Cloning and characterization of PHIP, a novel insulin receptor substrate-1pleckstrin homology domain interacting protein

Insulin receptor substrate-1 (IRS-1) protein is a major substrate of the in sulin receptor tyrosine kinase and is essential for transducing many of the biological effects of insulin including mitogenesis, gene expression, and glucose transport. The N terminus of IRS-1 contains a pleckstrin homology ( PH) domain that is critical for recognition and subsequent phosphorylation of IRS-1 by the activated insulin receptor. Here we report the isolation of a novel protein, PHIP (PH-interacting protein), which selectively binds to the PH domain of IRS-1 in vitro and stably associates with IRS-1 in vivo, Importantly, mutants of the IRS-1 PH domain that disrupt the PH fold fail t o bind to PHIP. Anti-phosphotyrosine immunoblots of PHIP revealed no discer nible insulin receptor-regulated phosphorylation, suggesting that PHIP is n ot itself a substrate of the insulin receptor. In contrast to full-length P HIP, overexpression of the PH-binding region of PHIP has a pronounced inhib itory effect on insulin-induced IRS-1 tyrosine phosphorylation levels. Furt hermore, expression of this dominant-negative PHIP mutant leads to a marked attenuation of insulin-stimulated mitogen-activated protein kinase activit y. We conclude that PHIP represents a novel protein ligand of the IRS-1 PH domain that may serve to link IRS-1 to the insulin receptor.