Fibroblast growth factor-induced signal transduction

Abstract: Fibroblast growth factors (FGFs) evoke cellular responses by binding to FGF receptors (FGFRs). FGFRs are cell surface expressed membrane-spanning receptors with intrinsic stimulatable tyrosine kinase activity. This thesis describes the cell proliferation or differentiation. Two new FGFR-1 substrates were identified, and intracellular signal transduction elicited by binding of FGF-2 to FGFR-1, leading to signal transduction molecules critical for FGF-2-stimulated endothelial cell differentiation were characterized.The GTP-binding protein Ras is ubiquitously activated by growth factor receptors via recruitment of the Grb2/Sos complex. The adaptor protein Grb2 did not bind directly to FGFR-1. Instead, activation of FGFR-1 led to binding of Grb2 to either the Shc adaptor or to a novel adaptor protein, p89, both of which were tyrosine phosphorylated upon FGF-2 stimulation. Tyr766 in FGFR-1 was needed for a full mitogenic response, possibly involving binding of Shc. While Shc was found in the cytosol, p89 was targeted to the membrane. FGF-2, but not EGF or PDGF-BB,stimulation resulted in Grb2 binding to p89. Thus, FGFR-1 was shown to activate Ras via two distinct adaptor proteins, Shc and a novel FGFR-1 substrate, p89. In the search for proteins potentially involved in FGFR-mediated cell-cycle regulation, we identified association between a Cdk-binding protein, p13suc, and a tyrosine phosphorylated component of about 70 kDa. The 70 kDa protein was purified and shown to correspond to the RNA-binding protein TLS (translocated in liposarcoma).TLS was bound to p13suc independent of growth factor stimulation but was tyrosine phosphorylated only after stimulation of cells with FGF-2, EGF, and PDGF-BB.FGF stimulates angiogenesis in vitro and in vivo. The endothelial cell line IBEC presents an in vitro model for angiogenesis, in which the FGF-2-stimulated cells either proliferate or differentiate into tube-like structures, dependent on culture condition. We compared FGF-2-induced signal transduction in these proliferating and differentiating endothelial cells. The adaptor protein Shc linked FGFR-1 to Ras activation in both cellular responses, while the p89 adaptor coupled the receptor to Ras activation only in proliferating cells. Ras was shown by pharmacological inhibition or by expression of a dominant-negative mutant Ras, to be crucial for FGF-2-induced endothelial cell proliferation and differentiation, while expression of a constitutively active Ras mutant showed that Ras by itself, is not sufficient for endothelial cell differentiation. An inhibitor of the MEK and MAPK enzymes abrogated FGF-2-stimulated endothelial cell proliferation, but was without effect on differentiation of those cells. Inhibition of the serine kinase Raf-1 function by introduction of Raf-1 antisense oligonucleotides, or pharmacological inhibition of the tyrosine kinase Src, showed these two kinases to be essential for FGF-2-induced endothelial cell differentiation.

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