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J. Biol. Chem., Vol. 283, Issue 24, 16320-16331, June 13, 2008

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Insulin-like Growth Factor-I Stimulates Shc-dependent Phosphatidylinositol 3-Kinase Activation via Grb2-associated p85 in Vascular Smooth Muscle Cells^*

Yashwanth Radhakrishnan, Laura A. Maile, Yan Ling, Lee M. Graves, and David R. Clemmons¹

From the Departments of Medicine and Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Insulin-like growth factor-I (IGF-I) stimulates vascular smooth muscle cell proliferation and migration by activating both MAPK and phosphatidylinositol 3-kinase (PI3K). Vascular smooth muscle cells (VSMCs) maintained in 25 mM glucose sustain MAPK activation via increased Shc phosphorylation and Grb2 association resulting in an enhanced mitogenic response compared with cells grown in 5 mM glucose. PI3K plays a major role in IGF-I-stimulated VSMC migration, and hyperglycemia augments this response. In contrast to MAPK activation the role of Shc in modulating PI3K in response to IGF-I has not been determined. In this study we show that impaired Shc association with Grb2 results in decreased Grb2-p85 association, SHPS-1-p85 recruitment, and PI3K activation in response to IGF-I. Exposure of VSMCs to cell-permeable peptides, which contained polyproline sequences from p85 proposed to mediate Grb2 association, resulted in inhibition of Grb2-p85 binding and AKT phosphorylation. Transfected cells that expressed p85 mutant that had specific prolines mutated to alanines resulted in less Grb2-p85 association, and a Grb2 mutant (W36A/W193A) that attenuated p85 binding showed decreased association of p85 with SHPS-1, PI3K activation, AKT phosphorylation, cell proliferation, and migration in response to IGF-I. Cellular exposure to 25 mM glucose, which is required for Shc phosphorylation in response to IGF-I, resulted in enhanced Grb2 binding to p85, activation of PI3K activity, and increased AKT phosphorylation as compared with cells exposed to 5 mM glucose. We conclude that in VSMCs exposed to hyperglycemia, IGF-I stimulation of Shc facilitates the transfer of Grb2 to p85 resulting in enhanced PI3K activation and AKT phosphorylation leading to enhanced cell proliferation and migration.

Received for publication, March 3, 2008 , and in revised form, April 15, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant HL56850. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Division of Endocrinology, University of North Carolina, CB 7170, 5030 Burnett-Womack, Chapel Hill, NC 27599-7170. Tel.: 919-966-4735; Fax: 919-966-6025; E-mail: endo{at}med.unc.edu.

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