Potential involvement of FRS2 in insulin signaling

Shp-2 is implicated in several tyrosine kinase receptor signaling pathways. This phosphotyrosine phosphatase is composed of a catalytic domain in its C-terminus and two SH2 domains in its N-terminus. Shp-2 becomes activated u pon binding through one or both SH2 domains to tyrosine phosphorylated mole cules such as Shc or insulin receptor substrates. We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor ((IR), that could interact with Shp-2. To do so, we s creened a human placenta complementary DNA (cDNA) Library with the SH2 doma in-containing part of Shp-2 using a modified yeast two-hybrid system. In th is system we induce or repress the expression of a constitutive active IR p -subunit. When expressed, IR phosphorylates proteins produced from the libr ary that can then associate with Shp-2. Using this approach, we isolated FRS2 as a potential target for tyrosine ph osphorylation by the IR. After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion depend ent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin lea ds to an increase in FRS2 association with the phosphatase. We next wanted to determine whether FRS2 could be a direct substrate for IR . In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein. Finally, i n intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2. In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 a s a possible substrate for IR. We found that IR can directly phosphorylate FRS2. Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2. Taken together these re sults suggest that FRS2 could participate in insulin signaling by recruitin g Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.