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

• Published in: The Journal of biological chemistry 2000-12, Vol.275 (51), p.40492-40497
• Main Authors: Farhang-Fallah, J, Yin, X, Trentin, G, Cheng, A M, Rozakis-Adcock, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Cloning, Molecular; DNA, Complementary; Enzyme Activation; Humans; Insulin - pharmacology; Insulin Receptor Substrate Proteins; Insulin receptor substrate-1; insulin receptors; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases - metabolism; Molecular Sequence Data; PH-interacting protein; Phosphoproteins - metabolism; Phosphorylation; Rats; receptor-tyrosine kinase; Sequence Homology, Amino Acid; Tyrosine - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.C000611200

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.