The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells

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.

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p66shc Negatively Regulates Insulin-Like Growth Factor I Signal Transduction via Inhibition of p52shc Binding to Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Leading to Impaired Growth Factor Receptor-Bound Protein-2 Membrane Recruitment

Molecular Endocrinology ◽

10.1210/me.2008-0079 ◽

2008 ◽

Vol 22 (9) ◽

pp. 2162-2175 ◽

Cited By ~ 37

Author(s):

Gang Xi ◽

Xinchun Shen ◽

David R. Clemmons

Keyword(s):

Growth Factor ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Growth Factor Receptor ◽

Membrane Recruitment ◽

Factor I ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Growth Factor Receptor Bound

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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

Journal of Biological Chemistry ◽

10.1074/jbc.m109.092270 ◽

2010 ◽

Vol 285 (21) ◽

pp. 15682-15695 ◽

Cited By ~ 18

Author(s):

Yashwanth Radhakrishnan ◽

Walker H. Busby ◽

Xinchun Shen ◽

Laura A. Maile ◽

David R. Clemmons

Keyword(s):

Smooth Muscle ◽

Growth Factor ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Insulin Receptor Substrate ◽

Factor I ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Phosphatase Substrate

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Recruitment of the Tyrosine Phosphatase Src Homology 2 Domain Tyrosine Phosphatase-2 to the p85 Subunit of Phosphatidylinositol-3 (PI-3) Kinase Is Required for Insulin-Like Growth Factor-I-Dependent PI-3 Kinase Activation in Smooth Muscle Cells

Endocrinology ◽

10.1210/en.2005-1115 ◽

2006 ◽

Vol 147 (3) ◽

pp. 1458-1465 ◽

Cited By ~ 15

Author(s):

Mijin Kwon ◽

Yan Ling ◽

Laura A. Maile ◽

Jane Badley-Clark ◽

David R. Clemmons

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Tyrosine Phosphatase ◽

Insulin Receptor Substrate ◽

Kinase Activation ◽

Src Homology 2 ◽

Igf I ◽

Src Homology ◽

Src Homology 2 Domain ◽

Kinase Pathway

IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.

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The αVβ3 Integrin Regulates Insulin-Like Growth Factor I (IGF-I) Receptor Phosphorylation by Altering the Rate of Recruitment of the Src-Homology 2-Containing Phosphotyrosine Phosphatase-2 to the Activated IGF-I Receptor

Endocrinology ◽

10.1210/en.2002-220395 ◽

2002 ◽

Vol 143 (11) ◽

pp. 4259-4264 ◽

Cited By ~ 49

Author(s):

Laura A. Maile ◽

David R. Clemmons

Keyword(s):

Growth Factor ◽

Αvβ3 Integrin ◽

Insulin Like Growth Factor ◽

Phosphotyrosine Phosphatase ◽

Receptor Phosphorylation ◽

Factor I ◽

Src Homology 2 ◽

Igf I ◽

Src Homology ◽

Growth Factor I

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SHP-2-Induced Activation of c-Myc Is Involved in PDGF-B-Regulated Cell Proliferation and Angiogenesis in RMECs

Frontiers in Physiology ◽

10.3389/fphys.2020.555006 ◽

2020 ◽

Vol 11 ◽

Author(s):

Jun Ma ◽

Wenyi Tang ◽

Ruiping Gu ◽

Fangyuan Hu ◽

Lei Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Molecular Mechanisms ◽

Tyrosine Phosphatase ◽

Tube Formation ◽

Proliferative Retinopathy ◽

Cell Counting ◽

Brdu Incorporation ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

Background: Aberrant neovascularization resulting from inappropriate angiogenic signaling is closely related to many diseases, such as cancer, cardiovascular disease, and proliferative retinopathy. Although some factors involved in regulating pathogenic angiogenesis have been identified, the molecular mechanisms of proliferative retinopathy remain largely unknown. In the present study, we determined the role of platelet-derived growth factor-B (PDGF-B), one of the HIF-1-responsive gene products, in cell proliferation and angiogenesis in retinal microvascular endothelial cells (RMECs) and explored its regulatory mechanism.Methods: Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, tube formation, cell migration, and Western blot assays were used in our study.Results: Our results showed that PDGF-B promoted cell proliferation and angiogenesis by increasing the activity of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) in RMECs, which was attenuated by the inhibition of PDGF receptor (PDGFR) or SHP-2 knockdown. Moreover, activation of c-Myc was involved in the processes of PDGF-B/SHP-2-driven cell proliferation in RMECs. The promoting effects of PDGF-B/SHP-2 on c-Myc expression were mediated by the Erk pathway.Conclusion: These results indicate that PDGF-B facilitates cell proliferation and angiogenesis, at least in part, via the SHP-2/Erk/c-Myc pathway in RMECs, implying new potential treatment candidates for retinal microangiopathy.

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Integrin-Associated Protein Association With Src Homology 2 Domain Containing Tyrosine Phosphatase Substrate 1 Regulates IGF-I Signaling In Vivo

Diabetes ◽

10.2337/db08-0326 ◽

2008 ◽

Vol 57 (10) ◽

pp. 2637-2643 ◽

Cited By ~ 25

Author(s):

L. A. Maile ◽

B. E. Capps ◽

E. C. Miller ◽

A. W. Aday ◽

D. R. Clemmons

Keyword(s):

Tyrosine Phosphatase ◽

Protein Association ◽

Src Homology 2 ◽

Igf I ◽

Src Homology ◽

Src Homology 2 Domain ◽

Phosphatase Substrate

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Role of SHPS-1 in the Regulation of Insulin-like Growth Factor I–stimulated Shc and Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0918 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3353-3364 ◽

Cited By ~ 48

Author(s):

Yan Ling ◽

Laura A. Maile ◽

Jaroslava Lieskovska ◽

Jane Badley-Clarke ◽

David R. Clemmons

Keyword(s):

Tyrosine Phosphatase ◽

Mitogen Activated Protein Kinase ◽

Mutant Form ◽

Mapk Phosphorylation ◽

Src Homology 2 ◽

Igf I ◽

Mitogen Activated Protein ◽

Src Homology ◽

Src Homology 2 Domain

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 β3 subunit of the αVβ3, 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-I–dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.

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