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PTP-1B is an essential positive regulator of platelet integrin signaling.

Arias-Salgado EG, Haj F, Dubois C, Moran B, Kasirer-Friede A, Furie BC, Furie B, Neel BG, Shattil SJ - J. Cell Biol. (2005)

Bottom Line: In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process.Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B.Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

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Interactions between PTP-1B, αIIbβ3, and c-Src in platelets. (a) Washed human platelets were incubated for 15 min at RT with 250 μg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2. Some samples were preincubated for 15 min with 2 μM SU6656, 5 μM PP2, or 5 μM PP3; the latter is an inactive congener of PP2. Clarified lysates were immunoprecipitated (IP) and probed on immunoblots as indicated. Vertical lines in the blots indicate grouping of images from different parts of the same gel. (b) Washed mouse platelets were incubated with MnCl2 and fibrinogen, and immunoblots of immunoprecipitates were probed as in a. Control immunoprecipitations used normal rabbit serum (NRS) or rat IgG (IgG). (c) Role of PTP-1B in platelet tyrosine phosphorylation. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b. Lysates were immunoblotted with antibodies to phosphotyrosine (pTyr) or c-Src phosphotyrosine 418 and reprobed with antibodies to c-Src. (d) αIIbβ3 surface expression in PTP-1B+/+ (black bar) and PTP-1B−/− (hatched bar) platelets was quantified by flow cytometry. Mean fluorescence intensities are depicted in arbitrary units, and error bars represent means ± SEM of three experiments. (e) PTP-1B is required for activation of integrin-associated c-Src. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and αIIbβ3 immunoprecipitates were probed on immunoblots as indicated. (f) PTP-1B is required for dissociation of Csk from the αIIbβ3–c-Src complex. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and Csk immunoprecipitates were probed on immunoblots as indicated. Each immunoblot panel is representative of three to five independent experiments.
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fig1: Interactions between PTP-1B, αIIbβ3, and c-Src in platelets. (a) Washed human platelets were incubated for 15 min at RT with 250 μg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2. Some samples were preincubated for 15 min with 2 μM SU6656, 5 μM PP2, or 5 μM PP3; the latter is an inactive congener of PP2. Clarified lysates were immunoprecipitated (IP) and probed on immunoblots as indicated. Vertical lines in the blots indicate grouping of images from different parts of the same gel. (b) Washed mouse platelets were incubated with MnCl2 and fibrinogen, and immunoblots of immunoprecipitates were probed as in a. Control immunoprecipitations used normal rabbit serum (NRS) or rat IgG (IgG). (c) Role of PTP-1B in platelet tyrosine phosphorylation. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b. Lysates were immunoblotted with antibodies to phosphotyrosine (pTyr) or c-Src phosphotyrosine 418 and reprobed with antibodies to c-Src. (d) αIIbβ3 surface expression in PTP-1B+/+ (black bar) and PTP-1B−/− (hatched bar) platelets was quantified by flow cytometry. Mean fluorescence intensities are depicted in arbitrary units, and error bars represent means ± SEM of three experiments. (e) PTP-1B is required for activation of integrin-associated c-Src. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and αIIbβ3 immunoprecipitates were probed on immunoblots as indicated. (f) PTP-1B is required for dissociation of Csk from the αIIbβ3–c-Src complex. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and Csk immunoprecipitates were probed on immunoblots as indicated. Each immunoblot panel is representative of three to five independent experiments.

Mentions: To explore how αIIbβ3 regulates c-Src, we sought to identify a PTP that localizes to the αIIbβ3–c-Src complex in response to fibrinogen binding to platelets. We reasoned that this might reverse phosphorylation of c-Src tyrosine 529 by Csk and, thereby, help to promote c-Src activation (Obergfell et al., 2002; Arias-Salgado et al., 2003). A previous study has demonstrated that PTP-1B is localized to internal membranes of resting platelets and is cleaved by calpain in a platelet aggregation–dependent manner (Frangioni et al., 1993). We found that PTP-1B coimmunoprecipitated with αIIbβ3 and c-Src from detergent lysates of human and mouse platelets. However, unlike the associations of c-Src and Csk with αIIbβ3, which are observed in resting platelets (Obergfell et al., 2002), the association of PTP-1B with αIIbβ3 and c-Src required fibrinogen binding to platelets. This was induced either by MnCl2, which activates αIIbβ3 directly (Fig. 1 a; Litvinov et al., 2004), or by plating the cells on fibrinogen (not depicted). PTP-1B recruitment to αIIbβ3 in response to MnCl2 and fibrinogen did not require PTP-1B cleavage by calpain because platelet aggregation was avoided under these unstirred conditions, and no such cleavage was observed. The interaction of PTP-1B with αIIbβ3 was specific and was observed whether immunoprecipitation was performed with antibodies to PTP-1B or αIIbβ3 (Fig. 1 b). The interactions of PTP-1B with αIIbβ3 and c-Src were prevented by pretreatment of platelets with 2 μM SU6656 or 5 μM PP2 to block Src kinase activity (Fig. 1 a) or with 2 mM RGDS (Arg-Gly-Asp-Ser) to inhibit fibrinogen binding.


PTP-1B is an essential positive regulator of platelet integrin signaling.

