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• W2008359273 abstract "Phosphorylation of αB-crystallin, a member of the hsp27 family, in human glioma (U373 MG) cells was stimulated by exposure of the cells to various stimuli, which included heat, arsenite, phorbol 12-myristate 13-acetate (PMA), okadaic acid, H2O2, anisomycin, and high concentrations of NaCl or sorbitol, but not in response to agents that elevated intracellular levels of cyclic AMP. Cells exposed to PMA together with okadaic acid yielded three bands of 32P-labeled αB-crystallin when immunoprecipitated samples were subjected to electrophoresis on an isoelectric focusing gel. All of the phosphorylated residues were identified as serine, an indication that three different serine residues can act as sites of phosphorylation in αB-crystallin. Structural analysis by mass spectrometry revealed that phosphorylation of αB-crystallin occurred at serines 19, 45, and 59. Dithiothreitol and staurosporine selectively inhibited the phosphorylation induced by arsenite and the phorbol ester, respectively. SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, suppressed the phosphorylation induced by arsenite, anisomycin, H2O2, sorbitol, NaCl, and heat shock, but not that induced by PMA and okadaic acid. The PMA-induced phosphorylation was selectively suppressed by an inhibitor of p44 MAP kinase kinase, PD98059. Although PMA and arsenite preferentially stimulated the phosphorylation of Ser-45 and Ser-59, respectively, as determined with antibodies that recognized the respective phosphorylated forms of αB-crystallin, all three sites were phosphorylated in response to each stimulus. These results suggest that p38 MAP kinase or p44 MAP kinase might be involved in the signal transduction cascade that leads to the phosphorylation of αB-crystallin. The phosphorylation of αB-crystallin was also enhanced in the heart and diaphragm when rats were exposed to heat stress (42 °C for 20 min). Phosphorylation of αB-crystallin, a member of the hsp27 family, in human glioma (U373 MG) cells was stimulated by exposure of the cells to various stimuli, which included heat, arsenite, phorbol 12-myristate 13-acetate (PMA), okadaic acid, H2O2, anisomycin, and high concentrations of NaCl or sorbitol, but not in response to agents that elevated intracellular levels of cyclic AMP. Cells exposed to PMA together with okadaic acid yielded three bands of 32P-labeled αB-crystallin when immunoprecipitated samples were subjected to electrophoresis on an isoelectric focusing gel. All of the phosphorylated residues were identified as serine, an indication that three different serine residues can act as sites of phosphorylation in αB-crystallin. Structural analysis by mass spectrometry revealed that phosphorylation of αB-crystallin occurred at serines 19, 45, and 59. Dithiothreitol and staurosporine selectively inhibited the phosphorylation induced by arsenite and the phorbol ester, respectively. SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, suppressed the phosphorylation induced by arsenite, anisomycin, H2O2, sorbitol, NaCl, and heat shock, but not that induced by PMA and okadaic acid. The PMA-induced phosphorylation was selectively suppressed by an inhibitor of p44 MAP kinase kinase, PD98059. Although PMA and arsenite preferentially stimulated the phosphorylation of Ser-45 and Ser-59, respectively, as determined with antibodies that recognized the respective phosphorylated forms of αB-crystallin, all three sites were phosphorylated in response to each stimulus. These results suggest that p38 MAP kinase or p44 MAP kinase might be involved in the signal transduction cascade that leads to the phosphorylation of αB-crystallin. The phosphorylation of αB-crystallin was also enhanced in the heart and diaphragm when rats were exposed to heat stress (42 °C for 20 min). A major portion in the eye lens of vertebrates is α-crystallin, which is found as large aggregates of two closely related subunits, αA and αB (1Wistow G. Piatigorsky J. Annu. Rev. Biochem. 1988; 57: 479-504Crossref PubMed Scopus (675) Google Scholar). Both αA-crystallin and αB-crystallin are also present in other tissues (2Bhat S.P. Nagineni C.N. Biochem. Biophys. Res. Commun. 1989; 158: 319-325Crossref PubMed Scopus (496) Google Scholar, 3Iwaki T. Kume-Iwaki A. Liem R.K.H. Goldman J.E. Cell. 1989; 57: 71-78Abstract Full Text PDF PubMed Scopus (494) Google Scholar, 4Nagineni C.N. Bhat S.P. FEBS Lett. 1989; 249: 89-94Crossref PubMed Scopus (38) Google Scholar, 5Dubin R.A. Wawrousek E.F. Piatigorsky J. Mol. Cell. Biol. 1989; 9: 1083-1091Crossref PubMed Scopus (335) Google Scholar, 6Iwaki T. Kume-Iwaki A. Goldman J.E. J. Histochem. Cytochem. 