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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 PTP-1B that are required for interactions with αIIbβ3 and c-Src. (a) αIIbβ3-SYF cells were transiently cotransfected with c-Src and wild-type or mutant forms of HA-tagged human PTP-1B. After 48 h, transfected cells were incubated with or without MnCl2 and fibrinogen as described in Fig. 2. Clarified lysates were immunoprecipitated with antibodies to the HA tag, and precipitates were probed on immunoblots as indicated. (b and c) PTP-1B is tyrosine phosphorylated in response to fibrinogen binding. αIIbβ3-SYF cells transfected with c-Src and empty vector (b) or human platelets (c) were incubated with or without 0.5 mM MnCl2 and 250 μg/ml fibrinogen for 10 min. Some platelet samples were preincubated for 15 min with c-Src inhibitors (5 μM PP2 or 2 μM SU6656) or 5 μM PP3 as a control. Clarified lysates were immunoprecipitated with an antibody to PTP-1B, and immunoprecipitates and lysates were probed on immunoblots. Data are from a single experiment that was representative of three that were performed. NRS, normal rabbit serum.
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fig3: Structural features of PTP-1B that are required for interactions with αIIbβ3 and c-Src. (a) αIIbβ3-SYF cells were transiently cotransfected with c-Src and wild-type or mutant forms of HA-tagged human PTP-1B. After 48 h, transfected cells were incubated with or without MnCl2 and fibrinogen as described in Fig. 2. Clarified lysates were immunoprecipitated with antibodies to the HA tag, and precipitates were probed on immunoblots as indicated. (b and c) PTP-1B is tyrosine phosphorylated in response to fibrinogen binding. αIIbβ3-SYF cells transfected with c-Src and empty vector (b) or human platelets (c) were incubated with or without 0.5 mM MnCl2 and 250 μg/ml fibrinogen for 10 min. Some platelet samples were preincubated for 15 min with c-Src inhibitors (5 μM PP2 or 2 μM SU6656) or 5 μM PP3 as a control. Clarified lysates were immunoprecipitated with an antibody to PTP-1B, and immunoprecipitates and lysates were probed on immunoblots. Data are from a single experiment that was representative of three that were performed. NRS, normal rabbit serum.

Mentions: To establish what regions of PTP-1B are required for these interactions, HA-tagged PTP-1B was cotransfected with c-Src into αIIbβ3-SYF cells. Wild-type PTP-1B and two different phosphatase-inactive “substrate-trapping” mutants (C215S and D181A) each interacted with αIIbβ3 and c-Src (Fig. 3 a). Interestingly, the interaction with c-Src was somewhat greater with the D181A PTP-1B mutant, which is known to exhibit a higher affinity for binding to PTP-1B substrates than the C215S mutant (Flint et al., 1997). These data are consistent with a direct dephosphorylation of c-Src tyrosine 529 by PTP-1B. In contrast to substrate-trapping mutants, the double mutation of proline 309 and 310 to alanine prevented PTP-1B interaction with αIIbβ3 and c-Src, as did the double mutation of tyrosine 152 and 153 to phenylalanine. These amino acid residues may help to mediate interactions of PTP-1B with one or more members of the integrin signaling complex during the early phase of outside-in signaling (Dadke and Chernoff, 2002). In addition, they may enable the phosphorylation of PTP-1B by c-Src because PTP-1B can phosphorylate c-Src in vitro (Jung et al., 1998), and fibrinogen binding to αIIbβ3-SYF cells (Fig. 3 b) or platelets (Fig. 3 c) stimulated tyrosine phosphorylation of PTP-1B in a Src-dependent manner.


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 PTP-1B that are required for interactions with αIIbβ3 and c-Src. (a) αIIbβ3-SYF cells were transiently cotransfected with c-Src and wild-type or mutant forms of HA-tagged human PTP-1B. After 48 h, transfected cells were incubated with or without MnCl2 and fibrinogen as described in Fig. 2. Clarified lysates were immunoprecipitated with antibodies to the HA tag, and precipitates were probed on immunoblots as indicated. (b and c) PTP-1B is tyrosine phosphorylated in response to fibrinogen binding. αIIbβ3-SYF cells transfected with c-Src and empty vector (b) or human platelets (c) were incubated with or without 0.5 mM MnCl2 and 250 μg/ml fibrinogen for 10 min. Some platelet samples were preincubated for 15 min with c-Src inhibitors (5 μM PP2 or 2 μM SU6656) or 5 μM PP3 as a control. Clarified lysates were immunoprecipitated with an antibody to PTP-1B, and immunoprecipitates and lysates were probed on immunoblots. Data are from a single experiment that was representative of three that were performed. NRS, normal rabbit serum.
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Related In: Results  -  Collection

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fig3: Structural features of PTP-1B that are required for interactions with αIIbβ3 and c-Src. (a) αIIbβ3-SYF cells were transiently cotransfected with c-Src and wild-type or mutant forms of HA-tagged human PTP-1B. After 48 h, transfected cells were incubated with or without MnCl2 and fibrinogen as described in Fig. 2. Clarified lysates were immunoprecipitated with antibodies to the HA tag, and precipitates were probed on immunoblots as indicated. (b and c) PTP-1B is tyrosine phosphorylated in response to fibrinogen binding. αIIbβ3-SYF cells transfected with c-Src and empty vector (b) or human platelets (c) were incubated with or without 0.5 mM MnCl2 and 250 μg/ml fibrinogen for 10 min. Some platelet samples were preincubated for 15 min with c-Src inhibitors (5 μM PP2 or 2 μM SU6656) or 5 μM PP3 as a control. Clarified lysates were immunoprecipitated with an antibody to PTP-1B, and immunoprecipitates and lysates were probed on immunoblots. Data are from a single experiment that was representative of three that were performed. NRS, normal rabbit serum.
Mentions: To establish what regions of PTP-1B are required for these interactions, HA-tagged PTP-1B was cotransfected with c-Src into αIIbβ3-SYF cells. Wild-type PTP-1B and two different phosphatase-inactive “substrate-trapping” mutants (C215S and D181A) each interacted with αIIbβ3 and c-Src (Fig. 3 a). Interestingly, the interaction with c-Src was somewhat greater with the D181A PTP-1B mutant, which is known to exhibit a higher affinity for binding to PTP-1B substrates than the C215S mutant (Flint et al., 1997). These data are consistent with a direct dephosphorylation of c-Src tyrosine 529 by PTP-1B. In contrast to substrate-trapping mutants, the double mutation of proline 309 and 310 to alanine prevented PTP-1B interaction with αIIbβ3 and c-Src, as did the double mutation of tyrosine 152 and 153 to phenylalanine. These amino acid residues may help to mediate interactions of PTP-1B with one or more members of the integrin signaling complex during the early phase of outside-in signaling (Dadke and Chernoff, 2002). In addition, they may enable the phosphorylation of PTP-1B by c-Src because PTP-1B can phosphorylate c-Src in vitro (Jung et al., 1998), and fibrinogen binding to αIIbβ3-SYF cells (Fig. 3 b) or platelets (Fig. 3 c) stimulated tyrosine phosphorylation of PTP-1B in a Src-dependent manner.

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