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

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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A Filarial Nematode-Secreted Phosphorylcholine-Containing Glycoprotein Uncouples the B Cell Antigen Receptor from Extracellular Signal-Regulated Kinase-Mitogen-Activated Protein Kinase by Promoting the Surface Ig-Mediated Recruitment of Src Homology 2 Domain-Containing Tyrosine Phosphatase-1 and Pac-1 Mitogen-Activated Kinase-Phosphatase

The Journal of Immunology ◽

10.4049/jimmunol.166.12.7462 ◽

2001 ◽

Vol 166 (12) ◽

pp. 7462-7468 ◽

Cited By ~ 33

Author(s):

Maureen R. Deehan ◽

William Harnett ◽

Margaret M. Harnett

Keyword(s):

Protein Kinase ◽

Tyrosine Phosphatase ◽

Mitogen Activated Protein Kinase ◽

B Cell Antigen Receptor ◽

Extracellular Signal Regulated Kinase ◽

Src Homology 2 ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Src Homology ◽

Src Homology 2 Domain

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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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Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Promotes Inflammation and Accelerates Osteoarthritis by Activating β-Catenin

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.646386 ◽

2021 ◽

Vol 9 ◽

Author(s):

Tenghui Tao ◽

Danni Luo ◽

Chenghao Gao ◽

Hui Liu ◽

Zehua Lei ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Cartilage Degradation ◽

Mitogen Activated Protein Kinase ◽

Osteophyte Formation ◽

Protein Tyrosine ◽

Src Homology 2 ◽

Primary Mouse ◽

Src Homology ◽

Src Homology 2 Domain

Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation, subchondral bone remodeling and osteophyte formation. Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) has not been fully investigated in the pathogenesis of OA. In this study, we found that SHP2 expression was significantly increased after interleukin-1β (IL-1β) treatment in primary mouse chondrocytes. Inhibition of SHP2 using siRNA reduced MMP3, MMP13 levels, but increased AGGRECAN, COL2A1, SOX9 expression in vitro. On the contrary, overexpression of SHP2 exerted the opposite results and promoted cartilage degradation. Mechanistically, SHP2 activated Wnt/β-catenin signaling possibly through directly binding to β-catenin. SHP2 also induced inflammation through activating Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Our in vivo studies showed that SHP2 knockdown effectively delayed cartilage destruction and reduced osteophyte formation in the mouse model of OA induced by destabilization of the medial meniscus (DMM). Altogether, our study identifies that SHP2 is a novel and potential therapeutic target of OA.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e03-04-0239 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3519-3528 ◽

Cited By ~ 38

Author(s):

Laura A. Maile ◽

Jane Badley-Clarke ◽

David R. Clemmons

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Tyrosine Phosphatase ◽

Growth Factor Signaling ◽

Factor I ◽

Src Homology 2 ◽

Igf I ◽

Important Regulator ◽

Src Homology ◽

Src Homology 2 Domain

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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v-Crk Modulation of Growth Factor-induced PC12 Cell Differentiation Involves the Src Homology 2 Domain of v-Crk and Sustained Activation of the Ras/Mitogen-activated Protein Kinase Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.270.35.20677 ◽

1995 ◽

Vol 270 (35) ◽

pp. 20677-20685 ◽

Cited By ~ 100

Author(s):

Kenneth K. Teng ◽

Harry Lander ◽

J. Eduardo Fajardo ◽

Hidesaburo Hanafusa ◽

Barbara L. Hempstead ◽

...

Keyword(s):

Cell Differentiation ◽

Growth Factor ◽

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Src Homology 2 ◽

Mitogen Activated Protein ◽

Src Homology ◽

Src Homology 2 Domain ◽

Kinase Pathway ◽

Sustained Activation

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p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4–p56lck association-dependent manner

Biochemical Journal ◽

10.1042/bj20061061 ◽

2007 ◽

Vol 402 (3) ◽

pp. 471-481 ◽

Cited By ~ 16

Author(s):

Christiane Barbat ◽

Maylis Trucy ◽

Maurizio Sorice ◽

Tina Garofalo ◽

Valeria Manganelli ◽

...

Keyword(s):

Tyrosine Phosphatase ◽

Structural Diversity ◽

Mutant Form ◽

Lymphocyte Function ◽

Dependent Manner ◽

Down Regulation ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Phosphoinositide 3 Kinase

We previously showed that the association of CD4 and GM3 ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the GM1 ganglioside that partially co-localized with GM3 in these domains. In the present study, we show that CD4–p56lck association in CD4 signalling is required for the redistribution of p56lck, PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56lck. In addition, we show that although these proteins associated in different ways with GM1 and GM3, all of the associations were dependent on CD4–p56lck association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56lck in detergent-insoluble membranes without association with GM1 or GM3. We propose that CD4 ligation and binding with p56lck and their interaction with GM3 and/or GM1 gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.

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