Interaction of Bruton's Tyrosine Kinase and Protein Kinase Cθ in Platelets
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m108965200
ISSN1083-351X
AutoresDavid A. Crosby, Alastair W. Poole,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoThe nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCθ in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCθ upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCθ in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCθ activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCθ in platelets, in which PKCθ positively modulates activity of Btk, which in turn feeds back negatively upon PKCθ. The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCθ in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCθ upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCθ in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCθ activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCθ in platelets, in which PKCθ positively modulates activity of Btk, which in turn feeds back negatively upon PKCθ. Bruton's tyrosine kinase bisindolylmaleimide 1 glycoprotein glutathione S-transferase 5-hydroxytryptamine myelin basic protein pleckstrin homology protein kinase C radioimmune precipitation assay Tec tyrosine kinase von Willebrand factor Bruton's tyrosine kinase (Btk)1 is a member of the Tec family of nonreceptor tyrosine kinases, which includes Itk, Tec, Txk, and Bmx and is of pathological significance when deficient or functionally mutated in the severe immunodeficiency syndrome X-linked agammaglobulinemia (1Satterthwaite A.B. Li Z. Witte O.N. Semin. Immunol. 1998; 10: 309-316Crossref PubMed Scopus (158) Google Scholar). Tec family kinases are characterized by a C-terminal proline-rich region, Src homology 1, 2, and 3 domains, and an N-terminal pleckstrin homology (PH) and Tec homology domains. Btk is expressed in cells of hematopoietic origin including platelets and has been shown to mediate calcium influx in these cells (2Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 3Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Crossref PubMed Scopus (75) Google Scholar, 4Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (388) Google Scholar, 5Genevier H.C. Callard R.E. Clin. Exp. Immunol. 1997; 110: 386-391Crossref PubMed Scopus (18) Google Scholar). Btk has been shown to play an important role in platelet activation by collagen because collagen stimulation induces tyrosine phosphorylation and activation of Btk (6Oda A. Ikeda Y. Ochs H.D. Druker B.J. Ozaki K. Handa M. Ariga T. Sakiyama Y. Witte O.N. Wahl M.I. Blood. 2000; 95: 1663-1670PubMed Google Scholar), and in platelets lacking functional Btk, collagen-induced platelet aggregation and calcium influx are impaired significantly (7Quek L.S. Bolen J. Watson S.P. Curr. Biol. 1998; 8: 1137-1140Abstract Full Text Full Text PDF PubMed Google Scholar). The activity of Btk is controlled by several factors including phosphorylation. Btk has been shown to be phosphorylated at tyrosine 551 within the catalytic domain by associated Src family kinases, leading to autophosphorylation at tyrosine 223 within the Src homology 3 domain, which is necessary for full activation (8Wahl M.I. Fluckiger A.C. Kato R.M. Park H. Witte O.N. Rawlings D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11526-11533Crossref PubMed Scopus (109) Google Scholar, 9Park H. Wahl M.I. Afar D.E. Turck C.W. Rawlings D.J. Tam C. Scharenberg A.M. Kinet J.P. Witte O.N. Immunity. 1996; 4: 515-525Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 10Afar D.E. Park H. Howell B.W. Rawlings D.J. Cooper J. Witte O.N. Mol. Cell. Biol. 1996; 16: 3465-3471Crossref PubMed Scopus (106) Google Scholar, 11Rawlings D.J. Scharenberg A.M. Park H. Wahl M.I. Lin S. Kato R.M. Fluckiger A.C. Witte O.N. Kinet J.P. Science. 