Cloning and characterization of PHIP, a novel insulin receptor substrate-1 pleckstrin homology domain interacting protein.
Insulin receptor substrate-1 (IRS-1) protein is a major substrate of the insulin 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 to bind to PHIP. Anti-phosphotyrosine immunoblots of PHIP revealed no discernible insulin receptor-regulated phosphorylation, suggesting that PHIP is not itself a substrate of the insulin receptor. In contrast to full-length PHIP, overexpression of the PH-binding region of PHIP has a pronounced inhibitory effect on insulin-induced IRS-1 tyrosine phosphorylation levels. Furthermore, expression of this dominant-negative PHIP mutant leads to a marked attenuation of insulin-stimulated mitogen-activated protein kinase activity. 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.
Pubmed ID: 11018022 RIS Download
Amino Acid Sequence | Animals | Base Sequence | Carrier Proteins | Cell Line | Cloning, Molecular | DNA, Complementary | Enzyme Activation | Humans | Insulin | Insulin Receptor Substrate Proteins | Intracellular Signaling Peptides and Proteins | Mitogen-Activated Protein Kinases | Molecular Sequence Data | Phosphoproteins | Phosphorylation | Rats | Sequence Homology, Amino Acid | Tyrosine