK and is thought to lead to the phosphorylation of 18 FAK on a number of carboxy terminal sites including tyrosines; 407, 576, 577, 861 and 925 (Schaller et al., 1994). Both v-Src, and its cellular homologue c-Src, have the ability to modulate the actin cytoskeleton and cell adhesions, by phosphorylating substrates, within the focal adhesion, that induce adhesion turnover and actin remodelling (Frame et al., 2002). One major difference between v-Src and c-Src is the lack of the negative regulatory site, Tyr 527 in v-Src.
c-Src tyrosine kinase (CSK) is a ubiquitously expressed enzyme structurally related to c-Src, which phosphorylates Src at Tyr 527, that negatively regulates the kinase activity of Src by inducing a closed conformation. Maximal catalytic activity of Src requires phosphorylation of its autophosphorylation site, tyrosine 416. The SH2 and SH3 domains play an important role in regulating the kinase activity. The SH2 domain interact intermolecularly with the phosphorylated tyrosine 527 and the SH3 domain with a polyproline helical structure between the SH2- and the kinase domain, locking Src in an inactive conformation. Dephosphorylation of Tyr 527 induces activation of Src. In summary, Src is activated when Tyr 527 is unphosphorylated and Tyr 416 is phosphorylated. Src has been implied in a variety of processes, including cancer and metastasis (Russello et al., 2003).
Cytoskeleton
Several factors have the ability to regulate the cytoskeleton, including Rho, Rac and Cdc42 (Ridley et al., 2001). Rho, which stands for Ras-homologous, was cloned in 1985. There are at least three identified human homologs; RhoA, RhoB and RhoC. Later, it was shown that RhoA/B/C were modified by Clostridium botulinum toxins. In fact, recombinant RhoA was identified as a substrate for C. botulinum C3 transferase (Aktories et al., 1992). By microinjecting C3 transferase into cells, Ridley and co-workers demonstrated that RhoA/B/C were required for serum-induced stress fiber formation (Ridley et al., 1992).
19 Stress fibers are described as axial bundles of F-actin that underlie the cell. Another study revealed that stress fibers and the formation of focal adhesions could also be induced by the addition of lysophosphatidic acid (LPA), which activates G-coupled protein receptor. Thus, Rho can be activated through both growth factor, LPA or bombesin stimulation. LPA activates an Edg receptor, coupled to GD12/13 and an unknown intermediate, which activates Rho. Active Rho-GTP activates Rho kinase and Myosin light chain kinase (MLCK), leading to actomyosin contraction and stress fiber formation.
Several factors, including PDGF, EGF, insulin and bombesin can activate Rac, leading to lamellipodia formation also known as membrane ruffling (Ridley et al., 1992), Lamellipodia are flattened, sheet-like structures, which are composed of cross-linked F-actin meshwork, that project from the surface of a cell. Lamellipodia initiates the process of cellular migration. There are at least three isoforms of Rac, Rac1,2 and 3. Activation of Cdc42 by addition of bradykinin or TNFD, leads to filopodia formation, also known as microspikes. Filopodia is often described as long, thin protrusions at the periphery of cells, which are composed of F-actin bundles. There is evidence implicating a cross talk between different Rho family members. Cdc42 can activate Rac, which in turn can affect Rho (Tapon et al., 1997).
The activity of the Rho family proteins is regulated by guanine exchange factor (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). To date, there are about 30 GEFs, 20 GAPs and a number of GDIs identified. These small GTPases (20-30 kDa) switch between an active GTP-binding form and the inactive GDP-binding state. GEFs facilitate the exchange of hydrolysed GDP in favour for GTP-binding, while GAPs speed up the hydrolysis procedure of the GTPase. GDIs inhibit the dissociation of GDP from the GTPase (Aspenström et al., 1999).
The cell migration process involves several steps. It starts with an extension of lamellipodium at the front of the migrating cell, which is stabilised by the formation of new focal adhesions with the 20 surrounding extracellular matrix. Next, the cell moves forward by actomyosin contraction. The tail of the cell is then detached from the substratum, the focal adhesions disassembled and the cell retracts. The migrating cell secretes proteases, that digest the extracellular matrix proteins and thereby facilitate the migration process (Ridley et al., 2001).
RNA interference
RNA interference (RNAi) is a relatively new technique, which can selectively lower protein expression. RNAi was originally discovered in C. elegans (Fire et al., 1998). The addition of double stranded RNA (dsRNA) results in sequence specific gene silencing by the action of the dicer enzyme, which processes dsRNA into 22 nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multicomponent nuclease RNA induced silencing complex (RISC), which becomes activated by unwinding of siRNAs. The activated RISC complexes can regulate gene expression on many levels, either by promoting RNA degradation, translational inhibition or target chromatin remodelling (reviewed in Hannon 2002). RNAi has recently shown great promise as a therapeutic delivery model, leading to suppression of the pathologically expressed genes (Wilson et al., 2003).
