The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase
2001; Elsevier BV; Volume: 276; Issue: 31 Linguagem: Inglês
10.1074/jbc.m101678200
ISSN1083-351X
AutoresGonzalo Izaguirre, Lina Aguirre, Ya-Ping Hu, Hwa Young Lee, David D. Schlaepfer, Brian J. Aneskievich, Beatrice Haimovich,
Tópico(s)Advanced Proteomics Techniques and Applications
Resumoα-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012–37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that α-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His6 tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant α-actinin protein containing a Tyr → Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored α-actinin phosphorylation. Purified wild type α-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of α-actinin that cosedimented with actin filaments. These results establish that α-actinin is a direct substrate for FAK and suggest that α-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton. α-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012–37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that α-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His6 tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant α-actinin protein containing a Tyr → Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored α-actinin phosphorylation. Purified wild type α-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of α-actinin that cosedimented with actin filaments. These results establish that α-actinin is a direct substrate for FAK and suggest that α-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton. reverse transcription polymerase chain reaction focal adhesion kinase base pair(s) hemagglutinin vasodilator-stimulated phosphoprotein α-Actinin is a ubiquitously expressed protein and a member of a large family of actin-cross-linking proteins that includes fimbrin, dystrophin, and spectrin (2Matsudaira P. Trends Biochem. Sci. 1991; 16: 87-92Abstract Full Text PDF PubMed Scopus (237) Google Scholar). α-Actinins form homodimers composed of two polypeptide subunits arranged in an antiparallel orientation. Three highly conserved domains have been identified in the protein (3Puius Y.A. Mahoney N.M. Almo S.C. Curr. Opin. Cell Biol. 1998; 10: 23-34Crossref PubMed Scopus (112) Google Scholar). The amino terminus contains two calponin-like actin-binding modules that can fold independently (4Goldsmith S.C. Pokala N. Shen W. Fedorov A.A. Matsudaira P. Almo S.C. Nat. Struct. Biol. 1997; 4: 708-712Crossref PubMed Scopus (110) Google Scholar). The central region is composed of four spectrin-like α-helical repeats that are involved in monomer-monomer interaction. The carboxyl terminus contains one or two calcium binding (EF) motifs depending on the isoform. At least four human α-actinin genes have been described. One gene (aac1) gives rise to two alternative splice variants; the cytoskeletal/non-muscle isoform contains two EF hand motifs, whereas the second variant, known as the smooth muscle isoform, has a single EF hand motif (5Millake D.B. Blanchard A.D. Patel B. Critchley D.R. Nucleic Acids Res. 1989; 17: 6725Crossref PubMed Scopus (56) Google Scholar, 6Waites G.T. Graham I.R. Jackson P. Millake D.B. Patel B. Blanchard A.D. Weller P.A. Eperon I.C. Critchley D.R. J. Biol. Chem. 1992; 267: 6263-6271Abstract Full Text PDF PubMed Google Scholar, 7Youssoufian H. McAfee M. Kwiatkowski D.J. Am. J. Hum. Genet. 1990; 47: 62-71PubMed Google Scholar). The binding of the cytoskeletal/non-muscle isoform to actin is inhibited by calcium, whereas the binding of the smooth muscle isoform to actin is calcium-insensitive. An additional α-actinin isoform (aac4) that exhibits 80% sequence identity to theaac1 gene products was cloned from a tumor cell line (8Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (390) Google Scholar). The two remaining genes, aac2 and aac3, encode several skeletal muscle isoforms (9Beggs A.H. Byers T.J. Knoll J.H. Boyce F.M. Bruns G.A. Kunkel L.M. J. Biol. Chem. 1992; 267: 9281-9288Abstract Full Text PDF PubMed Google Scholar). In non-muscle cells α-actinin colocalizes with actin and stabilizes the actin filament web. α-Actinin is also found in focal adhesion plaques where the actin filaments originate (10Lazarides E. Burridge K. Cell. 1975; 6: 289-298Abstract Full Text PDF PubMed Scopus (411) Google Scholar). The localization of α-actinin in focal adhesion plaques suggested that it might serve to anchor the network of actin filaments to the plasma membrane. This possibility was substantiated by the finding that α-actinin associates with the cytoplasmic tail of members of several adhesion receptors families including integrins (11Otey C.A. Pavalko F.M. Burridge K. J. 