p66shc inhibits insulin-like growth factor-I signaling via direct binding to Src through its polyproline and Src homology 2 domains, resulting in impairment of Src kinase activation

Abstract

p66shc is increased in response to cell stress, and these increases regulate growth factor actions. These studies were conducted to determine how p66shc alters IGF-I-stimulated Src activation, leading to decreased IGF-I actions. Our results show that p66shc binds to Src through a polyproline sequence motif contained in the CH2 domain, a unique domain in p66shc, and IGF-I stimulates this interaction. Disruption of this interaction using a synthetic peptide containing the p66shc polyproline domain or expression of a p66shc mutant containing substitutions for the proline residues (P47A/P48A/P50A) resulted in enhanced Src kinase activity, p52shc phosphorylation, MAPK activation, and cell proliferation in response to IGF-I. To determine the mechanism of inhibition, the full-length CH2 domain and intact p66shc were tested for their ability to directly inhibit Src kinase activation in vitro. The CH2 domain peptide was clearly inhibitory, but full-length p66shc had a greater effect. Deletion of the C-terminal Src homology 2 domain in p66shc reduced its ability to inhibit Src kinase activation. These findings demonstrate that p66shc utilizes a novel mechanism for modulating Src kinase activation and that this interaction is mediated through both its collagen homologous region 2 and Src homology 2 domains. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.