1996; 271: 822-825Crossref PubMed Scopus (381) Google Scholar). It has also been shown that for full activation, the PH domain of Btk must interact with the lipid product of phosphatidylinositol 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (12Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar). It has been hypothesized that the function of this interaction is to recruit Btk from the cytosol to the plasma membrane, where it can be subsequently activated by Src family kinases. Btk has been shown to associate with members of the protein kinase C (PKC) family of serine/threonine kinases through an interaction between the Btk PH domain and PKC C1 domain (13Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar, 14Yao L. Suzuki H. Ozawa K. Deng J. Lehel C. Fukamachi H. Anderson W.B. Kawakami Y. Kawakami T. J. Biol. Chem. 1997; 272: 13033-13039Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A constitutive association has been shown between Btk and PKCμ in B cells (15Johannes F.J. Hausser A. Storz P. Truckenmuller L. Link G. Kawakami T. Pfizenmaier K. FEBS Lett. 1999; 461: 68-72Crossref PubMed Scopus (39) Google Scholar) and between Btk and members of the classical and atypical families of PKC in murine mast cells (13Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar). Btk has been shown in these studies to be a substrate of PKC isoforms and its enzymatic activity to be down-regulated by PKC-mediated phosphorylation (13Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar). In turn, Btk has been shown to regulate the membrane translocation and activation of PKCβ1 by direct interaction with this kinase (16Kawakami Y. Kitaura J. Hartman S.E. Lowell C.A. Siraganian R.P. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7423-7428Crossref PubMed Scopus (80) Google Scholar). For the initiation of arrest of bleeding and subsequent vascular repair, the ability of platelets to adhere to subendothelial structures is critical. Collagen and von Willebrand factor (vWF) form the two most important structures to which platelets adhere through several surface glycoprotein (GP) receptors. These two adhesion molecules induce signaling events in platelets primarily through GP VI and GP Ib-V-IX, respectively. The initial interaction between platelets and vWF occurs via the platelet glycoprotein receptor complex GP Ib-V-IX (GP Ib). vWF supports not only the initial transient phase of platelet adhesion through GP Ib but also mediates integrin αIIbβ3-based cell arrest (17Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). The signaling pathways induced by GP Ib and GP VI share a number of similarities including constitutive association with and tyrosine phosphorylation of Fc receptor γ chain and recruitment of multiple components of a tyrosine phosphorylation-dependent signaling pathway. These components include Src family kinases, Syk, and phospholipase Cγ2 (18Wu Y. Suzuki-Inoue K. Satoh K. Asazuma N. Yatomi Y. Berndt M.C. Ozaki Y. Blood. 2001; 97: 3836-3845Crossref PubMed Scopus (110) Google Scholar, 19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar, 20Ezumi Y. Shindoh K. Tsuji M. Takayama H. J. Exp. Med. 1998; 188: 267-276Crossref PubMed Scopus (186) Google Scholar). Activation of GP Ib and GP VI leads to the hydrolysis of phosphoinositides, a rise in cytosolic calcium, activation of PKC, cytoskeletal reorganization, and platelet 5-HT secretion and aggregation (17Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar, 21Andrews R.K. Lopez J.A. Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (181) Google Scholar, 22Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 23Jackson S.P. Schoenwaelder S.M. Yuan Y. Rabinowitz I. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 27093-27099Abstract Full Text PDF PubMed Google Scholar, 24Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (238) Google Scholar, 25Poole A.W. Watson S.P. Br. J. Pharmacol. 1995; 115: 101-106Crossref PubMed Scopus (51) Google Scholar). In this report, combined activation of GP Ib and GP VI is achieved using the snake venom component alboaggregin A (19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar, 21Andrews R.K. Lopez J.A. Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (181) Google Scholar, 26Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 929-936Crossref PubMed Scopus (65) Google Scholar, 27Asazuma N. Marshall S.J. Berlanga O. Snell D. Poole A.W. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar), and activation of GP Ib alone is achieved by vWF plus ristocetin in the presence of EGTA to block binding to integrin αIIbβ3. It is clear that many aspects of platelet activation are regulated by combined serine/threonine and tyrosine phosphorylation. We were interested in investigating points of cross-talk between these two signaling pathways. Here we have investigated the interaction between a member of the novel PKC isoforms, PKCθ, and Btk and show a role for this interaction in terms of functional feedback between the two kinases. Trimeresurus albolabris venom was a kind gift from Professor R. D. G. Theakston (Liverpool, U. K.). Anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Biotechnology Inc (TCS Biologicals Ltd., Bucks, U. K.). All anti-PKC antibodies and anti-phosphothreonine were from Transduction Laboratories (Affiniti Research Products, Exeter, U. K.). Anti-Btk antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Src family kinase inhibitor PP1 was from Alexis Corp (Nottingham, U. K.). PKC inhibitor bisindolylmaleimide I (BIM) was from Tocris (Bristol, U. K.). Btk-PH domain construct was from Dr. C. I. E. Smith (Stockholm, Sweden). LFM-A13 and Raytide were from Calbiochem (La Jolla, CA). Plasma vWF was a kind gift from Prof. J. J. Sixma and Dr. T. Vink (Utrecht, The Netherlands). All other reagents were analytical grade. Human blood was drawn from healthy, drug-free volunteers on the day of the experiment. Acid citrate dextrose (120 mm sodium citrate, 110 mm glucose, 80 mm citric acid, used at 1:7 v/v) was used as an anticoagulant. Platelet-rich plasma was prepared by centrifugation at 200 × g for 20 min. Platelets were then isolated by centrifugation of platelet-rich plasma for 10 min at 1,000 × g, in the presence of 40 ng/ml prostaglandin E1. The pellet was resuspended to a density of 4.108 platelets/ml in a modified Tyrode's-HEPES buffer (145 mm NaCl, 2.9 mm KCl, 10 mmHEPES, 1 mm MgCl2, 5 mm glucose, pH 7.3). To this platelet suspension, 10 μm indomethacin was added, and a 30-min resting period was allowed before stimulation. Stimulation of platelets was performed in an aggregometer at 37 °C, with continuous stirring at 800 rpm. Unless otherwise specified, all platelet stimulation occurred in the presence of 1 mm EGTA. Alboaggregin A was prepared from crude T. albolabris venom as described previously (28Peng M. Lu W. Kirby E.P. Biochemistry. 1991; 30: 11529-11536Crossref PubMed Scopus (97) Google Scholar, 29Peng M. Lu W. Kirby E.P. Thromb. Haemostasis. 1992; 67: 702-707Crossref PubMed Scopus (44) Google Scholar) by ion exchange chromatography. Alboaggregin A was used at 3.5 μg/ml, a concentration previously determined to be the EC50 value for induction of 5-HT release (19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar). Plasma vWF was used at a concentration of 10 μg/ml (previously shown to give a near maximal aggregatory response), in the presence of 1 mg/ml ristocetin. Reactions were stopped by lysis of platelets with an equal volume of either 2× Nonidet P-40 extraction buffer (1% Nonidet P40, 300 mm NaCl, 20 mm Tris, 1 mmphenylmethylsulfonyl fluoride, 10 mm EDTA, 2 mmNa3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/ml pepstatin, pH 7.3), or 2× radioimmune precipitation assay (RIPA) buffer (2% Triton X-100, 2% sodium deoxycholate, 0.2% SDS, 300 mm NaCl, 20 mmTris, 1 mm phenylmethylsulfonyl fluoride, 10 mmEDTA, 2 mm Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/ml pepstatin, pH 7.3). Nonidet P-40 was used to preserve protein-protein interaction, and RIPA buffer was used to abolish immunoprecipitation of associated proteins. Insoluble material was removed by centrifugation (13,000 ×g, 5 min). Supernatants were then precleared by incubation with protein A-Sepharose for immunoprecipitations, or glutathione agarose for GST precipitations, for 1 h at 4 °C. For immunoprecipitations, lysates were incubated with protein A-Sepharose plus 1–2 μg of immunoprecipitating antibody for 2 h at 4 °C. For GST precipitations, lysates were incubated with glutathione-agarose beads coupled to GST-Btk-PH for 2 h at 4 °C. Proteins were separated by SDS-PAGE on 5–15% gradient slab gels and were then transferred to polyvinylidene difluoride membrane for immunoblotting and detection by enhanced chemiluminescence (ECL). Btk or PKCθ was immunoprecipitated from platelets lysed into Nonidet P-40 buffer. Immunoprecipitated proteins were suspended in 20 μl of kinase assay buffer (5 mm MgCl2, 5 mm MnCl2, 100 mm NaCl, 10 μm ATP, 2 mmNa3VO4, 20 mm HEPES, pH 7.2), and the reaction was started by the addition of 250 μCi/ml [γ-32P]ATP. After incubation for 10 min at room temperature, the