21 AIMS
The aims of this study were to investigate the following;
* To elucidate the role of Shb in angiogenesis in vitro, using the tube formation assay.
* To characterise the role of Shb in the activation of specific angiogenic signalling cascades in IBE cells overexpressing wild-type and R522K Shb.
* To investigate the potential role of the Shb adaptor protein in the regulation of FAK activity and cell spreading.
* To determine the importance of Shb in the signal transduction and physiological response to VEGF.
22 METHODOLOGY
Cells (I, II and III)
Several cell lines were used in this thesis, including murine immortalised brain endothelial (IBE) cells (I and II), Rat 1 fibroblasts expressing temperature sensitive v-src (tsLA29) and control Rat 1 fibroblasts (II), Porcine aortic endothelial (PAE) cells and PAE cells stably expressing the human VEGF-receptor 2 (III), PAE cells expressing chimeric VEGFR-2 containing the extracellular domain of human CSF-1R/c-fms fused with the transmembrane and the cytoplasmatic domains of murine VEGFR-2 and PAE cells expressing chimeric VEGFR-2 with a point mutation at tyrosine 1173 (murine) in the cytoplasmatic domain (III), Telomerase-immortalised human microvascular endothelial (TIME) cells (III).
IBE cells overexpressing either wild-type, R522K Shb (inactivation of the SH2 domain) or empty vector were generated by use of retroviruses (I and II). Stable clones were selected using puromycin and overexpression on protein level was verified by Western blot analysis.
Protein assays; Immunoprecipitation (I, II and III)
Immunoprecipitation assays are used to detect protein complex formations. Serum starved cells were agonist (FGF, LPA, FCS, VEGF or colony stimulating factor (CSF)) stimulated, washed and lysed. Cleared lysates were either mixed with sample buffer or incubated with the appropriate antibody, on ice, for 2h before a final 1h mixing with Protein A or G Sepharose. The complexes were extensively washed, resuspended in sample buffer and boiled.
23 Western blot analysis (I, II and III)
Proteins were separated according to size (kDa) using sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). The proteins will then be electrophoretically transferred onto a membrane, which will be blocked and incubated with primary antibody. After washing, probing with the appropriate secondary horse radish peroxidase (HRP)-labelled antibody and extensive washing, the bound antibodies will be visualised by enhanced chemiluminescence. The bound antibody complex on the membranes was stripped before reprobing with another antibody.
Fusion proteins, peptides and pull-down experiments (I, II and III)
Fusion proteins can be used to determine domains of interaction between different proteins. The fusion protein consists of two parts, a Glutathione-S-Transferase (GST), which has a very high affinity for Glutathione-Sepharose beads and the protein or protein domain of interest, that are fused together. By use of the bacteria E. coli and Isopropyl-thiogalaktoside (IPTG), which drives the expression of the fusion protein, and the ability of the fusion protein to bind to the heavy Glutathione-Sepharose beads, large amounts of the fusion protein can be purified.
I have used fusion proteins corresponding to the SH2- and the PTB-domain of Shb and also the p55Shb-'SH2 in my studies to identify interaction-domains of Shb with FAK and the VEGFR-2. IBE or PAE/VEGFR-2 cells were serum starved, stimulated with nothing (control), FGF-2 or VEGF and lysed. The cleared supernatants were incubated, on ice, with or without phospho-tyrosine, HEPES, or peptide (phosphorylated or non-phosphorylated) and with the different fusion proteins. The fusion proteins with the bound proteins were extensively washed, sample buffer added and boiled. Western blot analysis was performed and the membrane was probed for phospho- tyrosine or FAK content. The peptides were added to compete the binding between Shb-FAK and Shb-VEGFR-2 and thereby identify specific sites of interaction. Short peptides of approximately 16 amino acids (phosphorylated and non-phosphorylated) were synthesised 24 containing the phosphorylated tyrosine in the middle of the peptide sequence. The peptides were purified with reversed phase chromatography and analysed with matrix assisted laser desorption ionization (MALDI) technique.
Rac1 and Rap1 assay (I)
Activation of Rac1 affects the cytoskeleton, by forming lamellopodia also known as membrane ruffling. Rap1 is a small GTPase, with a high similarity to Ras, that becomes active upon cell adhesion. The activity of Rac1 and Rap1 can be measured using the corresponding fusion proteins pre-coupled to Glutathione Sepharose beads. Serum starved IBE, IBE/Shb and IBE/R522K Shb cells were stimulated with FGF, lysed and incubated with either GST-PAK-CD fusion protein (Rac1) or RalGDS-RBD fusion protein (Rap1). The bound active Rac1GTP and RapGTP was determined by Western blot analysis.
