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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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Structural features of c-Src that are required for interactions with PTP-1B and αIIbβ3. (a) SYF cells or SYF cells stably expressing human αIIbβ3 (αIIbβ3-SYF) were transiently transfected with wild-type c-Src or empty vector. After 48 h, transfected cells were incubated at 37°C for 15 min in the presence or absence of 1 mM MnCl2 and 250 μg/ml fibrinogen. Clarified lysates were immunoprecipitated with antibodies to c-Src or β3, and immunoprecipitates were probed on immunoblots as indicated. Lysates were probed for PTP-1B as a loading control. (b and c) αIIbβ3-SYF cells were transfected with wild-type c-Src or an indicated c-Src mutant. After 48 h, transfected cells were incubated in the presence or absence of MnCl2 and fibrinogen as in a. Clarified lysates were immunoprecipitated with antibodies to β3 (b) or c-Src (c) and with the precipitates probed on immunoblots. Data are from a single experiment that was representative of three that were performed.
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fig2: Structural features of c-Src that are required for interactions with PTP-1B and αIIbβ3. (a) SYF cells or SYF cells stably expressing human αIIbβ3 (αIIbβ3-SYF) were transiently transfected with wild-type c-Src or empty vector. After 48 h, transfected cells were incubated at 37°C for 15 min in the presence or absence of 1 mM MnCl2 and 250 μg/ml fibrinogen. Clarified lysates were immunoprecipitated with antibodies to c-Src or β3, and immunoprecipitates were probed on immunoblots as indicated. Lysates were probed for PTP-1B as a loading control. (b and c) αIIbβ3-SYF cells were transfected with wild-type c-Src or an indicated c-Src mutant. After 48 h, transfected cells were incubated in the presence or absence of MnCl2 and fibrinogen as in a. Clarified lysates were immunoprecipitated with antibodies to β3 (b) or c-Src (c) and with the precipitates probed on immunoblots. Data are from a single experiment that was representative of three that were performed.

Mentions: To better understand the basis for interactions between PTP-1B, αIIbβ3, and c-Src during outside-in αIIbβ3 signaling, mouse fibroblasts that were deficient in the ubiquitous Src family kinases c-Src, c-Yes, and Fyn (SYF cells; Klinghoffer et al., 1999) were stably transfected with αIIbβ3. These αIIbβ3-SYF cells express PTP-1B endogenously, enabling examination of PTP-1B interactions after transient transfection of c-Src. As observed with platelets, αIIbβ3-SYF cells expressing c-Src showed a fibrinogen-inducible association of PTP-1B with c-Src and αIIbβ3 (Fig. 2 a). However, PTP-1B failed to associate with c-Src in cells lacking αIIbβ3 or with αIIbβ3 in cells lacking c-Src. Thus, the fibrinogen-dependent interaction of PTP-1B with αIIbβ3 or c-Src requires both integrin and tyrosine kinase.


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)

Structural features of c-Src that are required for interactions with PTP-1B and αIIbβ3. (a) SYF cells or SYF cells stably expressing human αIIbβ3 (αIIbβ3-SYF) were transiently transfected with wild-type c-Src or empty vector. After 48 h, transfected cells were incubated at 37°C for 15 min in the presence or absence of 1 mM MnCl2 and 250 μg/ml fibrinogen. Clarified lysates were immunoprecipitated with antibodies to c-Src or β3, and immunoprecipitates were probed on immunoblots as indicated. Lysates were probed for PTP-1B as a loading control. (b and c) αIIbβ3-SYF cells were transfected with wild-type c-Src or an indicated c-Src mutant. After 48 h, transfected cells were incubated in the presence or absence of MnCl2 and fibrinogen as in a. Clarified lysates were immunoprecipitated with antibodies to β3 (b) or c-Src (c) and with the precipitates probed on immunoblots. Data are from a single experiment that was representative of three that were performed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171339&req=5

fig2: Structural features of c-Src that are required for interactions with PTP-1B and αIIbβ3. (a) SYF cells or SYF cells stably expressing human αIIbβ3 (αIIbβ3-SYF) were transiently transfected with wild-type c-Src or empty vector. After 48 h, transfected cells were incubated at 37°C for 15 min in the presence or absence of 1 mM MnCl2 and 250 μg/ml fibrinogen. Clarified lysates were immunoprecipitated with antibodies to c-Src or β3, and immunoprecipitates were probed on immunoblots as indicated. Lysates were probed for PTP-1B as a loading control. (b and c) αIIbβ3-SYF cells were transfected with wild-type c-Src or an indicated c-Src mutant. After 48 h, transfected cells were incubated in the presence or absence of MnCl2 and fibrinogen as in a. Clarified lysates were immunoprecipitated with antibodies to β3 (b) or c-Src (c) and with the precipitates probed on immunoblots. Data are from a single experiment that was representative of three that were performed.
Mentions: To better understand the basis for interactions between PTP-1B, αIIbβ3, and c-Src during outside-in αIIbβ3 signaling, mouse fibroblasts that were deficient in the ubiquitous Src family kinases c-Src, c-Yes, and Fyn (SYF cells; Klinghoffer et al., 1999) were stably transfected with αIIbβ3. These αIIbβ3-SYF cells express PTP-1B endogenously, enabling examination of PTP-1B interactions after transient transfection of c-Src. As observed with platelets, αIIbβ3-SYF cells expressing c-Src showed a fibrinogen-inducible association of PTP-1B with c-Src and αIIbβ3 (Fig. 2 a). However, PTP-1B failed to associate with c-Src in cells lacking αIIbβ3 or with αIIbβ3 in cells lacking c-Src. Thus, the fibrinogen-dependent interaction of PTP-1B with αIIbβ3 or c-Src requires both integrin and tyrosine kinase.

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