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 stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62^dok, is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr³⁶² and Tyr³⁹⁸ with phenylalanine greatly decreased the IR-catalyzed p62^dok tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr³⁶² and Tyr³⁹⁸ only partially blocked insulin-stimulated 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 Tyr³⁶² with phenylalanine abolished the interaction between p62^dok and Nck. Mutations at Tyr^362/398 of p62^dok disrupted the interaction between p62^dok and GAP and decreased the inhibitory effect of p62^dok on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62^dok on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62 ^dok is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr³⁶² and Tyr³⁹⁸ plays an essential role for p62^dok to interact with its effectors and negatively regulate the insulin signaling pathway.