Abstract
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.
Highlights
Grants HL56850 and AG02331. □S The on-line version of this article contains supplemental Fig. 1. 1 To whom correspondence should be addressed: CB 7170, 5030 Burnett
Following insulin-like growth factor-I (IGF-I) receptor stimulation, the integral membrane protein SHPS-1 (Src homology 2 domain-containing protein-tyrosine phosphatase substrate-1) is tyrosine-phosphorylated, and a signaling complex composed of SHP-2 (Src homology 2 domain-containing protein-tyrosine phosphatase-2), c-Src, and p52shc is assembled on SHPS-1
Because both p52 and p66shc isoforms are induced in response to hyperglycemic stress yet they have been reported to mediate different actions, we investigated the role of p66shc in modulating p52shc activation and showed that its overexpression attenuated p52shc phosphorylation and inhibited the mitogenic response to IGF-I [10]
Summary
Human IGF-I was a gift from Genentech (South San Francisco, CA). Dulbecco’s modified Eagle’s medium containing. An aliquot of the individual TNT mixture (0.4 M total protein for each) or different combinations of them, as indicated, were incubated with 200 ng of purified active Src (Millipore Corp.) in binding buffer (HEPES, pH 7.6, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2% Triton X-100, and 10% glycerol) in the presence or absence of p189 or p226 (1 g/ml), using a 100-l final volume and rotation for 2 h at 4 °C. An aliquot of the TNT mixture of each (final concentration 0.4 M protein) or combinations of them as indicated were incubated with 2.5 units of purified active Src, 150 M Src kinase substrate peptide (KVEKIGEGTYGVVYK), and 10 Ci of [␥-32P]ATP in Src reaction buffer (25 mM Tris-HCl, pH 7.2, 25 mM MgCl2, 1.25 mM MnCl2, 0.5 mM EGTA, 60 M sodium orthovanadate, 0.5 mM dithiothreitol, and 100 m ATP) in the presence or absence of p189 or p226. Student’s t test was used to compare differences between control and treatment groups or control cells and cells expressing mutant proteins. p Յ 0.05 was considered statistically significant
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