reaction was terminated by the addition of 0.5 ml of ice-cold 100 mm EDTA. Immunoprecipitated proteins were then washed in RIPA buffer before separation by SDS-PAGE and detection of phosphorylated proteins by autoradiography. In addition to autophosphorylation, activity of PKCθ was assayed using MBP as an exogenous substrate. The reaction was carried out as for autophosphorylation but in the presence of 2 μg of MBP/sample. MBP bands were cut out, and the incorporation of 32P was quantified by liquid scintillation counting. Btk activity was also assayed using Raytide as an exogenous substrate. Immunoprecipitated Btk was resuspended in 20 μl of kinase assay buffer, and 10 μg of Raytide was added to each sample. The reaction was started by the addition of 10 μl of ATP buffer (0.15 mm ATP, 30 mmMgCl2, and 200 μCi/ml [γ-32P]ATP in kinase assay buffer). After incubation at 30 °C for 30 min the reaction was terminated by the addition of 10% phosphoric acid. Samples were applied to 2 × 2-cm squares of P81 ion exchange chromatography paper, washed extensively in 0.5% phosphoric acid followed by a wash in acetone. Papers were then dried, and labeled Raytide was quantified by liquid scintillation counting. This was performed as described previously (25Poole A.W. Watson S.P. Br. J. Pharmacol. 1995; 115: 101-106Crossref PubMed Scopus (51) Google Scholar). Briefly, 3 μm Fura 2-AM was added to platelet-rich plasma and incubated at 30 °C for 45 min in the presence of 10 μmindomethacin. Platelets were isolated as described above. Platelets were stimulated at room temperature in the absence of EGTA. Fluorescence excitation was made at 340 and 380 nm, and emission at 510 nm was measured using a PerkinElmer Life Sciences LS5 spectrofluorometer. Data are presented as the excitation fluorescence ratio (340:380 nm). Platelets were loaded by incubation of platelet-rich plasma with 0.2 μCi/ml 5-[3H]HT for 1 h at 37 °C. Platelets were preincubated with 1 mm EGTA before stimulation to prevent aggregation. Reactions were terminated by brief microcentrifugation, and 5-[3H]HT released into the supernatant was determined by liquid scintillation counting and expressed as a percentage of the total tissue content, as described previously (30Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar). Previous studies have shown that collagen and collagen-related peptides acting through GP VI induce tyrosine phosphorylation and activation of Btk (6Oda A. Ikeda Y. Ochs H.D. Druker B.J. Ozaki K. Handa M. Ariga T. Sakiyama Y. Witte O.N. Wahl M.I. Blood. 2000; 95: 1663-1670PubMed Google Scholar, 7Quek L.S. Bolen J. Watson S.P. Curr. Biol. 1998; 8: 1137-1140Abstract Full Text Full Text PDF PubMed Google Scholar). Fig.1 shows that upon combined stimulation of GP VI and GP Ib with alboaggregin A, Btk became rapidly tyrosine-phosphorylated and associated with a number of tyrosine-phosphorylated proteins when platelets were lysed into Nonidet P-40 (1%). In contrast, these associated proteins were lost when platelets were lysed in the more stringent RIPA lysis buffer. Fig.1 B shows that upon stimulation of platelets with alboaggregin A, one of the Btk-associated proteins was the Fc receptor γ chain, an accessory signaling molecule that has been shown to associate with both GP VI and GP Ib (19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar, 31Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (261) Google Scholar). The Src family kinases Fyn and Lyn have been shown to associate with the GP Ib receptor complex and to be essential for signaling downstream of the receptor (19Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar). The Src family kinase inhibitor PP1 is shown in Fig.2 to inhibit tyrosine phosphorylation of Btk dose dependently, with maximal inhibition by 20 μm. The appearance of tyrosine-phosphorylated associated proteins was also markedly reduced by PP1. Fig. 2 also shows that tyrosine phosphorylation of Btk is receptor-specific; stimulation of platelets with plasma vWF and ristocetin also induces tyrosine phosphorylation of Btk in contrast to a lack of response to the G protein-coupled receptor agonist thrombin. Btk has been shown to be activated in platelets after stimulation with collagen and CD32 cross-linking (6Oda A. Ikeda Y. Ochs H.D. Druker B.J. Ozaki K. Handa M. Ariga T. Sakiyama Y. Witte O.N. Wahl M.I. Blood. 2000; 95: 1663-1670PubMed Google Scholar, 7Quek L.S. Bolen J. Watson S.P. Curr. Biol. 1998; 8: 1137-1140Abstract Full Text Full Text PDF PubMed Google Scholar). Here, Btk activity was assayed in vitro either as autophosphorylation (Fig.3 A) or by phosphorylation of an exogenous peptide substrate, Raytide (Fig. 3 B). Fig.3 A shows that there is kinase activity in samples containing Btk immunoprecipitated from alboaggregin A-stimulated platelets, and a number of proteins became phosphorylated in vitro. After transfer of these proteins to polyvinylidene difluoride membrane and Western blotting, one of these proteins was identified as Btk. Fig. 3,A(iii) and B, shows that activation of platelets with alboaggregin A induces a marked increase in Btk activity, as assayed by phosphorylation of the tyrosine kinase substrate Raytide. The Btk inhibitor LFM-A13 (40 μm) abolished Btk activity when preincubated with platelets before stimulation. Also, preincubation of platelets with the PKC inhibitor BIM caused ablation of Btk activity. Addition of the Src family kinase inhibitor PP1 to the kinase assay buffer caused no change in the measured activity (data not shown), but addition of LFM-A13 to the kinase assay buffer caused ablation of activity, showing that the tyrosine kinase activity measured was the activity of Btk. It has been reported recently that in both platelets and B cells there is a pathway of Ca2+ entry which involves phosphatidylinositol 3,4,5-trisphosphate and Btk but is independent of phospholipase C activity (2Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 3Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Crossref PubMed Scopus (75) Google Scholar, 4Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (388) Google Scholar). Stimulation of platelets with alboaggregin A causes a rise in cytosolic calcium ([Ca2+]i) which is achieved by release of stored calcium and by influx of calcium from the extracellular medium. Fig. 4 i shows that upon incubation of platelets with 40 μm LFM-A13, the profile of the [Ca2+]i rise induced by alboaggregin A stimulation was altered markedly. Although the rapid rise in [Ca2+]i was similar to controls, in LFM-A13-treated platelets the [Ca2+]ideclined from a peak more rapidly than control platelets. In the presence of 1 mm EGTA to chelate extracellular calcium, LFM-A13 had no effect on alboaggregin A-induced calcium response (Fig.4 ii), supporting the hypothesis that Btk regulated calcium entry. LFM-A13 had no effect on the calcium response to thrombin stimulation (Fig. 4 iii). Fig. 4 iv shows the secretion of stored 5-HT in response to alboaggregin A stimulation. 5-HT release was increased by preincubation of platelets with the Btk inhibitor LFM-A13 (40 μm) and was decreased by preincubation with the PKC inhibitor BIM (20 μm). In the presence of both inhibitors, secretion was enhanced relative to control samples but less than that in the presence of LFM-A13 alone. Fig.5 A shows that in addition to tyrosine phosphorylation, alboaggregin A stimulation of platelets induces rapid threonine phosphorylation of Btk, with phosphorylation occurring by 15 s of stimulation and furthermore that this phosphorylation is ablated by preincubating platelets with 20 μm BIM before stimulation. Preincubation of platelets with BIM had no effect on the tyrosine phosphorylation of Btk (data not shown). An association between Btk and PKC has been demonstrated in mast cells (13Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar), with the α, β1, β2, ε, and ζ isoforms able to bind to Btk in vitro and the β1 isoform associatingin vivo. In vivo associations between Btk and PKCs ε, ζ, and μ have also been demonstrated in B cells (14Yao L. Suzuki H. Ozawa K. Deng J. Lehel C. Fukamachi H. Anderson W.B. Kawakami Y. Kawakami T. J. Biol. Chem. 1997; 272: 13033-13039Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Kawakami Y. Kitaura J. Hartman S.E. Lowell C.A. Siraganian R.P. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7423-7428Crossref PubMed Scopus (80) Google Scholar). Using immunoblotting we examined platelet expression of PKC isoforms α, β, γ, δ, ι, ζ, ε, μ, and θ, and in Fig. 5 Bwe demonstrate the presence of all isoforms except ζ. Fig.5 B also examines association between Btk and PKC isoforms and shows that only PKCθ associates with Btk, in an alboaggregin A stimulation-dependent manner. Fig. 5 C shows that PKC θ coprecipitates with Btk from alboaggregin A-stimulated platelets lysed in Nonidet P-40 lysis buffer but not with those lysed in RIPA buffer. RIPA buffer has therefore been used in this study to demonstrate definitively PKCθ phosphorylation in gels where Btk has not been coprecipitated. Fig.6 A shows a time course of stimulation-dependent association between Btk and PKCθ, with association occurring by 15 s stimulation and becoming maximal by 30 s. This association is also induced by stimulation of platelet GP Ib-V-IX with vWF/ristocetin in the presence of 1 mm EGTA to block activation of integrin αIIbβ3.Figure 6Alboaggregin A induces time-dependent association between Btk and PKC θ; association occurs via the PH domain of Btk. Panel A(i), Btk was immunoprecipitated from Nonidet P-40 lysates of basal platelets or platelets stimulated with 3.5 μg/ml alboaggregin A for various time periods as indicated.Panel A(ii), Btk was immunoprecipitated from Nonidet P-40 lysates of basal platelets (WCL), platelets stimulated with alboaggregin A for 60 s, or platelets stimulated with 10 μg/ml plasma vWF and 1 mg/ml ristocetin for 180 s. Samples were Western blotted with anti-PKCθ antibody. Panel B, Nonidet P-40 lysates were prepared from basal platelets or platelets stimulated with 3.5 μg/ml alboaggregin A for various time periods as indicated. GST-Btk-PH coupled to glutathione-agarose beads was used to precipitate associating proteins. Samples, including a whole cell lysate, were Western blotted with anti-PKCθ antibody. Results shown are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been shown previously that Btk/PKC association is mediated by interaction between the N-terminal PH domain of Btk and the C1 domain of PKC in mast cell lysates (13Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar). Fig. 6 B shows that PKCθ associates with the GST-Btk PH domain under basal conditions, and association increases in an alboaggregin A stimulation-dependent manner. Activation of T lymphocytes through the T cell receptor leads to rapid tyrosine phosphorylation of PKCθ (32Liu Y. Witte S. Liu Y.C. Doyle M. Elly C. Altman A. J. Biol. Chem. 2000; 275: 3603-3609Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Consequently, it was decided to investigate the state of tyrosine phosphorylation of PKCθ in platelets, before and after stimulation with alboaggregin A. As can be seen in Fig. 7 A, PKCθ is not tyrosine-phosphorylated under basal conditions but becomes so upon platelet activation by alboaggregin A by 15 s. As with Btk, the Src family kinase inhibitor PP1 was found to inhibit tyrosine phosphorylation of PKCθ dose-dependently (Fig.7 B). vWF/ristocetin (in the presence of 1 mmEGTA to prevent binding to αIIbβ3) also induced phosphorylation of PKCθ (Fig. 7 B). In addition to Src family kinase inhibition, Btk inhibition with LFM-A13 was also shown to reduce PKCθ tyrosine phosphorylation dose-dependently (Fig. 7 C). PKCθ activity was assayed either as autophosphorylation (Fig.8 A) or as the phosphorylation of MBP, an exogenous PKC substrate (Fig. 8 B). Western blotting revealed that this latter procedure induced no tyrosine phosphorylation of MBP (data not shown), i.e. the activity measured in Fig. 8 B is a serine/threonine kinase. Using either method, stimulation of platelets with alboaggregin A is shown to induce activation of PKCθ. Addition of 20 μm BIM to the kinase assay buffer caused ablation of the measured activity. However, preincubation of platelets with either BIM or LFM-A13 before stimulation with alboaggregin A caused a marked potentiation of PKCθ activity assayed in vitro. Binding of vWF to the GP Ib-V-IX receptor complex and of collagen to GP VI induces a signaling cascade in platelets involving many divergent and convergent pathways including events such as a transient rise in cytosolic calcium levels, hydrolysis of phosphoinositides, synthesis of thromboxane A2, activation of PKC, cytoskeletal reorganization, and the activation of the binding function of integrin αIIbβ3 (22Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 24Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (23
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