Tyrosine Phosphorylation of α-Actinin in Activated Platelets
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.37012
ISSN1083-351X
AutoresGonzalo Izaguirre, Lina Aguirre, Ping Ji, Brian J. Aneskievich, Beatrice Haimovich,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe integrin αIIbβ3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human α-actinin sequences deposited in the Swiss Protein Database. α-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with α-actinin and anti-phosphotyrosine antibodies. The distribution of α-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained α-actinin. The purified α-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. α-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with α-actinin. Taken together, these data demonstrate that a fraction of α-actinin is tyrosine-phosphorylated in activated platelets. The integrin αIIbβ3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human α-actinin sequences deposited in the Swiss Protein Database. α-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with α-actinin and anti-phosphotyrosine antibodies. The distribution of α-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained α-actinin. The purified α-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. α-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with α-actinin. Taken together, these data demonstrate that a fraction of α-actinin is tyrosine-phosphorylated in activated platelets. phorbol 12-myristate 13-acetate monoclonal antibody phosphatidylinositol 3-kinase high pressure liquid chromatography polyacrylamide gel electrophoresis Tyrosine phosphorylation of protein substrates is central to all cellular processes that are regulated by the integrin family of extracellular matrix receptors (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar) including cell shape change, migration, growth, and survival (for recent reviews see Refs. 2Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (302) Google Scholar, 3Giancotti F.G. Curr. Opin. Cell Biol. 1997; 9: 691-700Crossref PubMed Scopus (407) Google Scholar, 4Sheetz M.P. Felsenfeld D.P. Calbraith C.G. Trends Cell Biol. 1998; 8: 51-54Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 5Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (647) Google Scholar, 6Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1019) Google Scholar). The array of responses regulated by integrins is greatly simplified in platelets that are enucleated, terminally differentiated cells. Platelets, therefore, provide a useful model system to study integrin-mediated protein tyrosine phosphorylation events that are closely linked to cell shape change and the underlying cytoskeleton organization. A rapid increase in protein tyrosine phosphorylation is detected within seconds to minutes of platelet activation by soluble agonists such as thrombin or phorbol 12-myristate 13-acetate (PMA)1 (7Shattil S.J. Ginsberg M.H. Brugge J.S. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar, 8Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). Platelet adhesion to immobilized matrix proteins such as fibrinogen triggers similar tyrosine phosphorylation events (9Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). Included in the first group of proteins that become tyrosine-phosphorylated are the non-receptor tyrosine kinases pp72syk (10Taniguchi T. Kitagawa H. Yanagi S. Sakai K. Aashi M. Ohta S. Takeuchi F. NaKamura S. Yananura H. J. Biol. Chem. 1993; 268: 2277-2279Abstract Full Text PDF PubMed Google Scholar) and RFTK/PYK (11Raja S. Avraham S. Avraham H. J. Biol. Chem. 1997; 272: 10941-10947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the guanine nucleotide exchange factor Vav (12Cichowski K. Brugge J.S. Brass L.F. J. Biol. Chem. 1996; 271: 7544-7550Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and the mitogen-activated protein (MAP) kinase family members extracellular signal-regulated kinase 2 (ERK2) and p38 (13Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 14Papkoff J. Chen R.-H. Blenis J. Forsman J. Mol. Biol. Cell. 1994; 14: 463-472Crossref Google Scholar). In platelets stimulated by thrombin, PMA, or adhesion to fibrinogen, a second wave of protein tyrosine phosphorylation that is strictly dependent on both ligand binding to the αIIbβ3 integrin receptor and the cytoskeleton organization is observed (7Shattil S.J. Ginsberg M.H. Brugge J.S. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar). As such, tyrosine phosphorylation of this subset of protein substrates is effectively blocked by either αIIbβ3 receptor antagonists or cytochalasin D, an inhibitor of actin filament assembly. The protease calpain (15Fox J.E.B. Taylor R.G. Taffarel M. Boyles J.K. Goll D.E. J. Cell Biol. 1993; 120: 1501-1507Crossref PubMed Scopus (133) Google Scholar), the phosphatase SHIP (16Giuriato S. Payrastre B. Drayer A.L. Plantavid M. Woscholski R. Parker P. Erneux C. Chap H. J. Biol. Chem. 1997; 272: 26857-26863Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and the non-receptor protein tyrosine kinases Tec (17Hamazaki Y. Kojima H. Mano H. Nagata Y. Todokoro K. Abe T. Nagasawa T. Oncogene. 1998; 16: 2773-2779Crossref PubMed Scopus (35) Google Scholar) and pp125FAK(18Lipfert L. Haimovich B. Schaller M. Cobb B.S. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (624) Google Scholar, 19Shattil S.J. Haimovich B. Cunningham M. Lipfert L. Parsons J.T. Ginsberg M.H. Brugge J.S. J. Biol. Chem. 1994; 269: 14738-14745Abstract Full Text PDF PubMed Google Scholar) are among the proteins that become phosphorylated at this stage. Tyrosine phosphorylation of two additional proteins of 101 and 105 kDa (pp101 and pp105) is also detected in platelets that aggregate following stimulation with an agonist such as PMA and in platelets adherent and spread on fibrinogen (9Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 20Golden A. Brugge J.S. Shattil S.J. J. Cell Biol. 1990; 111: 3117-3127Crossref PubMed Scopus (184) Google Scholar, 21Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar, 22Fox J.E.B. Lipfer L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar). Both platelet aggregation and adhesion to fibrinogen are mediated by the integrin αIIbβ3. Platelet spreading on fibrinogen and tyrosine phosphorylation of pp125FAK, pp101, and pp105 are inhibited by protein kinase C, PI3-kinase, and phospholipase A2 enzyme(s) (21Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar, 23Ji P. Haimovich B. Biochim. Biophys. Acta. 1999; 1448: 543-552Crossref PubMed Scopus (22) Google Scholar, 24Haimovich B. Ji P. Ginalis E. Kramer R. Greco R. Thromb. Haemost. 1999; 81: 618-624Crossref PubMed Scopus (15) Google Scholar). However, in platelets adherent to IgG, an FcγRIIA receptor-dependent interaction, protein kinase C (23Ji P. Haimovich B. Biochim. Biophys. Acta. 1999; 1448: 543-552Crossref PubMed Scopus (22) Google Scholar, 24Haimovich B. Ji P. Ginalis E. Kramer R. Greco R. Thromb. Haemost. 1999; 81: 618-624Crossref PubMed Scopus (15) Google Scholar, 25Haimovich B. Regan C. DiFazio L. Ginalis E. Ji P. Purohit U. Rowley R.B. Bolen J. Greco R. J. Biol. Chem. 1996; 271: 16332-16337Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) inhibitors but not PI3-kinase or phospholipase A2 enzyme inhibitors affect the phosphorylation of these proteins, demonstrating that multiple signaling mechanisms converge to govern the phosphorylation of these proteins. The goal of this study was to isolate and characterize pp105. To this end, a 105-kDa tyrosine-phosphorylated protein was partially purified from platelets stimulated with PMA and subjected to microsequencing. The sequence of an internal 12-mer peptide derived from the partially purified protein was identical to human α-actinin sequences deposited in the Swiss Protein Database. Consistent with the possibility that α-actinin is tyrosine-phosphorylated in platelets, α-actinin tyrosine phosphorylation was recently reported in other experimental systems (26Egerton M. Moritz R.L. Druker B. Kelso A. Simpson R.J. Biochem. Biophys. Res. Commun. 1996; 224: 666-674Crossref PubMed Scopus (28) Google Scholar, 27Mukai H. Toshimori M. Shibata H. Takanaga H. Kitagawa M. Miyahara M. Shimakawa M. Ono Y. J. Biol. Chem. 1997; 272: 4740-4746Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To further confirm the identity of pp105 as α-actinin, α-actinin was purified from activated platelets by four sequential chromatographic steps. The purified protein was recognized and immunoprecipitated by antibodies to phosphotyrosine. In addition, the antibodies to phosphotyrosine recognized α-actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates. Collectively, these data demonstrate that a fraction of α-actinin is tyrosine phosphorylated in activated platelets. Human platelets were isolated by gel filtration from freshly drawn blood anticoagulated with 0.15 volumes