The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells

Abstract: 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. 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. Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors. My aim has been to identify the role of Shb in FGF- and VEGF-signalling in endothelial cells. Shb was found to be phosphorylated in a Src-dependent manner upon both FGF- and VEGF-stimulation. This was confirmed using fibroblasts overexpressing temperature sensitive v-Src. Furthermore, Shb-induced cell spreading on collagen of immortalised brain endothelial (IBE) cells was also Src-dependent. FGF stimulation led to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding domain of Shb. IBE cells overexpressing wild-type or R522K Shb (inactive SH2 domain) displayed increased FAK activation on collagen.The SH2-domain of Shb was found to bind to tyrosine 1175 in the VEGFR-2 in a phosphotyrosine dependent manner using PAE cells expressing VEGFR-2. Furthermore, by use of siRNA, Shb knock-down experiments revealed that Shb regulates FAK activity, cellular migration and stress fiber formation in response to VEGF stimulation of VEGFR-2. In summary, Shb binds to both FGFR-1 and VEGFR-2 and regulates the activity of FAK and thereby stress fiber formation and cellular migration, which are necessary for formation of new blood vessels. IBE cells with an inactive SH2 domain of Shb displayed disorganised formation of tubular structures in the tube formation assay, while overexpression of wild-type Shb led to accelerated tubular morphogenesis.Taken together, my data show that the adaptor protein Shb plays an important role in the process angiogenesis, in response to angiogenic tyrosine kinase receptors, by interacting with FAK and regulating spreading, stress fiber formation and cellular migration.