Rho assay (II)
This assay measures the relative amount of the GTP-bound Rho, which correlates with its activity. Rho is a small GTPase, which is important in stress fiber formation. Serum starved IBE, IBE/Shb and IBE/R522K Shb cells were seeded on collagen for 3h and stimulated with nothing, FGF or LPA. Cells were washed, lysed and the supernatants were incubated with GST-Rhotekin Rho binding domain fusion protein on ice. The beads were extensively washed, sample buffer added and boiled. The samples were run on a gel and transferred onto Immobilon membrane and probed for the presence of Rho.
DNA synthesis (I)
IBE, IBE/Shb and IBE/R522K Shb cells were seeded in 15% serum for 48h, serum starved for 48h and then cultured for another 25 18h in 15%. The cells were labelled with 1 PCi/ml (3H) thymidine for 4h, washed, sonicated and TCA precipitated prior to scintillation counting. The DNA content of the samples were measured in parallel using a fluorometric assay.
Proliferation and Viability (I)
In order to further understand the role of Shb in FGFR-1 signalling, proliferation and apoptosis were investigated. IBE cells overexpressing wild-type and R522K Shb were compared with IBE cells. The cells were counted using haemocytometer (Bürker chambers), seeded in serum-containing media, and recounted after different time-points.
An assay that detects programmed cell death or apoptosis is a cell viability test, which measures Annexin V, a marker for apoptosis. IBE cells expressing either empty vector (control), wild-type or R522K Shb were seeded and starved for 48h. Cells were trypsinised, resuspended in Annexin V Fluorescein and propidium iodide, incubated and analysed by flow cytometri at 488 nm excitation. The criteria for apoptotic cells were defined as enhanced Annexin V fluorescence while displaying normal propidium iodide staining.
siRNA (III)
Short interfering RNA (siRNA) was used to knock-down Shb expression, enabling us to investigate the role of Shb in VEGF signalling. A few sequences for siRNA directed against Shb and a control sequence were designed by use of the Ambion web site. PAE- VEGFR-2 cells were transfected using Lipofectin£ reagent, Opti- MEM media and siRNA duplex. The Shb and actin content were assessed after 24, 48 and 72h, by lysing the cells in sample buffer, sonication and performing Western blot analysis.
Alternatively, 24h post transfection the cells were serum starved, and the next day, stimulated with VEGF or FCS. The cells were then washed, lysed in sample buffer, sonicated and Western blot 26 performed. The membranes were probed with the appropriate antibodies.
Alternatively, PAE/VEGFR-2 cells were transfected with fluorescently labelled oligonucleotides and after different time-points, the cells were trypsinised and analysed using a FACS Calibur.
Physiological assays; Tube formation (I)
The tube formation assay is an in vitro assay, where the ability of certain proteins or factors to induce tubular structures can be measured. Serum starved IBE, IBE/Shb and IBE/R522K Shb cells were added to collagen gels and allowed to attach for 3h. Another layer of collagen was added and after 1h, medium with or without growth factor was given to the cells. Photos were taken of tubular- like structures at different time-points.
Morphology assay (I and II)
This method assesses the ability of cells to spread on a collagen or fibronectin matrix. Serum starved IBE, IBE/Shb and IBE/R522K Shb cells were added to collagen gels or fibronectin and allowed to attach for 3h, after which photos were taken and the spreading was quantified using NIH Image program.
Actin staining (I and III)
Staining for the cytoskeleton/actin gives clues as to the role of, in our case, Shb, in the following processes; stress fiber formation, lamellopodia and membrane ruffles, and filopodia and microspikes. PAE/VEGFR-2 cells were transfected with different siRNA sequences, serum starved and stimulated or not with VEGF for 5 or 15 min, 48h post transfection. The cells were washed, fixed and permeabilised. The slides were dried, blocked and treated with fluorescently labelled rhodamine phalloidin. The cells were 27 extensively washed, Fluoromount G was added and the slides were mounted. The samples were examined and photos were taken using the x40 lens in a Nikon microscope.
Migration assay (I and III)
The ability of cells to migrate towards a chemotactic gradient can be measured in a Boyden chamber. The role of Shb in migration was investigated using siRNA treated cells. PAE/VEGFR-2 cells were transfected with either Shb1 siRNA or scrambled sequence or kept untransfected (control). Forty-eight hours later, the cells were seeded out on a collagen coated membrane in a Boyden chamber. After another 4h, the cells were fixed, stained and washed. The non- migrating cells were wiped off, the migrated cells were mounted on a coverglas using EUKITT and counted.
