Farhang-Fallah J, Yin X, Trentin G, Cheng A M, Rozakis-Adcock M
Departments of Biology and Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
J Biol Chem. 2000 Dec 22;275(51):40492-7. 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.
胰岛素受体底物-1（IRS-1）蛋白是胰岛素受体酪氨酸激酶的主要底物，对于转导胰岛素的许多生物学效应至关重要，这些效应包括有丝分裂、基因表达和葡萄糖转运。IRS-1的N末端包含一个普列克底物蛋白同源（PH）结构域，该结构域对于活化的胰岛素受体识别并随后磷酸化IRS-1至关重要。在此我们报告分离出一种新型蛋白质，即PHIP（PH相互作用蛋白），它在体外能选择性地与IRS-1的PH结构域结合，并在体内与IRS-1稳定缔合。重要的是，破坏PH折叠的IRS-1 PH结构域突变体无法与PHIP结合。对PHIP进行的抗磷酸酪氨酸免疫印迹显示没有可识别的胰岛素受体调节的磷酸化，这表明PHIP本身不是胰岛素受体的底物。与全长PHIP相反，过表达PHIP的PH结合区域对胰岛素诱导的IRS-1酪氨酸磷酸化水平具有明显的抑制作用。此外，这种显性负性PHIP突变体的表达导致胰岛素刺激的丝裂原活化蛋白激酶活性显著减弱。我们得出结论，PHIP代表IRS-1 PH结构域的一种新型蛋白质配体，可能有助于将IRS-1与胰岛素受体联系起来。