of NIH formula A acid-citrate-dextrose solution supplemented with 1 μm prostaglandin E1 and 1 unit/ml apyrase as described previously (9Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). Platelet concentration was adjusted to 2–5 × 108 platelets/ml in an incubation buffer containing 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 5.6 mm glucose, 1 mg/ml bovine serum albumin, 3.3 mmNaH2PO4, and 20 mm HEPES, pH 7.4. Platelet adhesion to fibrinogen (100 μg/ml) was studied in electrically untreated polystyrene plates pre-coated with the specific protein and blocked with bovine serum albumin as described previously (9Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). To purify large amounts of protein, platelets were isolated as described above from 1 to 2 units of outdated platelets that were purchased from the New Jersey Blood Bank. The isolated platelets were activated for 20 min with 10 nm PMA. Platelet aggregates were collected by 5 min of centrifugation at 800 × g. The platelet pellet from each unit was resuspended in 20 ml of buffer containing 0.02% Triton X-100, 1 mm EGTA, 50 mm Tris-HCl, pH 8.0, 1 mm Na3VO4, and 1 mmphenylmethylsulfonyl fluoride (lysis buffer) and lysed by sonication. All subsequent steps were carried out at 4 °C. Insoluble material was removed by 15 min of centrifugation at 15,000 × g. Ammonium sulfate was added sequentially to the supernatant to yield final concentrations of 10, 20, 30, and 40%. At each step the solution was mixed for at least 45 min prior to a 15-min centrifugation at 15,000 × g. Proteins precipitated in the presence of 10, 20, 30, and 40% ammonium sulfate were reconstituted in lysis buffer and dialyzed extensively. All fractions were subsequently centrifuged for 15 min at 15,000 × g. Duplicate samples from each fraction were analyzed by Western blots probed with the mAb to phosphotyrosine, 4G10, and by silver staining of gels. Most, if not all tyrosine-phosphorylated proteins precipitated in the presence of less than 40% ammonium sulfate. The proteins re-solubilized and dialyzed following precipitation with 30% and 40% ammonium sulfate were combined and applied to a DEAE-Sepharose ion exchange column (1.0 × 4 cm) pre-equilibrated with lysis buffer. Proteins retained on the column were eluted with a step gradient of 0.2, 0.4, 0.6, 0.8, and 1 m NaCl prepared in lysis buffer (two column volumes were used for each NaCl concentration). Proteins eluted off the DEAE column were dialyzed, concentrated, and analyzed by Western blots probed with mAb 4G10. Subsequently, proteins contained in the fractions eluted with 0.8 and 1 m NaCl were combined and Western-blotted onto an Immobilon polyvinylidene difluoride membrane. The membrane was stained with India ink, and the 105-kDa region was excised. Protein digestion with endoproteinase Lys-C, HPLC fractionation, and internal peptide sequencing were carried out by the microsequencing facility at the Rockefeller University (28Fernandez J. DeMott M. Atherton D. Mische S.M. Anal. Biochem. 1992; 201: 255-264Crossref PubMed Scopus (247) Google Scholar). α-actinin was purified from two units of outdated platelets. Platelets were isolated by gel filtration and stimulated for 20 min with 50 nm PMA. A purification flow chart is shown in Fig. 3. Platelet aggregates were collected by 5 min of centrifugation at 800 × g. The resulting pellet was resuspended in 5 ml of buffer containing 50 mmTris-HCl, pH 9.0, 200 mm NaCl, 10 mmCaCl2, 1 mm Na3VO4 and 1 mm phenylmethylsulfonyl fluoride (buffer 1). The activated platelets were lysed by sonication. The lysate was cleared by 20 min of centrifugation at 17,000 × g. The resulting supernatant was resolved by gel filtration on a Sephacryl 300-HR column (1.6 × 70 cm) at a constant flow of 0.32 ml/min in buffer 1. Fractions containing α-actinin were identified by Western blots and probing with mAb 4G10 and an α-actinin antiserum and were combined. KCl was added to the protein solution to reach 0.6 m. The solution was vigorously vortexed and dialysis-concentrated against buffer containing 50 mm Tris-HCl, pH 9.0, 0.6 mKCl, 10 mm CaCl2, and 1 mmNa3VO4 (buffer 2) utilizing a concentrator apparatus (Schleicher & Schuell) equipped with a collodion bag with a molecular mass cut-off of 75 kDa. The volume was reduced to 5 ml or less. The sample was centrifuged for 60 min at 100,000 ×g (20 °C; SW Ti55 Beckman rotor). The supernatant was further dialysis-concentrated to 2 ml and then subjected to gel filtration on a Sephacryl 300-HR column (1.6 × 70 cm) pre-equilibrated with buffer 2 at a constant flow of 0.32 ml/min. Fractions containing α-actinin were identified by Western blots, as described above, and combined. The protein solution was dialysis-concentrated down to 2 ml against buffer containing 50 mm Tris-HCl, pH 7.6, 10 mm CaCl2, 1 mm Na3VO4 (buffer 3). This sample was passed through a CM-Sepharose column (1.2 × 14 cm) at a flow of 1 ml/min. α-Actinin was not retained on this column. α-Actinin-containing fractions were pooled and loaded onto a DEAE-Sepharose column (1.2 × 14 cm) pre-equilibrated with buffer 3. The column was washed with 30 ml of the loading buffer, and proteins were eluted off the column with a linear increase in NaCl concentration from 0–0.5 m in 40 ml of buffer 3. Fractions containing α-actinin were identified by Western blots and combined. All chromatographic analyzes were performed at room temperature.FIG. 3α-Actinin purification from PMA-stimulated platelets. Purified outdated platelets were activated with PMA and lysed by sonication. Soluble proteins were subjected to gel filtration on a Sephacryl 300-HR column (Aand B). Panel A shows the chromatography profile of this column, and arrows point to the first and last fractions that were subsequently combined (fractions 3–12). Fractions containing α-actinin and tyrosine-phosphorylated proteins were identified, respectively, by Western blots and probing with mAb 4G10 and α-actinin antiserum (B). Most of the tyrosine-phosphorylated proteins, including the 105-kDa protein, were recovered in the α-actinin-containing fractions. The combined fractions were brought to 0.6 m KCl. The resulting sample was treated as described under ”Materials and Methods.“ The sample was next resolved by gel filtration on a Sephacryl 300-HR column in the presence of 0.6 m KCl. C shows the chromatography profile of the column. Arrows point to the first and last fractions that were subsequently combined (fractions 4–6). The migration profile of α-actinin and the tyrosine-phosphorylated proteins were determined by Western blots (D). The combined fractions were next applied to a CM-Sepharose column. α-Actinin was not retained on this column (data not shown). The sample was next loaded onto a DEAE-Sepharose column. α-Actinin was eluted off the column with a linear gradient of 0–0.5M NaCl (E). Fractions containing α-actinin and tyrosine-phosphorylated proteins were identified by Western blots as described above. An outline of the purification scheme, including the protein amount recovered at each step, is shown in F.G, duplicate samples from the various purification steps marked in F were loaded onto two gels. One gel was silver-stained. The second gel was Western-blotted and probed with mAb 4G10 and α-actinin antiserum. Lanes 2-7 were loaded with 10 μg of protein, and lanes 8 and 9were loaded with 5 μg of protein.View Large Image Figure ViewerDownload (PPT) Tyrosine-phosphorylated proteins were detected in most experiments using mAb 4G10 (Upstate Biotechnology). In some experiments, mAb PY-20 (Transduction Laboratories) or mAb PY-99 (Santa Cruz Biotechnology) were used yielding indistinguishable results. Two α-actinin antiserums were used. One antiserum was purchased from Sigma (A-2543). This antiserum was used to generate the data shown in Fig. 1. A second antiserum was generated by us by immunizing rabbits with a 14-mer peptide derived from residues 25–38 of human α-actinin (Swiss Protein Database accession number P12814,aac1_human). Attempts to precipitate α-actinin with either α-actinin antiserum were unsuccessful. Antibody reactivity was detected using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Phosphotyrosine-containing proteins and α-actinin were visualized by chemiluminescent detection with ECL (Amersham Pharmacia Biotech). To immunoprecipitate tyrosine-phosphorylated proteins, samples were pre-cleared for 1 h with 20 μl of protein A/G agarose beads (Santa Cruz Biotechnology) followed by an overnight incubation with mAb PY-20 (4 μg/sample) in the presence or absence of 50 μm phenyl phosphate. Antibody-antigen complexes were immunoprecipitated with 20 μl of protein A/G-agarose beads, eluted with sample buffer, and analyzed by SDS-PAGE. Fibrinogen-adherent platelets were lysed in buffer containing 20 mm Tris-HCl, pH 8.0, 0.02% Triton X-100, 1 mm EGTA, 1 mmNa3VO4, and 1 mmphenylmethylsulfonyl fluoride. Lysates containing 60 μg protein/lane were resolved by gel electrophoresis and transferred onto nitrocellulose. The membrane was blocked from 2 h to overnight with 2% albumin (Fraction V, ICN) in buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, and 0.03% sodium azide. The membrane was separated into strips each representing one lane of the gel. The strips were submerged in solution containing 20 mm Tris-HCl, pH 7.2, 150 mm NaCl, 0.1% mercaptoethanol, and 1 mg/ml bovine serum albumin with or without 25 milliunits/ml of protein tyrosine phosphatase (PTPase), a recombinant 34-kDa fragment from Yersinia enterocolitica (from Roche Molecular Biochemicals). Dephosphorylation was carried out at room temperature. At times 0 and 60 min, one strip from each group was removed, rinsed in water, and placed in 50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.02% Nonidet P-40, 1 mg/ml bovine serum albumin, and 1 mm Na3VO4 overnight. The strips were probed with mAb 4G10 and the antiserum to α-actinin. Two approaches were used for protein separation in the first (pI) dimension. The first approach involved lysis of fibrinogen adherent platelets or PMA-stimulated platelets in boiling 2% SDS in 66 mm Tris, pH 8.0. In some experiments, platelets were treated for 1 h with 12 μm bisindolylmaleimide, a protein kinase C inhibitor, (29Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar) to prevent platelet spreading and pp105 tyrosine phosphorylation (21Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar). Isoelectric focusing of samples containing 50 μg of protein was carried out in a glass tube using 2% pH 4–8 ampholines (BDH from Hoefer Scientific Instruments, as described in Ref. 30O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Second-dimension SDS-PAGE was carried out on 8% acrylamide gels. The two-dimensional gel analysis was performed by Kendrick Laboratories Inc. (Madison, WI). The second two-dimensional gel analysis performed by us entailed the use of polyacrylamide gels that are pre-cast on plastic support film and contain an immobilized pH gradient of 3–10 (Immobiline DryStrips, Amersham Pharmacia Biotech). Unstimulated, fibrinogen-adherent, and PMA-stimulated platelets were lysed in 9m urea, 2% Triton X-100, and 10 mmdithiothreitol. 50–100 μg of protein were loaded onto the Immobiline DryStrips. First-dimension electrophoresis was performed utilizing the Multiphor II apparatus (Amersham Pharmacia Biotech) following the procedure described by the manufacturer. Second-dimension SDS-PAGE was carried out on 7% acrylamide gels. Platelet activation with soluble agonists such as PMA as well as platelet adhesion to immobilized matrix proteins such as fibrinogen (9Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 31Ferrell J.E. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2234-2238Crossref PubMed Scopus (204) Google Scholar, 32Golden A. Brugge J.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 901-905Crossref PubMed Scopus (213) Google Scholar) triggers the induction of tyrosine phosphorylation of proteins that migrate with an apparent molecular mass of 101–105 kDa (pp101 and pp105) (Fig. 1 A). To begin to characterize these proteins, we set out to partially purify a 105-kDa protein(s) from PMA-activated outdated platelets. The activated platelets were lysed in a buffer containing 0.02% Triton X-100. Soluble proteins were precipitated with 10–40% ammonium sulfate. After re-solubilization, aliquots from each fraction containing an equal amount of protein (15 μg/lane) were resolved by SDS-PAGE. Gels were analyzed by silver staining or Western blotting and probed with the phosphotyrosine-specific mAb 4G10 (Fig. 1 C). A tyrosine-phosphorylated protein band of 105 kDa was enriched in 30 and 40% ammonium sulfate precipitates. These fractions were pooled and applied to a DEAE-Sepharose ion exchange column. Proteins retained on the column were eluted with a step gradient of 0.2–1 mNaCl. Duplicate samples containing an equal protein amount were subjected to SDS-PAGE and analyzed by silver staining or Western blotting and probing with mAb 4G10 (Fig. 1 D). Proteins eluted off the DEAE column with 0.8 and 1 m NaCl were pooled and Western-blotted onto an Immobilon polyvinylidene difluoride membrane and visualized with India ink. The 105-kDa region was cut out and HPLC purified peptides derived from endoproteinase Lys-C digestion were microsequenced (28Fernandez J. DeMott M. Atherton D. Mische S.M. Anal. Biochem. 1992; 201: 255-264Crossref PubMed Scopus (247) Google Scholar). A 12-amino acid internal sequence, QLEINFNTLQTK, was identified. The sequence was identical to human α-actinin sequences aac1_human, aac2_human, andaac3_human deposited in the Swiss Protein Database. Based on these data, the blots shown in Fig. 1, A, C, andD, were reprobed with a polyclonal antiserum to α-actinin. In all three cases (Fig. 1, B, C, andD), the α-actinin antiserum reacted with a single protein band of 105-kDa that co-migrated with the 105-kDa tyrosine-phosphorylated protein identified with mAb 4G10. Lysates from activated platelets were Western-blotted onto nitrocelullose and dephosphrylated with a recombinant tyrosine phosphatase (PTPase) fromY. enterocolitica. Dephosphorylation of the proteins eliminated reactivity with mAb 4G10 but had no effect on the reactivity of the samples with the antiserum to α-actinin (Fig. 1 E). Collectively, these data suggested that α-actinin may be the 105-kDa tyrosine-phosphorylated protein detected in activated platelets. To further demonstrate that α-actinin is tyrosine-phosphorylated in activated platelets, an α-actinin purification protocol was developed. Prior α-actinin purification protocols relied on the relatively low solubility of this cytoskeletal protein in buffers containing 1% Triton X-100. For example, Rosenberg et al.(33Rosenberg S. Stracher A. Burridge K. J. Biol. Chem. 1981; 256: 12986-12991Abstract Full Text PDF PubMed Google Scholar) reported that more than 90% of platelet α-actinin was recovered in the Triton-insoluble pellet fraction. To further investigate the cellular partitioning of α-actinin and pp105, platelets were activated with PMA for 0, 1, 5, 10, 15, and 20 min before lysis (Fig. 2). At the indicated time points, the platelets were lysed by the addition of an equal volume of buffer containing 2% Triton X-100 as described by Fox et al. (22Fox J.E.B. Lipfer L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar). Lysates were spun for 4 min at 12,000 × g, yielding the low speed pellet. The resulting supernatants were sedimented for 2.5 h at 100,000 × g, resulting in a soluble and an insoluble high speed pellet for each time point. The low speed and high speed fractions contain, respectively, cytoskeleton- and membrane-cytoskeleton-associated proteins (22Fox J.E.B. Lipfer L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar). Proteins contained in each fraction were Western-blotted and analyzed with mAb 4G10 and the α-actinin antiserum (Fig. 2, A and B). PMA stimulation triggered a time-dependent decrease in the amount of α-actinin recovered in the soluble (supernatant fractions) and the membrane-cytoskeleton (high speed pellet) pools with a simultaneous increase in α-actinin localized to the cytoskeleton fraction (low speed pellet). Whereas in resting platelets 18% of α-actinin was recovered in the cytoskeleton fraction, 56% of α-actinin was recovered in the cytoskeleton fraction of platelets stimulated with PMA for 20 min (Fig. 2 C). In contrast, the fraction of soluble α-actinin dropped from 53 to 38%. Most dramatic, however, was the change in α-actinin partitioning to the high speed/membrane-cytoskeleton fraction, which dropped from 29 to 4% over the same time period. Many other platelet proteins have been shown to move from the soluble to the cytoskeleton fraction of activated platelets (22Fox J.E.B. Lipfer L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar, 34Schoenwaelder S.M. Yuan Y. Cooray P. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 1694-1702Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In contrast with this trend, a time-dependent increase in the tyrosine-phosphorylated 105-kDa protein and its predominant enrichment in the detergent-soluble fractions was observed. After a much longer exposure of the gel shown in Fig. 2 A, we did note that α-actinin localized to the membrane-cytoskeleton compartment at the 20-min time point was also tyrosine-phosphorylated. However, no tyrosine phosphorylation signal associated with α-actinin in the cytoskeleton compartment was detected. Similar result were obtained with thrombin-stimulated platelets (22Fox J.E.B. Lipfer L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar). These data suggested that most of the phosphorylated 105-kDa protein was concentrated in fractions that contained soluble α-actinin. To enrich for the
Referência(s)