Statistical analysis (I, II and III)
Proliferation assay (I); Means r SEM were given for three observations and were based on two separate clones in each group.
DNA synthesis assay (I); *, P 0.05 when compared with control. Means r SEM for six to nine observations were given and were based on three separate clones in each group.
Apoptosis assay (I); *, P 0.05 when compared with the control. Means r SEM for nine observations were given for three different clones in each group.
Migration assay (I); Data were shown as mean r SEM for five independent experiments, *, P 0.05.
Morphology assay (II); Results are expressed as mean r SEM. Statistical analysis were performed using one way repeated measurements ANOVA and Bonferroni t-test.
28 Migration data (III); Results are expressed as percentage of basal response (mean r SEM) in three independent experiments. * Indicate significantly different compared with basal condition, p 0.05 using Student's t-test for unpaired data.
29 RESULTS AND DISCUSSION
The role of Shb in endothelial cell proliferation and differentiation (Paper I).
In order to understand the mode of action of the adaptor protein Shb in endothelial cells, IBE cells were stably transfected, using retro-viral mediated gene transfer, with either wild-type Shb, SH2-domain mutated (R522K) Shb or empty vector and characterised. Clones were isolated exhibiting a 2-4 fold increase in Shb content compared with control clones. The role of Shb in these cells was investigated using various assays.
No significant differences were apparent in the proliferation rate between the different clones. However, the wild-type Shb cells displayed a significantly lower rate of DNA synthesis, in presence of serum, and an increased rate of apoptosis, in the absence of serum, compared with control cells. In order to determine the mechanism, resulting in the increased apoptosis, activation of the serine/threonine kinase PKB/Akt was monitored. PKB/Akt is downstream of PI3- kinase and phosphorylation of PKB/Akt is known to transmit survival signals. Several growth factors, such as PDGF, EGF and FGF-2, can activate PKB/Akt (Burgering et al., 1995). FGF-2 induced rapid and transient phosphorylation of PKB/Akt in all cells, indicating that the apoptotic signalling is not related to changes in PKB/Akt activation. There was no difference in the cell cycle profiles between the IBE/Shb, IBE/R522K Shb and control cells, when cultured in FCS or in the presence of FGF-2. The IBE/Shb cells demonstrated a decreased percentage of cells in the S phase of the cell cycle, compared with the control cells upon serum withdrawal.
The role of Shb in cell morphology was also investigated. The different cells were plated on gelatin in serum-containing medium and examined after 2h. Overexpression of Shb caused increased cell spreading compared with control cells. The R522K Shb cells displayed increased spreading, but not to the same degree as the wild- type Shb cells. We also tested the different cells in two other physiological assays; stress fiber formation and chemotaxis. FGF-2 30 stimulation of IBE cells overexpressing Shb leads to a slight increase in stress fiber formation and the formation of irregular, densely stained patches. The R522K Shb cells displayed the same structures, but to a lesser extent. To further characterise the signalling pathway leading to stress fiber formation, cells were pre-incubated with the PI3-kinase inhibitor LY294002. There was no apparent effect on the stress fiber formation, suggesting it was not dependent on PI3- kinase/Rac. Addition of PP2, a Src family kinase inhibitor, reverted all of the FGF-2-induced changes in the control cells and also prevented Shb dependent patchy staining pattern in the wild-type Shb cells. It appears that overexpression of Shb augmented Src signalling leading to cytoskeletal rearrangements.
In the migration assay, FGF-2 failed to promote chemotaxis in wild-type and R522K Shb cells, while FGF-2 caused an approximate doubling in migration rate in the control cells. However, the wild-type and R522K Shb cells displayed an elevated basal rate of migration compared with the control cells. Activation of Rac1 and Rap1 was also assessed. FGF-2 stimulation led to a slight increase in Rac activity in control cells, but not in wild-type Shb cells. However, in R522K Shb cells, the basal Rac activity was decreased, with a small increase in its activity upon FGF-2 stimulation. The activity of Rap1 was decreased upon FGF-2 treatment in control cells. FGF-2 stimulation led to an increased Rap1 activity in both wild-type and R522K Shb cells, mainly due to a decreased basal Rap1 activity compared with the control cells.
Shb is necessary for tubular morphogenesis (Paper I).
