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

Harvard University, Cambridge, Massachusetts, United States
The Journal of Cell Biology (Impact Factor: 9.83). 09/2005; 170(5):837-45. DOI: 10.1083/jcb.200503125
Source: PubMed


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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Available from: Barry Moran
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    • "Consistent with this notion, lack of PTP1B strongly affected protrusion persistence times but not retraction times. Our results provide mechanistic insights to previous findings suggesting a positive regulation of cell matrix– adhesion and spreading by PTP1B in many cell types (Arregui et al., 1998; Cheng et al., 2001; Pathre et al., 2001; Arias-Salgado et al., 2005; Liang et al., 2005; Fuentes and Arregui, 2009). The increased paxillin disassembly kinetics observed in KO cells may contribute to shorten the lifetime of focal complexes. "
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    ABSTRACT: Previous findings established that ER-bound PTP1B targets peripheral cell-matrix adhesions and regulates positively cell adhesion to fibronectin. Here we show that PTP1B enhances focal complex lifetime at the lamellipodium base, delaying their turnover and facilitating α-actinin incorporation. We demonstrate the presence of catalytic PTP1BD181A-α-actinin complexes at focal complexes. Kymograph analysis reveals that PTP1B contributes to lamellar protrusion persistence and directional cell migration. Pull down and FRET analysis also shows that PTP1B is required for efficient integrin-dependent downregulation of RhoA and upregulation of Rac1 during spreading. A substrate trap strategy revealed that FAK/Src recruitment and Src activity were essential for the generation of PTP1B substrates in adhesions. PTP1B targets the negative regulatory site of Src (phosphotyrosine 529), paxillin and p130Cas at peripheral cell-matrix adhesions. We postulate that PTP1B modulates more than one pathway required for focal complex maturation and membrane protrusion, including α-actinin-mediated cytoskeletal anchorage, integrin-dependent activation of the FAK/Src signaling pathway, and RhoA and Rac1 GTPase activity. By doing so, PTP1B contributes to coordinate adhesion turnover, lamellar stability and directional cell migration.
    Full-text · Article · Feb 2013 · Journal of Cell Science
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    • "Previous work from our laboratory showed that PTP1B-D181A colocalized with Src family members at peripheral puncta, most likely associated with cell-matrix adhesion sites [8]. In addition, both PTP1B-D181A and wild type PTP1B co-immunoprecipitate with Src family kinases [8], [23], [24]. Although these studies suggest that PTP1B and Src may be engaged in common protein complexes they do not provide compelling evidence of direct physical interaction at the membrane-substrate interface. "
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    ABSTRACT: PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.
    Full-text · Article · Jun 2012 · PLoS ONE
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    • "Other signaling molecules, involved in outside-in signaling are Protein Tyrosine Phosphatase-1B (PTP-1B) [25], Protein Phosphatase 1C (PP1c) [26], Calcium and integrin binding protein (CIB) [27] and Protein Kinase C-β (PKC-β) [28], [29]. "
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    ABSTRACT: We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G(12/13) signaling pathways. Hence, we evaluated the role of these pathways in TXA(2) generation. Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G(12/13) pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G(12/13) pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA(2) generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA(2) generation. Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G(12/13) pathways contributing to TXA(2) generation in platelets remains elusive.
    Full-text · Article · Feb 2011 · PLoS ONE
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