Insulin receptor-mediated p62(dok) tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulatio n. However, whether this protein is a direct in vivo substrate for the insu lin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show th at the insulin-stimulated tyrosine phosphorylation of the GAP-associated pr otein, now identified as p62(dok), is inhibited by Grb10, an adaptor protei n that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362). and Tyr(398) with phenylalanine greatly decrease d the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting t hat these two residues are the major IR-mediated phosphorylation sites. How ever, mutations at Tyr362 and Tyr398 only partially blocked insulin-stimula ted p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p6 2(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interac tion between p62(dok) and GAP and decreased the inhibitory effect of p62(do k) on the insulin-stimulated activation of Ras and Akt, but not mitogen-act ivated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Ak t phosphorylation could be blocked by coexpression of a constitutively acti ve Has. Taken together, our findings indicate that p62(dok) is a direct sub strate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effector s and negatively regulate the insulin signaling pathway.