Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the ␤3 subunit of the ␣V␤3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-Idependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs. INTRODUCTIONBoth vascular smooth muscle cell proliferation and migration in response to growth factor stimulation play important roles in the formation of atherosclerotic plaques, and insulin-like growth factor I (IGF-I) is a potent stimulator of smooth muscle cell proliferation and migration (Jones et al., 1996). In primary cultured smooth muscle cells (pSMCs), IGF-I induces activation of both the phosphatidylinositol (PI)-3 kinase and the mitogen-activated protein (MAP) kinase (MAPK) pathways. Both pathways have been shown to play roles in mediating IGF-I-dependent cell migration and cell proliferation responses (Imai and Clemmons, 1999;Maile et al., 2003).Previous studies have shown that the protein tyrosine phosphatase SHP-2 plays an impor...

Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC).Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr 328 ) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr 328 / Tyr 358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phosphorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2⌬10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2⌬10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response. IGF-I2 stimulation of vascular smooth muscle cells (SMC) leads to activation of two major signaling pathways, e.g. the MAP kinase (MAPK) and PI 3-kinase pathways (1). Activation of the MAPK pathway is required for IGF-I-dependent proliferation, whereas activation of the PI 3-kinase pathway is the predominant determinant of IGF-I-dependent SMC migration. BindingofIGF-ItotheIGF-Ireceptorleadstoreceptorautophosphorylation followed by tyrosine phosphorylation of substrates such as IRS-1 and Shc (2). These adaptor proteins bind Grb2/ SOS and activate the Ras/MAPK pathway (3). Previous studies in SMC have shown that IGF-I-induced Shc phosphorylation and its association with Grb-2 are necessary for sustained phosphorylation of Erk1/2 MAPK and IGF-I-dependent cell proliferation (4). The requirement of Shc phosphorylation for growth factor-dependent mitogenesis has been demonstrated in other cell types as well (5, 6).Src family kinases (SFK) have been implicated in mediating the mitogenic effect of several growth factors (7, 8); however, the mechanism by which SFK function is not completely understood. Src has also been implicated i...

Growth factor signaling is usually analyzed in isolation without considering the effect of ligand occupancy of transmembrane proteins other than the growth factor receptors themselves. In smooth muscle cells, the transmembrane protein Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) has been shown to be an important regulator of insulin-like growth factor-I (IGF-I) signaling. SHPS-1 is phosphorylated in response to IGF-I, leading to recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2). Subsequently, SHP-2 is transferred to IGF-I receptor and regulates the duration of IGF-I receptor phosphorylation. Whether ligand occupancy of SHPS-1 influences SHPS-1 phosphorylation or SHP-2 recruitment, thereby altering growth factor signaling, is unknown. Previous studies have shown that integrin associated protein (IAP) associates with SHPS-1. We undertook these studies to determine whether this interaction controlled SHPS-1 phosphorylation and/or SHP-2 recruitment and thereby regulated IGF-I signaling. Disruption of IAP-SHPS-1 binding, by using an IAP monoclonal antibody or cells expressing mutant forms of IAP that did not bind to SHPS-1, inhibited IGF-I-stimulated SHPS-1 phosphorylation and SHP-2 recruitment. This was associated with a lack of SHP-2 transfer to IGF-I receptor and sustained receptor phosphorylation. This resulted in an inability of IGF-I to stimulate sustained mitogen-activated protein kinase activation, cell proliferation, and cell migration. The effect was specific for IGF-I because disruption of the IAP-SHPS-1 interaction had no effect on platelet-derived growth factor-stimulated SHPS-1 phosphorylation or cell migration. In summary, our results show that 1) ligand occupancy of SHPS-1 is a key determinant of its ability to be phosphorylated after IGF-I stimulation, and 2) the interaction between IAP and SHPS-1 is an important regulator of IGF-I signaling because disruption of the results in impaired SHP-2 recruitment and subsequent inhibition of IGF-I-stimulated cell proliferation and migration.

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.

Recruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated ␤3 subunit of the ␣V␤3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to ␤3, we attempted to identify a linker protein that could mediate SHP-2/␤3 association. DOK1 is a member of insulin receptor substrate protein family that binds ␤3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to ␤3 and to SHP-2. Preincubation of cells with synthetic peptides that blocked either DOK1/␤3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to ␤3. Expression of a DOK1 mutant that does not bind to ␤3 also disrupts SHP-2/␤3 association. As a result of SHP-2/␤3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/␤3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells. Vascular smooth muscle cell (SMC)1 migration and proliferation play significant roles in atherosclerotic plaque formation (1). Insulin-like growth factor I (IGF-I) is a potent stimulant of SMC migration and proliferation responses (2). We have shown previously that ligand occupancy of the ␣V␤3 integrin is required for SMC to respond appropriately to IGF-I (3). Blocking the ligand occupancy of ␣V␤3 inhibits IGF-I-dependent downstream signaling including phosphorylation of IRS-1 (3) and the trans-membrane, scaffolding protein Src homology 2 domain containing protein-tyrosine phosphatase substrate-1 (SHPS-1), as well as cell migration and proliferation (4). One important event that occurs in response to ligand occupancy of ␣V␤3 is the phosphorylation of the ␤3 subunit, and previous studies have shown that this is required for IGF-I-dependent signaling and biologic actions (5). SMCs expressing a mutant form of ␤3 in which the two tyrosines in the cytoplasmic domain of ␤3 were substituted with phenylalanines did not respond to IGF-I with an increase in DNA synthesis (5). Therefore in SMC phosphorylation of the ␤3 subunit of ␣V␤3 integrin plays a key role in regulating IGF-I dependent cellular responses.Our prior studies have shown that ligand occupancy of ␣V␤3 regulates IGF-IR signaling by regulating the transfer of the protein-tyrosine phosphatase SHP-2 (4, 5). In high density cultures, the ␤3 subunit is constitutively tyrosine phosphorylated and Src homology 2 domain tyrosine phosphatase (SHP-2) can be co-immunoprecipitated with phosphorylated ␤3 (5). This association correlates with membrane localization of SHP-2 and is required for the subsequent transfer of SHP-2 to its membrane substrate protein SHPS-1 following IGF-I stimulation (5). The disruption of SHP-2 and ␤3 association results in the elimination of...