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Lin S. Biochem. Biophys. Res. Commun. 1987; 146: 554-560Crossref PubMed Scopus (132) Google Scholar), zyxin (18Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar), and the newly described proteins palladin (19Parast M.M. Otey C.A. J. Cell Biol. 2000; 150: 643-656Crossref PubMed Scopus (171) Google Scholar) and CLP-36 (20Vallenius T. Luukko K. Makela T.P. J. Biol. Chem. 2000; 275: 11100-11105Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 21Bauer K. Kratzer M. Otte M. de Quintana K.L. Hagmann J. Arnold G.J. Eckerskorn C. Lottspeich F. Siess W. Blood. 2000; 96: 4236-4245Crossref PubMed Google Scholar). A third group of proteins that interact with α-actinin includes signaling molecules such as extracellular signal-regulated kinase 1 (22Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (125) Google Scholar), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (23Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskelet. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar), PKN, a fatty acid and Rho-activated serine/threonine protein kinase (24Mukai 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), and the p85 subunit of phosphatidylinositol 3-kinase (25Shibasaki F. Fukami K. Fukui Y. Takenawa T. Biochem. J. 1994; 302: 551-557Crossref PubMed Scopus (89) Google Scholar). The broad spectrum of molecules with which α-actinin interacts strongly suggests that in addition to its role as an actin cross-linking protein, α-actinin also functions as a scaffold to promote protein-protein interactions. As a scaffold protein that is closely associated with both transmembrane adhesion receptors and cytoskeletal proteins, α-actinin may be an attractive regulatory target. Calcium binding to the EF hand modules in α-actinin decreases the interaction between α-actinin and actin (26de Arruda M.V. Watson S. Lin C.S. Leavitt J. Matsudaira P. J. Cell Biol. 1990; 111: 1069-1079Crossref PubMed Scopus (158) Google Scholar, 27Witke W. Hofmann A. Koppel B. Schleicher M. Noegel A.A. J. Cell Biol. 1993; 121: 599-606Crossref PubMed Scopus (80) Google Scholar). Fukami et al. (28Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Crossref PubMed Scopus (304) Google Scholar, 29Fukami K. Sawada N. Endo T. Takenawa T. J. Biol. Chem. 1996; 271: 2646-2650Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) have shown that the skeletal muscle isoform of α-actinin binds phosphatidylinositol 4,5-bisphosphate and that the actin gelating activity of the protein was enhanced by the phospholipid. More recently, Greenwood et al. (30Greenwood J.A. Theibert A.B. Prestwich G.D. Murphy-Ullrich J.E. J. Cell Biol. 2000; 150: 627-642Crossref PubMed Scopus (103) Google Scholar) reported that in rat embryonic fibroblasts, phosphatidylinositol 3,4,5-triphosphate, a lipid product of phosphatidylinositol 3-kinase, bound to α-actinin. Immunoprecipitation studies suggested that the binding of phosphatidylinositol 3,4,5-triphosphate to α-actinin decreased the binding affinity of α-actinin for β3 and β1 integrins (30Greenwood J.A. Theibert A.B. Prestwich G.D. Murphy-Ullrich J.E. J. Cell Biol. 2000; 150: 627-642Crossref PubMed Scopus (103) Google Scholar). This correlated with a relocalization of α-actinin and actin to the cell cortex and was accompanied by dissolution of actin stress fibers. These data raise the possibility that the interaction between α-actinin and its ligands may be regulated by more than one mechanism. Protein phosphorylation is a common signaling relay mechanism in numerous cellular processes. We have recently reported that α-actinin is tyrosine-phosphorylated in activated platelets (1Izaguirre G. Aguirre L. Ji P. Aneskievich B. Haimovich B. J. Biol. Chem. 1999; 274: 37012-37020Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Tyrosine phosphorylation of α-actinin was also observed in activated T-cells (31Egerton M. Moritz R.L. Druker B. Kelso A. Simpson R.J. Biochem. Biophys. Res. Commun. 