Arias-Salgado EG, Haj F, Dubois C, Moran B, Kasirer-Friede A, Furie BC, Furie B, Neel BG, Shattil SJ - J. Cell Biol. (2005)

Interactions between PTP-1B, αIIbβ3, and c-Src in platelets. (a) Washed human platelets were incubated for 15 min at RT with 250 μg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2. Some samples were preincubated for 15 min with 2 μM SU6656, 5 μM PP2, or 5 μM PP3; the latter is an inactive congener of PP2. Clarified lysates were immunoprecipitated (IP) and probed on immunoblots as indicated. Vertical lines in the blots indicate grouping of images from different parts of the same gel. (b) Washed mouse platelets were incubated with MnCl2 and fibrinogen, and immunoblots of immunoprecipitates were probed as in a. Control immunoprecipitations used normal rabbit serum (NRS) or rat IgG (IgG). (c) Role of PTP-1B in platelet tyrosine phosphorylation. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b. Lysates were immunoblotted with antibodies to phosphotyrosine (pTyr) or c-Src phosphotyrosine 418 and reprobed with antibodies to c-Src. (d) αIIbβ3 surface expression in PTP-1B+/+ (black bar) and PTP-1B−/− (hatched bar) platelets was quantified by flow cytometry. Mean fluorescence intensities are depicted in arbitrary units, and error bars represent means ± SEM of three experiments. (e) PTP-1B is required for activation of integrin-associated c-Src. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and αIIbβ3 immunoprecipitates were probed on immunoblots as indicated. (f) PTP-1B is required for dissociation of Csk from the αIIbβ3–c-Src complex. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and Csk immunoprecipitates were probed on immunoblots as indicated. Each immunoblot panel is representative of three to five independent experiments.
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Related In: Results  -  Collection

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fig1: Interactions between PTP-1B, αIIbβ3, and c-Src in platelets. (a) Washed human platelets were incubated for 15 min at RT with 250 μg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2. Some samples were preincubated for 15 min with 2 μM SU6656, 5 μM PP2, or 5 μM PP3; the latter is an inactive congener of PP2. Clarified lysates were immunoprecipitated (IP) and probed on immunoblots as indicated. Vertical lines in the blots indicate grouping of images from different parts of the same gel. (b) Washed mouse platelets were incubated with MnCl2 and fibrinogen, and immunoblots of immunoprecipitates were probed as in a. Control immunoprecipitations used normal rabbit serum (NRS) or rat IgG (IgG). (c) Role of PTP-1B in platelet tyrosine phosphorylation. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b. Lysates were immunoblotted with antibodies to phosphotyrosine (pTyr) or c-Src phosphotyrosine 418 and reprobed with antibodies to c-Src. (d) αIIbβ3 surface expression in PTP-1B+/+ (black bar) and PTP-1B−/− (hatched bar) platelets was quantified by flow cytometry. Mean fluorescence intensities are depicted in arbitrary units, and error bars represent means ± SEM of three experiments. (e) PTP-1B is required for activation of integrin-associated c-Src. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and αIIbβ3 immunoprecipitates were probed on immunoblots as indicated. (f) PTP-1B is required for dissociation of Csk from the αIIbβ3–c-Src complex. Fibrinogen binding to PTP-1B+/+ and PTP-1B−/− platelets was induced as in b, and Csk immunoprecipitates were probed on immunoblots as indicated. Each immunoblot panel is representative of three to five independent experiments.
Mentions: To explore how αIIbβ3 regulates c-Src, we sought to identify a PTP that localizes to the αIIbβ3–c-Src complex in response to fibrinogen binding to platelets. We reasoned that this might reverse phosphorylation of c-Src tyrosine 529 by Csk and, thereby, help to promote c-Src activation (Obergfell et al., 2002; Arias-Salgado et al., 2003). A previous study has demonstrated that PTP-1B is localized to internal membranes of resting platelets and is cleaved by calpain in a platelet aggregation–dependent manner (Frangioni et al., 1993). We found that PTP-1B coimmunoprecipitated with αIIbβ3 and c-Src from detergent lysates of human and mouse platelets. However, unlike the associations of c-Src and Csk with αIIbβ3, which are observed in resting platelets (Obergfell et al., 2002), the association of PTP-1B with αIIbβ3 and c-Src required fibrinogen binding to platelets. This was induced either by MnCl2, which activates αIIbβ3 directly (Fig. 1 a; Litvinov et al., 2004), or by plating the cells on fibrinogen (not depicted). PTP-1B recruitment to αIIbβ3 in response to MnCl2 and fibrinogen did not require PTP-1B cleavage by calpain because platelet aggregation was avoided under these unstirred conditions, and no such cleavage was observed. The interaction of PTP-1B with αIIbβ3 was specific and was observed whether immunoprecipitation was performed with antibodies to PTP-1B or αIIbβ3 (Fig. 1 b). The interactions of PTP-1B with αIIbβ3 and c-Src were prevented by pretreatment of platelets with 2 μM SU6656 or 5 μM PP2 to block Src kinase activity (Fig. 1 a) or with 2 mM RGDS (Arg-Gly-Asp-Ser) to inhibit fibrinogen binding.

Bottom Line: In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process.Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B.Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

Show MeSH
Related in: MedlinePlus