|M.Sc Student||Dym Peles Naama|
|Subject||The Role of Tyrosine Phosphorylation in Mediating hSef|
|Department||Department of Biology||Supervisor||Professor Dina Ron|
|Full Thesis text|
Growth factor signaling by receptor tyrosine kinases (RTKs) is essential for controlling fundamental biological processes and is therefore subjected to multiple levels of regulation. Sef (Similar Expression to FGF genes) is a feedback antagonist of RTK signaling. Initially, Sef was identified as a fibroblast growth factor (FGF) inhibitor in zebrafish, but subsequent studies revealed that Sef can inhibit other RTKs mediated signaling. The mammalian Sef gene encodes several isoforms, including an integral membrane protein (hSef-a) and a cytosolic protein (hSef-b). hSef‑a is a multi-functional protein that can regulate at least three intracellular signaling pathways involved in cell cycle progression and cell survival including the PI-3K/Akt, p38- and ERK- MAPK pathways. hSef-a contain several putative sites for tyrosine phosphorylation, including an invariant tyrosine that is conserved throughout evolution. Given the known role of phosphorylated tyrosine in mediating signaling molecules interaction, it is likely that phosphorylated tyrosine might be important for mediating hSef function. Here, we show for the first time that hSef-a and hSef-b are phosphorylated on tyrosine upon FGFR1 activation and that an activated receptor is essential for this phosphorylation. Next, we have generated tyrosine mutants in which the tyrosines on positions 330, 343, 435 and 665 were replaced with phenylalanine (Y30F, Y435F, double mutant Y330 and Y435F, triple Y330-343-435F mutant and a quadruple Y330-343-435-665F mutant). We have tested the effect of these mutations on hSef-a ability to inhibit the RAS/MAPK pathway in human-embryonic kidney cells, HEK 293, and the effect on hSef-a capacity to suppress colony formation in both MDA-MB 435 and NIH/3T3 cells. Furthermore, we have examined the mutants phosphorylation state upon growth factor stimulation. We showed that the mutations had no effect on the ability of hSef-a to inhibit the RAS/MAPK pathway in HEK 293 cells. Moreover, Y330F and Y435F mutants, each, retained the capacity to suppress colony formation in both MDA-MB 435 and NIH/3T3 cells. Furthermore, all of the tyrosine mutants we have generated were still recognized by an anti-phosphotyrosine antibody, suggesting the existence of other tyrosines residues that can undergo tyrosine phosphorylation on hSef-a. These results show that neither the conserved tyrosine (Y330) nor the other mutations are essential for hSef-a mediated MAPK inhibition.