1996; 224: 666-674Crossref PubMed Scopus (28) Google Scholar). The question of whether α-actinin is phosphorylated in non-hematopoietic cells is currently unresolved. We considered the possibility that the robust phosphorylation of α-actinin in platelets may be due to the expression of a distinct platelet α-actinin isoform. Analysis of platelet RNA by RT-PCR1 and sequencing of the resulting cDNA revealed that the α-actinin isoform expressed in platelets is identical to the human cytoskeletal/non-muscle isoform. Next, we generated and expressed a recombinant His-tagged construct of the human cytoskeletal/non-muscle isoform in various cell types. Using the recombinant protein we show that α-actinin is phosphorylated in non-hematopoietic cells and that the tyrosine residue at position 12 in α-actinin is the site of phosphorylation. In platelets, tyrosine phosphorylation of α-actinin and the focal adhesion kinase (FAK) are closely regulated events raising the possibility that α-actinin is a FAK substrate (32Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 33Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar, 34Lipfert L. Haimovich B. Schaller M. Cobb B.S. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (632) Google Scholar). Consistent with this possibility we show that α-actinin is not phosphorylated in cells that lack FAK and that re-expression of FAK restored the phosphorylation of wild type α-actinin in these cells. Furthermore, wild type α-actinin, but not a mutant protein carrying a Tyr → Phe substitution at position 12 (Y12F), was phosphorylated by FAK in vitro, whereas a kinase-dead FAK mutant protein did not stimulate phosphorylation. Phosphorylation reduced the binding of α-actinin to actin. These data establish that α-actinin is a novel FAK substrate and as such is likely to transduce FAK-dependent signals that regulate the organization of the cytoskeleton. Human platelets were isolated by gel filtration from freshly drawn blood anticoagulated with 0.15 volumes of National Institutes of Health formula A acid-citrate-dextrose solution supplemented with 1 µm prostaglandin E1 and 1 unit/ml apyrase as described previously (32Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). The 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 mm NaH2PO4, and 20 mm HEPES, pH 7.4. Platelet adhesion to fibrinogen (100 µg/ml) was studied in electrically untreated polystyrene plates precoated with fibrinogen and blocked with bovine serum albumin as described previously (32Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar). Cell spreading was promoted by the addition of 10 nm phorbol 12-myristate 13-acetate. Platelets were isolated from 100 ml of fresh blood, pelleted by centrifugation, and resuspended in 3 ml of TRIzol (Life Technologies, Inc.). Reverse transcription of RNA and amplification of cDNA were carried out utilizing the one-step RT-PCR System. Approximately 200 ng of total RNA were used in a 50-µl reaction mixture with the reverse transcription step at 50 °C for 30 min followed by 2 min at 94 °C. cDNA amplification was accomplished in 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 15 s, and extension at 72 °C for 1 min with a final one-step extension for 7 min at 72 °C. Two sets of primers that matched the sequence of the human non-muscle isoform of α-actinin (GenBankTM accession number X15804) (5Millake D.B. Blanchard A.D. Patel B. Critchley D.R. Nucleic Acids Res. 1989; 17: 6725Crossref PubMed Scopus (56) Google Scholar) resulted in PCR products with predicted sizes of 1051 bp (primer 1, 5′-gag aag gtg gag gaa gaa-3′ (forward) and 5′-cag cgt gtt gaa gtt gat-3′ (reverse)) and 1745 bp (primer 2, 5′-gca caa gcc gcc caa ggt-3′ (forward) and 5′-gtg caa ggc agg gca cgg-3′ (reverse) (see Fig. 1). When combined, the two PCR products contained the complete sequence of the cDNA. The nucleotide sequence from the PCR products was determined using primers that hybridized to the cytoskeletal/non-muscle isoform of α-actinin. Using these primers, the entire platelet α-actinin cDNA product was sequenced. The cell line MCR-5 was obtained from the American Type Culture Collection. RNA from MCR-5 cells was extracted and analyzed by RT-PCR as described above. One set of primers was used to amplify the smooth muscle α-actinin isoform (primer 3, 5′-gag atc aat ggc aaa tgg-3′ (forward) and 5′-aca atc cat cat acc agt ctt c (reverse)) (see Fig. 1). The latter primer (primer 3, reverse) matched the sequence unique to the human smooth muscle isoform (6Waites G.T. Graham I.R. Jackson P. Millake D.B. Patel B. Blanchard A.D. Weller P.A. Eperon I.C. Critchley D.R. J. Biol. Chem. 1992; 267: 6263-6271Abstract Full Text PDF PubMed Google Scholar). The complete cDNA encoding for the cytoskeletal/nonmuscle isoform of α-actinin (7Youssoufian H. McAfee M. Kwiatkowski D.J. Am. J. Hum. Genet. 