Data from this paper show that Shb mediates signalling and physiological responses upon FGF-2 treatment. Therefore, the role of Shb in FGF-2-induced angiogenesis was investigated using the tube formation assay. Here, we demonstrate that IBE cells overexpressing the wild-type Shb form tubular structures in response to FGF-2. Furthermore, Shb, with a functional SH2 domain, was shown to be required for proper tubular morphogenesis, since IBE cells expressing R522K-mutant Shb, form disorganised tubes. Of the four clones repeatedly analysed, only one gave structures reminiscent of tubes but 31 to a lesser degree than in the control cells. These tubular structures were less regular and frequent than those of the control cells. The formation of tubular structures was prominent in IBE/Shb cells and could be detected at earlier time points compared with the control cells.
Taken together, our data strongly suggest that Shb is required for FGF-2-induced angiogenesis, since IBE cells overexpressing an SH2 domain mutation exhibit disorganised vessel formation.
Shb causes Src-dependent cell spreading and activation of FAK (Paper II).
We wanted to elucidate the molecular mechanism behind the angiogenic phenotype of Shb described in paper I. Therefore, the role of Shb in cell spreading and the regulation of FAK phosphorylation was investigated. Overexpression of Shb led to increased cell spreading in both untreated and FGF-2 stimulated cells, which was completely abolished upon pre-treatment with PP2 (a Src family kinase inhibitor). The Shb-induced cell spreading correlated with an increase in FAK phosphorylation. We also observed an interaction between Shb and FAK upon FGF-2 stimulation. To further define the nature of this interaction, GST pull-down experiments in the presence of phosphorylated peptides corresponding to the known phosphorylation sites in FAK (Tyr 397, 407, 576/577, 861 and 925) were carried out. We found that the PTB domain of Shb interacts with FAK in a phosphotyrosine-dependent manner. None of the phosphorylated peptides could block the Shb-FAK interaction. This could possibly be explained by the binding of Shb, to an as yet, uncharacterised phosphorylation site in FAK. Phosphorylation- dependent Shb-FAK interaction have also been observed in the insulinoma RINm5F cells (Welsh et al., 2002).
32 Shb and the downstream signalling of FGFR-1 (Paper II).
We were also interested in the down-stream signalling pathway from the FGFR-1 via Shb. Therefore, Shb phosphorylation assays with various inhibitors were performed. Our data show that the Src family kinases are necessary for FGF-2 induced Shb phosphorylation. In agreement with this, Rat-1 cells overexpressing a temperature sensitive v-Src mutant displayed increased Shb phosphorylation, compared with control Rat-1 fibroblasts. However, pre-treatment with cytochalasin D, which causes cytoskeletal disruption and inhibition of FAK, did not affect FGF-induced Shb phosphorylation. Taken together, this suggests that FGF-2-mediated Shb phosphorylation is downstream of Src but independent of FAK activity.
FAK has been known to interact with a number of proteins within the focal adhesions. The phosphorylation status of FAK, PLC-J and paxillin, all of which have been implicated in cell spreading, were investigated. Overexpression of wild-type and R522K Shb leads to an increased activation of FAK, PLC-J and paxillin in a Src-dependent manner, in both control and FGF-2 stimulated cells. This suggests that Src activity is necessary for a maximal phosphorylation response. However, in R522K Shb cells, FAK phosphorylation was stimulated to a significantly lower degree compared with the cells expressing wild-type Shb, suggesting that a functional SH2 domain was required for maximal stimulation of FAK activity.
Stimulation of Rho is known to activate FAK, which led us to investigate whether the Shb-induced FAK phosphorylation was downstream of elevated Rho activity. Analysis of the GTP-status of Rho, revealed that while IBE cells expressing wild-type Shb showed a small increase in basal Rho activity, cells expressing the R522K Shb did not result in enhanced Rho activity. FGF-2 stimulation did not display increased Rho activity, whereas stimulation with LPA, a known activator of Rho, led to elevated GTP-Rho in all cells. The data demonstrated that Rho is not responsible for the increased FAK phosphorylation in Shb overexpressing cells.
33 Taken together, the data indicate that Shb binds directly to FAK and regulates its phosphorylation, leading to enhanced cell spreading in a Src-dependent manner. This study shows that Shb, upon FGF stimulation in IBE cells, forms a complex with Src and FAK, leading to cellular spreading and angiogenesis (Fig. 5).
FGFR-1
Plasma Integrin FGF α β membrane
Src
Src FAK Pro PTB SH2 P Y766 Pro PTB SH2 Shb Shb
Erk
Cell spreading/ Tubular morphogenesis Proliferation
Angiogenesis
Figure 5. Overview of the role of Shb in FGFR-1 signalling.
Shb interacts with tyrosine 1175 in the VEGFR-2 (Paper III).
