Date of Award
Doctor of Philosophy (PhD)
Insulin receptor substrate- I (IRS-I) 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-terrninus of IRS-1 contains a pleckstrin homology (PH) domain, followed by a phosphotyrosine-binding (PTB) domain that have been shown to co-operatively contribute in mediating productive receptor/substrate interactions. The PH domain is critical for recognition and subsequent phosphorylation of lRS-1 by the activated insulin receptor. In this thesis, the cloning and biochemical characterization of a novel protein, named PHIP (PH-interacting protein), has been described. PHIP binds selectively to the PH domain of lRS-1 in vitro, and stably associates with IRS-l ill 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. By 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. Moreover, expression of this dominant-negative PHIP mutant (DN/PHIP) leads to a marked attenuation of insulin-stimulated mitogen activated protein (MAP) kinase activity. Furthermore, overexpression of PHIP enhances insulin-induced transcriptional responses in a MAP Kinase-dependent manner. By contrast, DN/PHIP mutants specifically block mitogenic signals elicited by insulin and not serum.In addition, DN/PHIP mutant proteins also inhibit insulin-triggered GLUT4 translocation to the cell surface as well as actin assembly. It is therefore concluded that PHIP represents a physiological ligand of the IRS-1 PH domain which plays a critical role in insulin-stimulated biological responses mediated by IRS-1.
Farhang-Fallah, Janet, "Cloning and characterization of PHIP, a novel protein ligand of the PH domain of IRS-1" (2002). Open Access Dissertations and Theses. Paper 1501.