1990; 47: 62-71PubMed Google Scholar) (a gift from Dr. Avri Ben-Z'eev) was subcloned in frame into pQE-31 vector (Qiagen, Inc. Chatsworth, CA), resulting in the addition of a His6 tag to the amino terminus. The gene was then subcloned into the vector pcDNA3.1(+) (Invitrogen Corp., San Diego, CA) for mammalian expression. Tyrosines at positions 4, 12, 193, and 319 were replaced with phenylalanines using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) and Pfu Turbo DNA polymerase to make single base changes from TAT and TAC to TTT and TTC, respectively. The mutation primers for Tyr-4 were: 5′-ccg cgc acc atc atg gac cat ttt gat tct cag caa acc-3′ (forward) and 5′-ggt ttg ctg aga atc aaa atg gtc cat gat ggt gcg cgg-3′ (reverse), and those for Tyr-12 were 5′-gca aac caa cga ttt cat gca gcc aga aga gga ctg gg-3′ (forward) and 5′-ccc agt cct ctt ctg gct gca tga aat cgt tgg ttt gc-3′ (reverse). The mutation primers for Tyr-193 were: 5′-ggc-ccg-agc-tca-ttg-act-tcg-gga-agc-tgc-gga-ag-3′ (forward) and 5′-ctt-ccg-cag-ctt-ccc-gaa-gtc-aat-gag-ctc-ggg-cc-3′ (reverse). The mutation primers for Tyr-319 were 5′-caa-cag-aag-ctt-gag-gac-ttc-cgg-gac-ttc-cgg-cgc-ctg-3′ (forward) and 5′-cag-gcg-ccg-gaa-gtc-ccg-gaa-gtc-ctc-aag-ctt-ctg-ttg-3′ (reverse). The tyrosine residue at position 12 was converted to a glutamic acid residue using the following primers: 5′-gca-aac-caa-cga-tga-gat-gca-gcc-aga-aga-gga-ctg-gg-3′ (forward) and 5′-ccc-agt-cct-ctt-ctg-gct-gca-tct-cat-cgt-tgg-ttt-gc-3′ (reverse). DNA sequencing was performed to confirm the correct introduction of the mutations. The synthesis of the primers and the DNA sequencing were carried out at Integrated DNA Technologies, Inc. (Coralville, IA). The fibroblasts established from FAK−/− and p53−/− mouse embryos (35Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar) were cultured as described (35Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar, 36Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar) and used at passages 15–25. Transfection of COS-7 cells and the FAK−/− fibroblasts was carried out using the LipofectAMINE Plus reagents (Life Technologies, Inc.) following the procedure recommended by the vendor. Where indicated, vanadate (sodium vanadate, Fisher catalog number S454-50) was added to the culture medium 48 h after transfection. Vanadate (100 mm stock solution) was dissolved in deionized water, boiled for 5 min, and used at a 1:200 dilution (500 µm). Unless otherwise indicated, the cells were cultured in the presence of vanadate for 24 h prior to analysis. To immunoprecipitate the recombinant proteins, adherent COS-7 cells were lysed in RIPA buffer (1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate, 158 mm NaCl, 10 mmTris, pH 7.2, 1 mm phenylmethylsulfonyl fluoride, and 1 mm vanadate). The samples were normalized for protein content (200–400 µg of protein/sample in 500 µl) and were precleared for 30 min with 20 µl of protein A/G-agarose beads (Santa Cruz Biotechnology. Santa Cruz, CA) followed by an incubation with 4 µg of an anti-His monoclonal antibody (Qiagen, Inc.) for 2 h. Antibody-antigen complexes were precipitated with 20 µl of protein A/G-agarose beads, washed with RIPA buffer, and eluted with Laemmli's loading buffer. The proteins were resolved by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Where indicated, the RIPA washes were followed by three washes with 50 mm Tris, pH 8, 0.25 mm MgCl2. The immune complexes were next incubated for 2 h with 50 mm Tris, pH 8, 0.25 mm MgCl2 in the absence or the presence ofEscherichia coli alkaline phosphatase (5 units/sample) (Calbiochem, San Diego, CA) prior to elution with Laemmli's loading buffer. A monoclonal antibody to tyrosine-phosphorylated proteins (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), an antiserum to α-actinin (antiserum generated by immunizing