Abstract
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.
Highlights
Insulin-like growth factor-I (IGF-I)2 stimulates proliferation and migration in vascular smooth muscle cells (VSMCs) [1]
Because hyperglycemia has been shown to enhance the ability of IGF-I to stimulate Shc activation and cell migration, this study was undertaken to determine whether Shc activation and subsequent Shc-Grb2 association played a role in phosphatidylinositol 3-kinase (PI3K) activation and whether Grb2 association with p85 was required for IGF-I-stimulated cell migration
Because cell proliferation is decreased in VSMCs expressing the Shc-3F mutant and PI3K activation is required for optimal cell proliferation, we wished to determine whether attenuation of Shc activation would alter the ability of IGF-I to activate PI3K
Summary
Insulin-like growth factor-I (IGF-I) stimulates proliferation and migration in vascular smooth muscle cells (VSMCs) [1]. Shc-dependent PI3K Activation by Grb2-p85 Binding tors themselves (e.g. platelet-derived growth factor or Fc receptors) or present on the receptor-associated proteins, termed adaptors (e.g. IRS-1, Gab, and Cbl). These phosphotyrosine residues act as binding sites for specific SH2 domain-containing effector molecules [15]. The SH2 domain of p85␣ binds to phosphotyrosine residues contained in the sequence pYXXM (where pY is phosphotyrosine) on receptor tyrosine kinases or adaptor molecules such as IRS-1 [16] This binding relieves both the basal inhibition of p110 by p85 and recruits the p85p110 heterodimer to the plasma membrane [17] where it phosphorylates phosphoinositide 4,5-bisphosphate generating phosphatidylinositol 3,4,5-trisphosphate [18]. Because hyperglycemia has been shown to enhance the ability of IGF-I to stimulate Shc activation and cell migration, this study was undertaken to determine whether Shc activation and subsequent Shc-Grb association played a role in PI3K activation and whether Grb association with p85 was required for IGF-I-stimulated cell migration
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