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Potential Involvement of FRS2 in Insulin Signaling¹

INSERM, U-145 and IFR 50, 06107 Nice, France

Address all correspondence and requests for reprints to: Dr. Laurent Delahaye, INSERM U-145 and IFR 50, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail: delahaye{at}unice.fr

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 upon binding through one or both SH2 domains to tyrosine phosphorylated molecules 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 screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system. In this system we induce or repress the expression of a constitutive active IR ß-subunit. When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2.

Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation 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 dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads 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, in 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 as 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 results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.

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