rabbits with a 14-mer peptide derived from residues 25–38 of human α-actinin), an anti-FAK antiserum directed against the FAK carboxyl-terminal region (36Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar), and anti-actinin antiserum (Sigma) and anti-HA monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA or Babco, Berkely, CA) were used for immunodetection of blotted proteins. Immunocomplexes were detected using horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies and visualized by chemiluminescence with enhanced chemiluminescence (PerkinElmer Life Sciences). Recombinant α-actinin was transiently expressed in COS-7 cells alone or in combination with recombinant HA-FAK. Unphosphorylated and phosphorylated His-α-actinin proteins were purified, respectively, from cells that were untreated or treated with vanadate for 24 h prior to cell lysis. The recombinant proteins were purified utilizing nickel-nitrilotriacetic acid agarose beads (Qiagen, Inc.) in a column setting. Adherent cells in 100-mm dishes were lysed in 1 ml of lysis buffer (50 mm Tris-HCl, pH 8.0, 1 m NaCl, 10 mm imidazole, 1 mm vanadate, 1 mmphenylmethylsulfonyl fluoride, and 1% Triton X-100) per dish. The cell lysate was passed several times through a 27G1/2 needle to disrupt the cells and to shear the DNA. The lysate was cleared by centrifugation and mixed with 1 ml of nickel-nitrilotriacetic acid agarose resin that was pre-equilibrated in wash buffer (same as lysis buffer but lacking Triton X-100). After 1 h of incubation with repeated resuspensions, the resin was washed with 50 ml of wash buffer supplemented with 3 m urea and then with 50 ml of 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 10 mm imidazole, and 1 mm vanadate. The proteins were eluted in 4 ml of 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 50 mm EDTA, and 1 mmvanadate. The sample was concentrated by dialysis concentration in a collodion bag with a molecular mass cut-off of 75 kDa (Schleicher & Schuell) against 10 mm Tris-HCl, pH 8.0, containing 1 mm vanadate. Platelet and recombinant α-actinin isolated from COS-7 cells were subjected to in gel proteolytic digestion. Platelet α-actinin was obtained from fibrinogen adherent platelets costimulated with 10 nm phorbol 12-myristate 13-acetate. The cells were lysed in Laemmli's loading buffer and electrophoresed under denaturing conditions in a 7.5% T acrylamide gel (18 cm long, 1.5 mm thick). A section of the gel was blotted onto nitrocellulose. The membrane was probed with antibodies to phosphotyrosine and to α-actinin to verify the phosphorylation of α-actinin and its position in the gel. The rest of the gel was stained with Coomassie Blue R-250 for 2 h and destained overnight. The gel was rehydrated in water until it regained its original dimensions. The α-actinin band was excised using a razor blade and placed in a microcentrifuge tube. Each gel piece was washed for 5 min with 300 µl of 50% acetonitrile and then for 30 min each with the same volumes of 50% acetonitrile, 50 mm NH4HCO3, pH 8.0, followed by 50% acetonitrile, 10 mmNH4HCO3, pH 8.0. After the last wash, the gel pieces were dried by lyophilization and stored below 0 °C. Recombinant proteins were resolved by electrophoresis under denaturing conditions in a 10% T acrylamide gel (5 cm in length, 1.5 mm thick). The protein bands were excised and washed as described above for the platelet α-actinin. In gel proteolytic digestions of α-actinin were carried out with thermolysin (0.01 µg/µl; Sigma) in 20 mmNH4HCO3, pH 8.0, 10 mmCaCl2, and 1 mm ZnCl2, or with aminopeptidase I (0.05 µg/µl; Sigma) in 20 mmNH4HCO3, pH 8.0. The dehydrated gel pieces were first rehydrated on ice for 30 min with 20 µl of buffer plus enzyme (2-fold concentrated). The volume was increased to 40 µl by adding buffer, and the samples were incubated at 30 °C for the indicated time. The digestion was terminated by the addition of 50 µl of Laemmli's loading buffer (4×) and by heating for 2 min at 100 °C. The samples (including the gel pieces) were loaded onto an acrylamide gel, and the proteins were electrophoresed and analyzed by Western blotting and immunodetection. Human 293 cells were cultured and transiently transfected with either HA-tagged wild type FAK, an autophosphorylation defective (Phe-397) FAK, or kinase inactive (Arg-454) FAK cDNAs as described