In paper I and II, we show that Shb plays a role in FGF-2- induced angiogenesis by regulating FAK activity. Therefore, the potential role of Shb in angiogenic signalling induced by VEGF was analysed in paper III. Here, we show that Shb is phosphorylated upon VEGF stimulation in a transient fashion in PAE cells stably expressing the human VEGFR-2 (KDR). VEGF-induced activation of 34 Shb was shown to be dependent on Src kinase activity, similar to that observed with FGFR-1 activation. The SH2 domain of Shb has been previously shown to bind to the consensus motif pY-T/V/I-X-L. In the human VEGFR-2 (KDR), tyrosine 1175 has the sequence pY-I-V-L, suggesting that it may be a potential Shb binding site.
An interaction between VEGFR-2 and Shb was observed in human telomerase-immortalised microvascular endothelial (TIME) cells, suggesting that Shb is activated in response to VEGFR-2 phosphorylation in a number of endothelial cells. Furthermore, in a GST pull-down assay, the SH2-domain of Shb was shown to interact with phosphorylated tyrosine 1175. We also found that Shb, with an SH2 domain deletion, appeared to bind to the VEGFR-2, although this association was only partly phospho-tyrosine dependent. The nature of this interaction remains unknown, but could result from a combined Shb PTB domain/proline rich interaction with the receptor, alternatively involving a third component.
In order to confirm the role of tyrosine 1175 in regulating Shb activation, we utilised PAE cells expressing a chimeric receptor composed of the extracellular domain of the human colony stimulating factor 1 (CSF-1) receptor and the intracellular domain of the murine VEGFR-2 (Flk-1) and also a murine receptor mutant in which tyrosine 1173 was mutated to phenylalanine (Y1173F) (Rahimi et al., 2000). The tyrosine 1175 (pY-I-V-L) site in the human VEGFR- 2 (KDR) is conserved and corresponds to tyrosine 1173 in the murine VEGFR-2 (Flk1).
Analysis of Shb phosphorylation revealed that while activation of the chimeric receptor resulted in Shb phosphorylation, activation of the Y1173F mutant receptor did not stimulate Shb phosphorylation, confirming that Shb binds and becomes activated upon binding to tyrosine 1175 in the VEGFR-2. Analysis of the degree of phosphorylation of other intracellular signalling proteins revealed that while the mutant receptor could evoke a normal activation of p44/42 MAPK, it was not able to stimulate the phosphorylation of FAK at position tyrosine 576.
35 Taken together, these results strongly suggest that Shb binds to the pY-I-V-L site in the VEGFR-2 (Flk-1/KDR) and regulates the VEGF-induced activation of FAK.
Shb regulates FAK activity, stress fiber formation and migration in VEGFR-2 (Paper III).
In order to determine the physiological role of Shb in VEGFR- 2 signalling, we decided to utilise short interfering RNA (siRNA) technology to knock-down Shb expression. Our data demonstrate that Shb protein expression was specifically decreased by 80% upon treatment with siRNA in a transient fashion. Since Shb has been shown to bind and regulate FAK phosphorylation, we investigated the phosphorylation status of FAK under these conditions. We found that VEGF-induced phosphorylation of FAK tyrosine 576 was reduced in the siRNA Shb1 treated cells by 40 %. In contrast, in the untransfected and in the scrambled oligonucleotide treated cells, VEGF increased FAK tyrosine 576 phosphorylation by 20 %. FCS caused increased FAK phosphorylation in all cells, indicating that in the siRNA Shb1 treated cells, FAK tyrosine 576 could still be phosphorylated.
FAK is phosphorylated in vitro by Src at a number of sites; Y407, Y576, Y577, Y861 and Y925. Phosphorylation of tyrosine 576, in the FAK kinase domain, enhances the activity of FAK. Therefore, it is possible that Shb is required for VEGF-dependent FAK activity by allowing Src to phosphorylate tyrosine 576. We also show that lowering Shb expression with the siRNA technique did not affect VEGFR-2 phosphorylation and the subsequent activation of PLC-J, Akt and MAPK. This data suggests that, in the case of VEGFR-2 signalling, Shb specifically regulates FAK activity. Taken together, it appears that Shb regulates FAK activity both in response to FGFR-1 and VEGFR-2 activation. However, unlike with the FGFR-1, Shb does not appear to regulate MAPK activity in response to VEGFR-2 activation, as shown in both the PAE CSF1/VEGFR-2 Y1173 cells and in the siRNA knock down. Therefore, we conclude that Shb is able to differentially modulate intracellular signalling from different receptor tyrosine kinases.
