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.
p66(shc) is increased in response to cell stress, and these increases regulate growth factor actions. These studies were conducted to determine how p66(shc) alters IGF-I-stimulated Src activation, leading to decreased IGF-I actions. Our results show that p66(shc) binds to Src through a polyproline sequence motif contained in the CH2 domain, a unique domain in p66(shc), and IGF-I stimulates this interaction. Disruption of this interaction using a synthetic peptide containing the p66(shc) polyproline domain or expression of a p66(shc) mutant containing substitutions for the proline residues (P47A/P48A/P50A) resulted in enhanced Src kinase activity, p52(shc) phosphorylation, MAPK activation, and cell proliferation in response to IGF-I. To determine the mechanism of inhibition, the full-length CH2 domain and intact p66(shc) were tested for their ability to directly inhibit Src kinase activation in vitro. The CH2 domain peptide was clearly inhibitory, but full-length p66(shc) had a greater effect. Deletion of the C-terminal Src homology 2 domain in p66(shc) reduced its ability to inhibit Src kinase activation. These findings demonstrate that p66(shc) 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.
Xi
Gang
G
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Shen
Xinchun
X
Clemmons
David R
DR
D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D018909:src Homology Domains; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000943:Antigens, Differentiation; D051380:GRB2 Adaptor Protein; D046912:Multiprotein Complexes; D010455:Peptides; D011971:Receptors, Immunologic; D055765:Shc Signaling Adaptor Proteins; D019000:Phosphotyrosine; C011083:polyproline; D007334:Insulin-Like Growth Factor I; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases