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• W2053465902 abstract "Human platelets express the receptor for immunoglobulin G, FcγRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcγRIIa cross-linking was followed by its complete dephosphorylation in an αIIb/β3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcγRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcγRIIa stimulation. Human platelets express the receptor for immunoglobulin G, FcγRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcγRIIa cross-linking was followed by its complete dephosphorylation in an αIIb/β3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcγRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcγRIIa stimulation. Human platelets possess only one Fc γ receptor for immunoglobulin G (FcγRIIa), which is a 40-kDa single-chain transmembrane glycoprotein also present in monocytes, neutrophils, and B lymphocytes (1Rosenfeld S.I. Looney R.J. Leddy J.P. Phipps D.C. Abraham G.N. Anderson C.L. J. Clin. Invest. 1985; 76: 2317-2322Crossref PubMed Scopus (172) Google Scholar). In platelets, clustering of FcγRIIa induces shape change, secretion, and aggregation, which are typical physiological responses required for efficient hemostatic function of these cells (2Anderson C.L. Chacko G.W. Osborne J.M. Brandt J.T. Semin. Thromb. Hemost. 1995; 21: 1-9Crossref PubMed Scopus (62) Google Scholar). This activating signal contributes to the rapid destruction of platelets during heparin-induced thrombocytopenia and in some autoimmune diseases (3KappersKlunne M.C. Boon D.M.S. Hop W.C.J. Michiels J.J. Stibbe J. vanderZwaan C. Koudstaal P.J. vanVliet H.H.D.M. Br. J. Haematol. 1997; 96: 442-446Crossref PubMed Scopus (85) Google Scholar, 4Newman P.M. Chong B.H. Blood. 2000; 96: 182-187Crossref PubMed Google Scholar). FcγRIIa-mediated signaling pathway implicates the cytoplasmic tail of the receptor, which presents an amino acid sequence called Immunoreceptor Tyrosine-based Activation Motif (ITAM). 1The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; FcγRIIa, Fc γ receptor for immunoglobulin G; GPVI, glycoprotein VI; GST, glutathione S-transferase; HA, hemagglutinin; LAT, linker for activation of T cell; PLC, phospholipase C; PTP, protein tyrosine phosphatase; SH2, Src homology 2; Inh, inhibitor; IP, immunoprecipitate. Mouse platelets lack this receptor but develop a similar signaling pathway downstream the glycoprotein VI (GPVI), also present in human platelets. Upon clustering, GPVI recruits and requires the Fc γ-chain sharing a strong homology with the human FcγRIIa receptor (5Poole A. Gibbins J.M. Turner M. Vanvugt M.J. Vandewinkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (396) Google Scholar, 6Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In human platelets, upon FcγRIIa receptor cross-linking, the tyrosine residues within the ITAM motif are rapidly phosphorylated and become docking sites for proteins containing Src homology 2 (SH2) domains (7Ibarrola I. Vossebeld P.J.M. Homburg C.H.E. Thelen M. Roos D. Verhoeven A.J. Biochim. Biophys. Acta. 1997; 1357: 348-358Crossref PubMed Scopus (55) Google Scholar). Activated GPVI is coupled with the Fc-γ-chain protein, which contains the similar ITAM motif. The phosphorylation of ITAM appears to be mediated by the Src-related kinases p59Fyn and p56Lyn and allows the recruitment and the activation of the p72Syk kinase and subsequently the tyrosine phosphorylation of phospholipase C γ2 (PLCγ2) (8Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, the links between Src kinases, Syk, and PLCγ2 are not clearly established in platelets and likely involve adaptor molecules. Linker for Activation of T cells (LAT), a transmembrane 36–38-kDa adaptor protein essential for T-cell receptor (TCR)-mediated activation, is also present in platelets (9Gross B.S. Melford S.K. Watson S.P. Eur. J. Biochem. 1999; 263: 612-623Crossref PubMed Scopus (57) Google Scholar). In T-cells, LAT is tyrosine-phosphorylated after TCR stimulation by the Syk-related kinase ZAP 70 (10Zhang W.G. Sloanlancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar) and contains in its intracellular part five optimal binding sequences for linking to SH2 domains containing proteins. In T-lymphocyte, numerous signaling molecules have been shown to associate with phosphorylated LAT, including PLCγ1, the p85 subunit of phosphatidylinositol (PI 3)-kinase, Grb2, and SLP76 (11Zhang W. Trible R.P. Zhu M. Liu S.K. McGlade C.J. Samelson L.E. J. Biol. Chem. 2000; 275: 23355-23361Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). In platelets, LAT is strongly tyrosine-phosphorylated downstream of GPVI clustering by collagen or convulxin stimulation. Several signaling proteins, including the p85 subunit of PI 3-kinase and PLCγ2, have been shown to interact with phosphorylated LAT in platelets (9Gross B.S. Melford S.K. Watson S.P. Eur. J. Biochem. 1999; 263: 612-623Crossref PubMed Scopus (57) Google Scholar, 12Gibbins J.M. Briddon S. Shutes A. van Vugt M.J. van de Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Asazuma N. Wilde J.I. Berlanga O. Leduc M. Leo A. Schweighoffer E. Tybulewicz V. Bon C. Liu S.K. McGlade C.J. Schraven B. Watson S.P. J. Biol. Chem. 2000; 275: 33427-33434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In collagen-stimulated platelets, the signaling complexes recruited by tyrosine-phosphorylated LAT are essential for PLCγ2 activation (14Pasquet J.M. Gross B. Quek L. Asazuma N. Zhang W. Sommers C.L. Schweighoffer E. Tybulewicz V. Judd B. Lee J.R. Koretzky G. Love P.E. Samelson L.E. Watson S.P. Mol. Cell. Biol. 1999; 19: 8326-8334Crossref PubMed Google Scholar). The tyrosine phosphorylation level of proteins is the result of a controlled balance between protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). The mechanisms involved in protein dephosphorylation are still poorly known in platelets. Until now, no transmembrane PTP has been described at the platelet surface but three cytosolic PTPs have already been identified in these cells (SHP-1, SHP-2, and PTP1B) (15Ezumi Y. Takayama H. Okuma M. J. Biol. Chem. 1995; 270: 11927-11934Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar, 17Li R.Y. Gaits F. Ragab A. Ragab-Thomas J.M. Chap H. FEBS Lett. 1994; 343: 89-93Crossref PubMed Scopus (34) Google Scholar). SHP-1, a PTP containing two SH2 domains in its N terminus, is highly expressed in hematopoietic cells, where it is often implicated in the negative regulation of a number of membrane receptors (18Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S.H. Thomas M. Ullrich A. Zhao Z.Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). SHP-1 is rapidly phosphorylated and translocated to the platelet cytoskeleton upon thrombin stimulation (17Li R.Y. Gaits F. Ragab A. Ragab-Thomas J.M. Chap H. FEBS Lett. 1994; 343: 89-93Crossref PubMed Scopus (34) Google Scholar, 19Li R.Y. Gaits F. Ragab A. Ragab-Thomas J.M.F. Chap H. EMBO J. 1995; 14: 2519-2526Crossref PubMed Scopus (63) Google Scholar, 20Rendu F. Bachelot C. Falet H. Platelets. 1996; 7 (361): 361Google Scholar). However, the role of SHP-1 in platelet functions is still unclear. SHP-2, another SH2 domain-containing PTP, is associated with the receptor PECAM 1 (CD31) in platelets and could be involved in the signaling pathway initiated by this adhesion molecule (21Edmead C.E. Crosby D.A. Southcott M. Poole A.W. FEBS Lett. 1999; 459: 27-32Crossref PubMed Scopus (27) Google Scholar, 22Cicmil M. Thomas J.M. Leduc M. Bon C. Gibbins J.M. Blood. 2002; 99: 137-144Crossref PubMed Scopus (132) Google Scholar). The 50-kDa PTP1B has also been described in human platelets (16Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar). In these cells, the amount of PTP1B is about 0.2% of total detergent-soluble proteins, a level comparable with that of pp60Src kinase. In resting platelets, the full-length PTP1B tightly associates with the endoplasmic reticulum via its C-terminal 35 amino acids (23Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (505) Google Scholar). When platelets are activated by thrombin, PTP1B undergoes a proteolytic cleavage in a region between its catalytic domain and its membrane-anchoring C-terminal targeting sequence. This process is dependent on integrin engagement and platelet aggregation and leads to enzymatic activation of PTP1B (16Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar). In other cells such as fibroblasts, PTP1B plays an important role in integrin-mediated cell adhesion and spreading (24Arregui C.O. Balsamo J. Lilien J. J. Cell Biol. 1998; 143: 861-873Crossref PubMed Scopus (130) Google Scholar, 25Balsamo J. Arregui C. Leung T.C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (144) Google Scholar). It has also been demonstrated that PTP1B can dephosphorylate p130Cas, suggesting that it might have a regulatory role in mitogen-mediated signal transduction pathway via integrin (26Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar). The tyrosine kinase pp60c-src has also been identified as a good substrate of PTP1B leading to an activation of this kinase (27Bjorge J.D. Pang A. Fujita D.J. J. Biol. Chem. 2000; 275: 41439-41446Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Recently, a critical role for PTP1B in the negative regulation of insulin signaling has been well documented (28Dadke S. Kusari J. Chernoff J. J. Biol. Chem. 2000; 275: 23642-23647Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). PTP1B appears as the major PTP responsible for insulin receptor regulation (29Goldstein B.J. Bittner-Kowalczyk A. White M.F. Harbeck M. J. Biol. Chem. 2000; 275: 4283-4289Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 30Mahadev K. Zilbering A. Zhu L. Goldstein B.J. J. Biol. Chem. 2001; 276: 21938-21942Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar), suggesting that PTP1B inhibitors may become new drugs for type 2 diabetes treatment (31Johnson T.O. Ermolieff J. Jirousek M.R. Nat. Rev. Drug Discov. 2002; 1: 696-709Crossref PubMed Scopus (563) Google Scholar, 32Tobin J.F. Tam S. Curr. Opin. Drug Discov. Devel. 2002; 5: 500-512PubMed Google Scholar). In platelets, the substrate and the role of PTP1B are still unknown. The aim of our study was to investigate the mechanisms involved in the tight control of LAT adaptor protein dephosphorylation in platelets activated by FcγRIIa clustering. We found that the adaptor LAT, for which phosphorylation appeared transient in this signaling cascade, was actually one of the PTP1B substrates. Indeed, our results demonstrate that LAT dephosphorylation requires PTP1B activation via calpains downstream of αIIb/β3 integrin engagement. Our data suggest a role of PTP1B in the coordination of signaling processes leading to irreversible platelet aggregation through dephosphorylation of proteins such as LAT. Reagents, Antibodies, and Fusion Proteins—The anti-FcγRIIa monoclonal antibody (mAb IV.3), the monoclonal PTP1B antibody, the polyclonal Src family kinase antibody (SRC-2), and the anti-phosphotyrosine 4G10 antibody were purchased from Upstate Biotechnology Inc. The specific F(ab′)2 fragment was from Jackson ImmunoResearch Laboratories. The monoclonal anti-HA antibody was from Eurogentec. The fluorescein-conjugated anti-mouse Ig secondary antibody (ALEXA 488) and rhodamine-conjugated phalloidin (ALEXA 594) were from Molecular Probe. Convulxin was purified from the venom of Crotalus durissus terrificus as previously described (33Francischetti I.M. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Enhanced chemiluminescence Western blotting reagents were from Amersham Biosciences. Poly-(Glu4-Tyr1)n, calpains inh1, RGDS, and other chemical products were from Sigma. The phosphatase inhibitor PTP InhI was from Calbiochem. [γ-32P]ATP (3000 Ci/mmol) was from PerkinElmer Life Sciences. Ni-ProBond resin was from Invitrogen. GST·LAT cDNA was generated by inserting the cytosolic domain of LAT containing amino acid 18–255 obtained by RT-PCR from megacaryocytic DAMI cells into pGEX-KG vector. The fusion protein was purified using glutathione-Sepharose beads using Amersham Biosciences instructions. Rabbit anti-LAT antibody was produced in our laboratory by immunizing rabbit with this GST·LAT fusion protein. The full-length human cDNA (GST·PTP1B), containing a Cys215 to Ser mutation (GST·PTP1B C215S) was a gift from Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA). The full-length cDNA construct PTP1B (D181A) obtained from Dr. N. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was subcloned in pTAT-HA-His vector (34Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (886) Google Scholar) as a NcoI/EcoR1 fragment. Isolation of TAT fusion protein was realized after sonication of bacteria in 8 m urea HEPES (pH 7.2), 100 mm NaCl buffer, 20 mm imidazole; the clarified sonicate was applied to Ni-ProBond resin. TAT fusion protein elution was performed by the same buffer containing 100 mm imidazole. Detection of the TAT·HA-His PTP1B (D181A) fusion protein was performed by Western blotting using anti-HA or anti-PTP1B antibodies. Quantification of the purified protein was done by BIO-RAD protein assay system. Platelet Preparation and Stimulation—Human blood platelet concentrates were obtained from the local blood bank (Etablissement de transfusion Sanguine, Toulouse, France). Platelet preparation and FcγRIIa cross-linking were performed as previously described (35Gratacap M.P. Payrastre B. Viala C. Mauco G. Plantavid M. Chap H. J. Biol. Chem. 1998; 273: 24314-24321Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Platelet aggregation was measured at 37 °C by a turbidimetric method in a dual-channel aggregometer (Payton Associates, Scarborough, Ontario, Canada). In some experiments, platelets were not shacked. To test the effect of inhibitors on LAT tyrosine phosphorylation, platelet suspensions (1 × 109 platelets/ml) were preincubated for 3 min at 37 °C with 500 μm RGDS or 10 μm calpains inh1. In some experiments, the phosphatase inhibitor (PTP InhI) was added at 12.5 μm final concentration on platelet suspensions after the indicated time of stimulation. To test the effect of PTP1B substrate trapping, 10 μg of TAT·HA-His-PTP1B (D181A) fusion protein were added to 500 μl of platelets, and cells were incubated at 37 °C for 20 min before stimulation. Mouse platelets were prepared according to the following protocol: C57/Bl6 animals were anesthetized with a mix of Imalgene (ketamine) Rompun, and blood was collected by cardiac puncture using heparin (100 units/ml) as anticoagulant. Pooled blood was centrifuged at 1500 rpm for 8 min at 22 °C, and platelet-rich plasma was removed and centrifuged at 3000 rpm for 8 min at 22 °C. The platelet pellet was washed twice in tyrode's buffer (134 mm NaCl, 2.9 mm KCl, 12 mm NaHCO3, 0.34 mm NaH2PO4,20mm HEPES, pH 7.3, 1 mm MgCl2,5mm glucose) and finally suspended in the same buffer at the density of 8 × 108 platelets/ml. Stimulations of mouse platelets were carried out with 5 nm convulxin at 37 °C under stirring conditions. Isolation of Platelet Cytoskeleton—500 μl of human platelets (1 × 109 cells/ml) were activated by FcγRIIA cross-linking for indicated times; stimulation was stopped by addition of cytoskeleton buffer (CSK) to give a final concentration of 50 mm Tris-HCL, pH 7.4, 10 mm EGTA, 1% TritonX-100, 1 mm sodium orthovanadate, 1 mm phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin. The lysate was incubated for 5 min at room temperature and 10 min at 4 °C under shaking. Cytoskeletal material was collected by centrifugation (12,000 × g, 10 min, 4 °C) and washed three times with CSK buffer. Cytoskeletal proteins were sonicated three times for 10 s in Lammeli sample buffer and then submitted to Western blotting using appropriate antibodies. Immunoprecipitation and Immunoblotting—500 μl of 1 × 109/ml resting or stimulated platelets were lysed in RIPA buffer at final concentrations of 150 mm NaCl, 20 mm Tris-HCl, pH 7.7, 4 mm EDTA, 0.5% Triton X-100, 1 mm sodium orthovanadate, 1 mm phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin. 5 μg of the indicated antibody was added to the clarified lysate in a final volume of 750 μl and placed on a rocking platform for 1 h at 4 °C. The immune complexes were collected by adding 35 μl of 50% (w/v) protein A/G-Sepharose beads for 1 h at 4 °C. After washing, the immunoprecipitated proteins were resolved by 10% SDS-PAGE, electrotransferred to nitrocellulose membranes, and detected by immunoblotting with the appropriate antibodies using the enhanced chemiluminescence lighting system. For re-immunoprecipitation technique, the first immunoprecipitate (IP-PTP1B) obtained from control or 5-min-stimulated platelets was incubated with 60 μl of buffer (50 mm Tris-HCl, pH 7.5, 10 mm dithiothreitol, 1% SDS) and boiled for 2 min. The final volume was brought to 1.5 ml by the addition of RIPA buffer containing 0.5% Triton X-100, and a second IP was performed with LAT antibody. In some experiments TAT·HA-PTP1B (D181A) treated or non-treated human platelets were subjected to HA immunoprecipitation as described above and analyzed by Western blot using polyclonal anti-Src kinases antibody (SRC-2). Binding Assays Using GST Fusion Proteins—For LAT re-immunoprecipitation from PTP1B (C215S) pulldown complex, 500 μl of platelet suspension (5 × 108) were stimulated for 5 min in the absence or in the presence of 12.5 μm PTP InhI added 1 min after FcγRIIa cross-linking. The reaction was stopped by 250 μl of 3× RIPA buffer, and the clarified lysate was incubated with GST·PTP1B (C215S) for 2 h at 4 °C. Beads were washed, and the protein complexes from pulldown assay were denatured by addition of 60 μl of buffer (50 mm Tris-HCl, pH 7.5, 10 mm dithiothreitol, 1% SDS) and boiled for 2 min. The final volume was brought to 1.5 ml by the addition of RIPA buffer containing 0.5% Triton X-100, and antiphosphotyrosine immunoprecipitation was performed with 4G10 antibody followed by Western blotting using LAT antibody. Immune Complex Protein Phosphatase Assay and in Vitro LAT Dephosphorylation—Enzymatic activity of PTP1B in platelets was determined using paranitrophenyl phosphate as substrate. PTP1B immune complexes were washed twice with PTP assay buffer (62 mm HEPES, pH.5, 6.25 mm EDTA, 12.5 mm dithiothreitol) and incubated with 25 mm final concentration of paranitrophenyl phosphate for 30 min at 30 °C under shaking. Reactions were terminated by adding 800 μl of 1N NaOH. After centrifugation at 13,000 rpm for 3 min, optical density of supernatants was measured at 410 nm. For LAT dephosphorylation in vitro, LAT immunoprecipitation was performed from platelets after 1 min of FcγRIIa stimulation as described above. PTP1B immune complex was realized at 5 min stimulation. The dephosphorylation reaction was performed by mixing the two washed immune complexes in 50 μlof the same buffer. After 30 min of incubation at 30 °C under shaking, the reactions were stopped by the addition of sample buffer and detection of the phosphoproteins was performed by SDS-PAGE, followed by anti-phosphotyrosine immunoblotting. In-gel Phosphatase Assay—These experiments were performed according to the procedure of Burridge and Nelson (36Burridge K. Nelson A. Anal. Biochem. 1995; 232: 56-64Crossref PubMed Scopus (85) Google Scholar) with some modifications. Briefly, 2 mg of poly(Glu4-Tyr1)n were phosphorylated by incubation overnight at 30 °C with pp60c-src kinase immunoprecipitated from thrombin-activated platelets and 20 μCi of [γ-32P]ATP. The kinase reaction was terminated by centrifugation at 4 °C (13,000 rpm; 5 min). The supernatant of agarose beads was mixed with an equal volume of 20% trichloroacetic acid. After 30 min on ice, labeled poly(Glu-Tyr) was sedimented. The precipitate was dissolved in 200 μl of Tris buffer 0.75 m, pH 8.8, and incorporated in SDS-polyacrylamide running gel prior to polymerization at ∼106 cpm/ml. Platelet lysates or LAT immunoprecipitates were submitted to SDS-PAGE according to the standard protocol. Confocal Immunofluorescence Microscopy—Resting human platelets were incubated or not with TAT·HA-PTP1B (D181A) fusion protein as described above. Platelets were allowed to adhere on fibrinogen-coated coverslips at the concentration of 100 μg/ml during 1 h at 37 °C. Cells were washed with phosphate-buffered saline and fixed with formalde-hyde 3%, 30 min at room temperature. Then they were permeabilized with 0.01% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. Nonspecific sites were saturated with 3% bovine serum albumin in phosphate-buffered saline for 30 min. Platelets were incubated with monoclonal anti-HA antibody followed by fluorescein-conjugated anti-mouse Ig secondary antibody mixed with rhodamine-conjugated phalloidin. Slides were examined under a Zeiss confocal microscope (LSM 510, Axiovert 100) using immersion objective ×63. The Rapid Tyrosine Phosphorylation of LAT Is Followed by an Integrin-mediated Dephosphorylation in FcγRIIa-stimulated Platelets—FcγRIIa cross-linking led to a rapid LAT phosphorylation reaching a maximum at 1 min, followed by a dephosphorylation that was complete after 5 min of stimulation (Fig. 1A). When platelet aggregation was prevented by the absence of shaking during stimulation (Fig. 1B) or by cell preincubation with RGDS peptide (Fig. 1C), we observed a sustained tyrosine phosphorylation of LAT. In some, but not all, experiments, a small delay in LAT phosphorylation was observed in non-shaking conditions of stimulation (Fig. 1B). Overall, these data indicate that the phosphorylation of LAT did not require platelet aggregation and integrin engagement, whereas its dephosphorylation was strongly dependent on these processes. Because calpains have been shown to regulate some tyrosine dephosphorylation events downstream of integrins in platelets, we investigated the effect of a calpains inhibitor (calpains inh1) on LAT phosphorylation status. As shown in Fig. 1D, LAT dephosphorylation was partly inhibited by this inhibitor, suggesting an implication of calpains-regulated PTPs downstream of integrin in this mechanism. PTP1B Is Activated in Human Platelets upon FcγRIIa Cross-linking—To investigate the PTPs involved in LAT dephosphorylation in human platelets via FcγRIIa cross-linking, lysates obtained from resting or stimulated platelets were submitted to an In-gel phosphatase assay as described under “Experimental Procedures.” Fig. 2A shows that resting platelets developed two basal PTP activities at 50 and 42 kDa. After 3 min of FcγRIIa cross-linking, the level of phosphatase activity of the 42-kDa species strongly increased with the appearance of several new bands detected at a molecular mass between 40 and 55 kDa. Some other PTPs presenting higher molecular mass were also detected during platelet aggregation with modest activation level (data not shown). Moreover, experiments performed with RGDS preincubated platelets show that the increase in the 42-kDa species at 5 min of stimulation was blocked, indicating a crucial role of integrins in this PTP activation. As expected, the tyrosine phosphatase inhibitor (PTP InhI) inhibited this enzymatic activation. The proteins detected in the In-gel phosphatase assay at about 40 and 50 kDa could be the active fragment of PTP1B and its full-length, respectively. To determine the identity of these proteins, we performed immunodepletion of the whole lysates obtained from 5-min-stimulated platelets. As shown in Fig. 2B, the supernatant of PTP1B immunoprecipitate (lane 2) exhibited only a weak phosphatase activity at 42 kDa and a significant decrease at 50 kDa (about 50%), indicating that these two bands corresponded to the two forms of PTP1B present in stimulated platelets. Moreover, Fig. 2C shows that, under basal conditions, PTP1B was essentially present as a full-length 50-kDa protein, whereas 3 min after FcγRIIa cross-linking, the truncated form of PTP1B (42 kDa) was generated. This cleavage decreased when platelets were preincubated with a calpains inhibitor (calpains inh1), suggesting that calpains catalyzed PTP1B proteolysis in FcγRIIa-stimulated platelets. Because some enzymes refold better than others, In-gel phosphatase assay is not always appropriate to quantify a specific PTP activity. Therefore, we immunoprecipitated PTP1B from platelet lysates and measured its activity by an in vitro phosphatase assay using paranitrophenyl phosphate as a substrate. PTP1B developed a 2.3-fold increase in enzymatic activity after 5 min of platelet stimulation (Fig. 2D). Moreover, pretreatment of cells with RGDS totally prevented integrin-induced PTP1B cleavage (not shown) and decreased the activity detected in PTP1B immunoprecipitate (Fig. 2D). These data demonstrate that αIIb/β3 integrin and calpains pathway regulate the cleavage of PTP1B and modulate its global activity measured in vitro in human platelets stimulated by FcγRIIa cross-linking, as previously observed in thrombin-activated platelets (16Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar). The actin cytoskeleton plays an important role in cell physiology, and numerous signaling proteins have been found to associate with this cellular compartment after platelet aggregation (17Li R.Y. Gaits F. Ragab A. Ragab-Thomas J.M. Chap H. FEBS Lett. 1994; 343: 89-93Crossref PubMed Scopus (34) Google Scholar, 37Clark E.A. Brugge J.S. Mol. Cell. Biol. 1993; 13: 1863-1871Crossref PubMed Scopus (198) Google Scholar, 38Fox J.E.B. Shattil S.J. Kinloughrathbone R.L. Richardson M. Packham M.A. Sanan D.A. J. Biol. Chem. 1996; 271: 7004-7011Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 39Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 40Kralisz U. Cierniewski C.S. IUBMB Life. 2000; 49: 33-42Crossref PubMed Google Scholar). As shown in Fig. 3, LAT associated with the cytoskeleton fraction upon FcγRIIa-mediated platelet activation. This association reached a maximum at 2 min and persisted until 5 min. Interestingly, PTP1B was found in the same compartment after 2 min of stimulation. The co-localization of LAT and PTP1B in the platelet cytoskeleton correlated with the time course of LAT dephosphorylation, suggesting that PTP1B could dephosphorylate LAT in this cell compartment. Phosphorylated LAT Is a PTP1B Substrate in Fc" @default.
• W2053465902 created "2016-06-24" @default.
• W2053465902 creator A5005609731 @default.
• W2053465902 creator A5016129398 @default.
• W2053465902 creator A5021757402 @default.
• W2053465902 creator A5064849353 @default.
• W2053465902 creator A5077349534 @default.
• W2053465902 creator A5081552914 @default.
• W2053465902 date "2003-10-01" @default.
• W2053465902 modified "2023-10-01" @default.
• W2053465902 title "The Tyrosine Phosphatase 1B Regulates Linker for Activation of T-cell Phosphorylation and Platelet Aggregation upon FcγRIIa Cross-linking" @default.
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• W2053465902 doi "https://doi.org/10.1074/jbc.m303602200" @default.
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