DETERMINATION OF GAB1 (GRB2-ASSOCIATED BINDER-1) INTERACTION WITH INSULIN RECEPTOR-SIGNALING MOLECULES

The newly identified insulin receptor (IR) substrate, Gab1 [growth fac tor receptor bound 2 (Grb2)associated binder-1] is rapidly phosphoryla ted on several tyrosine residues by the activated IR. Phosphorylated G ab1 acts as a docking protein for Src homology-2 (SH2) domain-containi ng proteins. These include the regulatory subunit p85 of phosphatidyli nositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this repor t, using a modified version of the yeast two-hybrid system, we localiz ed which Gab1 phospho-tyrosine residues are required for its interacti on with phosphatidylinositol 3-kinase and with SHP-2. Our results demo nstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be t yrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 5 89 are participating in this process. Concerning Gab1/SHP-2 interactio n, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Fin ally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for G ab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.