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

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. Insulin-like growth factor-I (IGF-I)2 stimulates proliferation and migration in vascular smooth muscle cells (VSMCs) (1). IGF-I-stimulated VSMC migration and/or proliferation requires ligand occupancy of the ␣V␤3 integrin (2). Activation of the IGF-I receptor is coupled to downstream signaling events via recruitment and phosphorylation of two major adaptor proteins, Shc or members of the insulin receptor substrate (IRS) family. In VSMCs IGF-I stimulation leads to recruitment of the signaling protein Shc to SHP substrate-1 (SHPS-1) allowing sustained MAPK activation (3). IGF-I stimulation of phosphatidylinositol 3-kinase (PI3K) is also required to induce VSMC migration (4). Hyperglycemia has been shown to increase the responsiveness of smooth muscle cells as well as other cell types to growth factor stimulation (5-9). Following IGF-I stimulation of porcine VSMCs maintained in hyperglycemic conditions (e.g. Ͼ12 mM glucose) there is an increase in Shc phosphorylation and subsequent Grb2 association resulting in sustained MAPK activation and enhanced cell proliferation. In contrast cells grown in 5 mM glucose do not show these changes (5). Mutation of three Shc tyrosine phosphorylation sites, Tyr-239/ 240/317, that are required for Grb2 associatio...

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…This review will concentrate on publications in the last two years on the SIRPα recognition and constraints on the functional outcome. Detailed analysis of signalling mechanisms is outside the scope of the review although there are recent data [2][3][4][5][6][7][8]; however molecular interactions that influence whether signalling will occur will be discussed.…”

Insulin-like Growth Factor-I-stimulated Insulin Receptor Substrate-1 Negatively Regulates Src Homology 2 Domain-containing Protein-tyrosine Phosphatase Substrate-1 Function in Vascular Smooth Muscle Cells

“…353046, Falcon) the day before transduction. The viral stocks were thawed, and the viral complexes were precipitated as described previously (2,3). The expression of the HA-tagged IRS-1-WT and IRS-1 mutants were detected by immunoblotting with a 1:1000 dilution of anti-HA antibody using 20 g of protein.…”

“…All of the transfections and viral stock generations were carried out as described previously (2,3).…”

“…IGF-I is a known stimulant of vascular smooth muscle cell (VSMC) proliferation and migration, and these effects are mediated through the phosphatidylinositol 3-kinase and MAPK pathways (1)(2)(3). Upon ligand occupancy, the receptor undergoes a conformational change that activates its tyrosine kinase, which phosphorylates downstream signaling molecules.…”

“…Our studies have shown that SHPS-1 phosphorylation and the subsequent assembly of a signaling complex that includes SHP-2, Src, Shc (Src homology 2 domain-containing protein), and Grb2 (growth factor receptor-bound protein 2) is essential for these cells to respond to hyperglycemic stress and that it enhances the ability of IGF-I to activate both MAPK and phosphatidylinositol 3-kinase pathways, leading to increased proliferation and migration (2,3,20). Following their tyrosine phosphorylation, IRS-1 and Shc bind independently to Grb2, which increases activation of MAPK/ extracellular signal-regulated kinase (ERK) (2,21,22).…”