36 We then decided to use siRNA treated PAE/VEGFR-2 cells to study the role of Shb in VEGF-induced stress fiber formation. Reduced Shb expression led to decreased stress fiber formation in response to both 5 and 15 min VEGF stimulation, which was not seen in the cells treated with the control oligonucleotide or in untransfected cells. To further define the physiological role of Shb in the VEGFR-2 response we decided to study cellular migration / chemotaxis. Stimulation with VEGF and FCS lead an approximate doubling in migration in untransfected and control oligonucleotide treated cells. In the siRNA Shb1 treated cells, VEGF failed to induce migration, however, FCS could still evoke a migratory response.
This suggests that Shb is required for VEGFR-2-mediated migration in endothelial cells. Therefore, by regulating FAK activation, Shb could influence cellular migration. Indeed, cell migration is an important in vivo physiological function of VEGF, as endothelial cells must migrate and proliferate to form new capillaries in the angiogenic response. The ability of Shb to regulate this process suggests that modulators of Shb signalling may have a therapeutic potential as anti-angiogenic therapy.
Interestingly, in the FGFR-1, both Shb and PLC-J can bind to the same site at tyrosine 766. In the case of the VEGFR-2, PLC-J has been previously reported to bind to tyrosine 1175. In addition, Shb and PLC-J can associate with each other. However, it appears that Shb and PLC-J do not compete for the common binding sites in FGFR-1 or in the VEGFR-2, as shown in the siRNA experiment, where VEGF- stimulated PLC-J phosphorylation was not altered upon lowered Shb expression. Another adaptor protein, Sck, has also been shown to interact with tyrosine 1175 in the VEGFR-2. This would suggest that this site has the potential to interact with multiple signalling molecules.
In conclusion, paper III demonstrates that Shb is important in VEGF signalling in endothelial cells. Upon VEGF stimulation, the VEGFR-2 is phosphorylated, Src associates with the receptor and the SH2-domain of Shb binds to phosphorylated tyrosine 1175 in the C- terminal tail of the VEGFR-2. Src phosphorylates Shb, which 37 promotes the subsequent phosphorylation of FAK at tyrosine 576 in the kinase domain. FAK is then able to regulate focal adhesion/ stress fiber formation allowing the spatial and temporal orchestration of the migratory response in endothelial cells.
VEGF
VEGFR-2 Plasma membrane
PIP PIP PIP DAG 3 2 2 PKC Ras 2+ + Ca PI3K Y951 VRAP IP3 SPK Y996 Rac Akt/PKB Src Y1054 Y1059 γ Raf PLC- p38MAPK Shb Y1175 Y1175 Y1214 Sck 2+ 2+ Ca BAD Ca 2+ FAK Ca 2+ 2+ Ca Ca 2+ 2+ Ca Caspase 9 Ca 2+ MEK eNOS Paxillin Ca
MAPKAPK2/3 Endoplasmic reticulum Erk
2+ Ca SURVIVAL NO production HSP27 Focal adhesion cPLA turn over 2
Actin PGI2 production reorganization
Nucleus Gene Transcription
PERMEABILITY MIGRATION PROLIFERATION
Figure 6. Overview of VEGFR-2 signalling.
38 CONCLUSIONS
* Shb is important in the angiogenic process, since Shb with a functional SH2 domain is required for proper tubular morphogenesis. IBE cells expressing an R522K-mutant Shb formed disorganised tubes.
* Overexpression of Shb causes Src-dependent cell spreading and activation of FAK in IBE cells seeded on collagen. The PTB-domain of Shb binds directly to FAK in a phosphotyrosine-dependent manner. The FGF-2 induced phosphorylation of Shb is Src-dependent.
* Shb is phosphorylated upon VEGF stimulation in a Src- dependent manner. The SH2-domain of Shb binds to tyrosine 1175 in the VEGFR-2 and regulates FAK activity, stress fiber formation and cellular migration.
39 FUTURE PERSPECTIVE
This work demonstrates the importance of the adaptor protein Shb in angiogenesis. The data suggest that in the case of FGFR-1 response, Shb can regulate the activity of both FAK, leading to cell spreading, and MAPK, via FRS2, leading to proliferation. However, in response to VEGFR-2 Shb has no effect on MAPK phosphorylation but regulates FAK activity, resulting in stress fiber formation and migration. Furthermore, the exact interaction between Shb and FAK and how Shb regulates FAK activity is not characterised in detail. What is the role of Src in this interaction?