previously (37Schlaepfer D.D. Hunter T. J. Biol. Chem. 1997; 272: 13189-13195Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). After 48 h, the cells were serum-starved overnight and were then stimulated with 100 ng/ml phorbol 12-myristate 13-acetate for 10 min. The cells were lysed in RIPA, and recombinant FAK proteins were isolated with antibodies to the HA tag (monoclonal antibody 16B12 from Babco, Berkeley, CA). Immunoprecipitated proteins were washed twice in Triton lysis buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, 1 mmEGTA, 1 mm sodium vanadate, 10 mm sodium pyrophosphate, 100 mm NaF, 1% Triton X-100, 10 µg/ml leupeptine, 10 units/ml aprotinin, and 1 mmphenylmethylsulfonyl fluoride), once with HNTG buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 0.1% Triton X-100, 10% glycerol), and once with kinase buffer (20 mmHEPES, pH 7.4, 10% glycerol, 10 mm MgCl2, 150 mm NaCl) and were then incubated with 1 µg/sample of recombinant wild type or Y12F mutant α-actinin proteins (purified as described above from transfected COS-7 cells using the nickel-nitrilotriacetic acid-agarose beads) along with 10 µCi of [γ-32P]ATP. Phosphorylation reactions were carried out for 15 min at 32 °C, and all products were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidene difluoride membrane. Direct exposures were used to visualize FAK autophosphorylation and α-actinin transphosphorylation. The amount of either α-actinin or FAK present in the reactions was visualized by immunoblotting of the same Immobilon membrane used for the direct exposure. The cosedimentation of α-actinin and actin was assayed using a method adapted from Refs.38Johnson R.P. Craig S.W. J. Biol. Chem. 2000; 275: 95-105Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 39Landon F. Gache Y. Touitou H. Olomucki A. Eur. J. Biochem. 1985; 153: 231-237Crossref PubMed Scopus (36) Google Scholar, 40Imamura M. Masaki T. J. Biol. Chem. 1992; 267: 25927-25933Abstract Full Text PDF PubMed Google Scholar. Actin purified from avian pectoral muscle and polymerized as described previously (41Hammell R.L. Hitchcock-DeGregori S.E. J. Biol. Chem. 1996; 271: 4236-4242Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) was kindly provided by Dr. Sarah Hitchcock-DeGregori (UMDNJ). Recombinant wild type His-α-actinin fusion proteins in the phosphorylated and nonphosphorylated form were purified, respectively, from COS-7 cells untreated or treated with vanadate as described above. Various concentrations of F-actin (0–0.8 µm) were mixed for 60 min at 25 °C with a constant concentration of recombinant His-α-actinin fusion proteins (0.1 or 0.2 µm dimer form) in buffer containing 50 mm NaCl, 5 mmimidazole, pH 7.0, 0.5 mm dithiothreitol, 0.2 mm EGTA, 0.1 mm ATP, and 1 mmNaN3. The mixes (total volume, 50 µl) were centrifuged at 140,000 × g for 25 min at 25 °C using a TLA-100 rotor in a Beckman TL-100 Ultracentrifuge. The supernatants were separated from the pellets, and all of the samples were analyzed by Western blotting. The blots were probed with antibodies to phosphotyrosine, α-actinin, or actin. The films were scan digitized using the program Adobe PhotoShop 5.0. The integrated densities of the bands were measured using the program NIH Image 1.6. We recently demonstrated that α-actinin is tyrosine-phosphorylated in activated platelets (1Izaguirre G. Aguirre L. Ji P. Aneskievich B. Haimovich B. J. Biol. Chem. 1999; 274: 37012-37020Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We considered the possibility that the α-actinin isoform expressed in platelets is distinct from the isoforms expressed in non-hematopoietic cells and therefore more readily phosphorylated. To resolve this issue we characterized the platelet α-actinin at the molecular level. To this end, RNA was extracted from freshly isolated platelets, and RT-PCR reactions were carried out using primers based on the DNA sequence of the cytoskeletal/non-muscle isoform (aac1 human; GenBankTM accession number X15804) (5Millake D.B. Blanchard A.D. Patel B. Critchley D.R. Nucleic Acids Res. 1989; 17: 6725Crossref PubMed Scopus (56) Google Scholar). Two sets of primers yielded two products, one 1051 bp long and the second 1745 bp long (Fig. 1). The combined nucleotide sequence of these two products covered the entire se
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