Another factor, LPA has been shown to induce angiogenesis and preliminary data indicate that Shb is phosphorylated by LPA in endothelial cells. My data demonstrate that both FGF- and VEGF- induced Shb phosphorylation is Src-dependent. Preliminary data suggest that LPA-induced Shb phosphorylation also is Src-dependent. It seems that Src is more important than the agonist in stimulating Shb activation. By use of immunoprecipitation assays and access to various inhibitors and cells carrying a mutation in the G-protein coupled receptor, it would be possible to elucidate the role of Shb in LPA-induced signalling. To date, there are no studies showing that Shb is activated by G-protein coupled receptors.
Furthermore, genes regulated by Shb upon agonist (FGF, VEGF and LPA) stimulation could be identified using siRNA treated cells in microarray assays.
40 ACKNOWLEDGEMENTS
This study was carried out in the Department of Medical Cell Biology, Uppsala University. I wish to express my sincere gratitude to;
My supervisor Professor Michael Welsh for giving me the opportunity to study Shb signalling in angiogenesis (including Rho assay and LPA study). For your impressive knowledge, memory and technical expertise that you so willingly share with us and for your sense of humour and all the extra curricular activities including food and excellent wine.
My co-supervisor; Professor Arne Andersson for support and creating a nice environment.
Head of the Department; Professor Godfried Roomans for continuos support and for your way of running the Department .
Department of Medical Biochemistry and Microbiology for financial support and for introducing me to graduate studies.
Professor Lena Claesson-Welsh for support and borrowing of equipment and reagents. The Ph.D students and postdocs (Mike, Peetra, Svante, Johan, Lars, Emma and the rest of you) in Lena's lab for help with computers, microscopes, conference attendance and fun extra curricular activities.
Special thanks to all my co-authors; Lingge Lu, Michael Cross, Debbie Riley, Robert Hägerkvist and Nader Rahimi.
Senior people at the Department; Professors Ulf Eriksson, Stellan Sandler, Claes Hellerström, Leif Jansson, Erik Gylfe, Bo Hellman and associate Professors Nils Welsh, Håkan Borg, Peter Bergsten, Eva Grapengiesser, Carina Carlsson, P-O Carlsson and Parri Wentzel. Thank you for pleasant company, interesting discussions and help with computer problems.
41 All technical staff at the Department for being nice and very helpful; Agneta Bäfwe, Karin Öberg and Birgitta Jönzén, Göran Ståhl, Birgitta Bodin, Eva Törnelius, Lisbeth Sagulin and Astrid Nordin.
Past and present Ph.D students of the Department for creating a good atmosphere, for support and friendship.
Past and present members of Michael's group; Lotta, Cecilia, Cecilia, Lingge, Vitek, Björn, Johan, Nina, Charlotte and the excellent technical staff; Ing-Marie and Ing-Britt.
My room mates through out the years; Göran, Andreea, Björn and Nina for interesting discussion, good friendship and help with watering my plants.
My old friend Eva for coffee-breaks, excellent travelling companion, shopping in various cities and cinema visits.
My oldest friend Anette for our long friendship (26 years), fun birthday celebrations and Stockholm visits.
My Kalmar friends, where it all started; My old best friend Patrik for sharing my interests; cooking food and drinking wines (Chateauneuf du pape), for always supporting me and for all the great laughs we have had through the (12) years.
Fredrik: I always enjoy our long but not so frequent telephone conversations; which basicly is at least one hour constant laughing.
Charlotta: for being a good friend and for our one day a year in the summer in Skåne. I will come and see your house.
and Sara for long lunches, discussions concerning life and cats. We need a shopping day in Stockholm.
Basil for your amazing belief in people. I am honoured to have meet you. Never forget your dreams! My soul-mate Inga-Lill for being quite similar to me.
Ulla, for your knowledge and wisdom.
42 My water training friends and Wet-Vest Uppland (Linda, Annelie, Gujje, Ellan, Karin, Annette, Catharina, Kristina, Marianne, Curre, Sven-Ola, Anna, Jenny and all the rest of you) for living my life with me, for laughs, crises and reality. You are always with me!
Annelie for being the best friend you could possibly be. For your way of finding solutions on any problem and for your positive attitude.
My little furry friends, the cats; Miss Daisy, Jasper and Lady who always make me happy.
Mike for emphasising that it's not how much you do, it's what you do that is important. For wanting cats, for taking me to shoppers paradise; NK, Harrods and Brown Thomas, for loving me and for helping me through difficult times.
My brother Lars-Göran for being who you are.
My parents Lars and Birgitta for always supporting me and my brother and for having the good taste in buying a summer house, in 1973, in my favourite place in Österlen, Kivik.
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51 Acta Universitatis Upsaliensis Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the sum- mary alone is distributed internationally through the series Comprehen- sive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to October, 1985, the series was published under the title “Abstracts of Uppsala Dissertations from the Faculty of Medicine”.)
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