1990; 38: 31-39Crossref PubMed Scopus (270) Google Scholar, 7Longoni S. Lattonen S. Bullock G. Chiesi M. Mol. Cell. Biochem. 1990; 97: 113-120Crossref Scopus (77) Google Scholar, 8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar, 9Kato K. Shinohara H. Kurobe N. Goto S. Inaguma Y. Oshima K. Biochim. Biophys. Acta. 1991; 1080: 173-180Crossref PubMed Scopus (197) Google Scholar). Each subunit is highly homologous in terms of amino acid sequence to a small heat shock protein, hsp27 (10Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (678) Google Scholar,11Hickey E. Brandon S.E. Potter R. Stein G. Stein J. Weber L.A. Nucleic Acids Res. 1986; 14: 4127-4145Crossref PubMed Scopus (195) Google Scholar), and furthermore, the synthesis of αB-crystallin is induced by the physiological and nonphysiological stimuli that induce the synthesis of hsp27 (12Klemenz R. Fröhli E. Steiger R.H. Schäfer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (479) Google Scholar, 13Inaguma Y. Shinohara H. Goto S. Kato K. Biochem. Biophys. Res. Commun. 1992; 182: 844-850Crossref PubMed Scopus (50) Google Scholar). We have shown that hsp27 copurifies with αB-crystallin as a large aggregate from skeletal muscle (14Kato K. Shinohara H. Goto S. Inaguma Y. Morishita R. Asano T. J. Biol. Chem. 1992; 267: 7718-7725Abstract Full Text PDF PubMed Google Scholar). Phosphorylation is one of the major types of post-transcriptional modification of hsp27, and this phenomenon has been intensively investigated. Phosphorylation of hsp27 is observed when cells are exposed to heat (15Landry J. Chretinen P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar, 16Arrigo A.-P. Welch W.J. J. Biol. Chem. 1987; 262: 15359-15369Abstract Full Text PDF PubMed Google Scholar), arsenite (17Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar), and phorbol ester, okadaic acid, tumor necrosis factor α, or interleukin-1α (IL-1α) 1The abbreviations used are: IL-1α, interleukin-1α; MAP, mitogen-activated protein; PMA, phorbol 12-myristate 13-acetate; 4α-PMA, 4α-phorbol 12-myristate 13-acetate; IEF, isoelectric focusing; HPLC-ESI/TSQMS, high-performance liquid chromatography-electrospray ionization/triple stage quadrupole mass spectrometry; MS/MS, tandem mass spectrometry. (17Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar, 18Arrigo A.-P. Mol. Cell. Biol. 1990; 10: 1276-1280Crossref PubMed Scopus (107) Google Scholar, 19Welch W.J. J. Biol. Chem. 1985; 260: 3058-3062Abstract Full Text PDF PubMed Google Scholar, 20Saklatvala J. Kaur P. Guesdon F. Biochem. J. 1991; 277: 635-642Crossref PubMed Scopus (71) Google Scholar, 21Guy G.R. Cao X. Chua S.P. Tan Y.H. J. Biol. Chem. 1992; 267: 1846-1852Abstract Full Text PDF PubMed Google Scholar, 22Kasahara K. Ikuta T. Chida K. Asakura R. Kuroki T. Eur. J. Biochem. 1993; 213: 1101-1107Crossref PubMed Scopus (11) Google Scholar). It has been suggested that phosphorylation of hsp27 is catalyzed by MAP kinase-activated protein kinase-2 (23Stokoe D. Caudwell B. Cohen P.T.W. Cohen P. Biochem. J. 1993; 296: 843-849Crossref PubMed Scopus (168) Google Scholar, 24Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (392) Google Scholar), which itself is activated by a novel protein kinase (p38 MAP kinase) cascade that can be triggered by stress (25Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 26Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar). However, the physiological relevance of the phosphorylation of hsp27 has not been elucidated. It has been reported that phosphorylation of hsp27 results in the dissociation of an aggregated form of hsp27 to oligomers (27Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar), and phosphorylated hsp27 no longer exhibits the ability to inhibit the polymerization of actin (28Benndorf R. Hayeß K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). Reports on the phosphorylation of αB-crystallin are mostly related to the phosphorylation of this protein in the lens. Phosphorylated forms of αB-crystallin (αB1-crystallin) have been found in bovine lens (29Chiesa R. Gawinowicz-Kolks M.A. Kleiman N.J. Spector A. Exp. Eye Res. 1988; 46: 199-208Crossref PubMed Scopus (54) Google Scholar, 30Voorter C.E.M. de Haard-Hoekman W.A. Roersma E.C. Meyer H.E. Bloemendal H. de Jong W.W. FEBS Lett. 1989; 259: 50-52Crossref PubMed Scopus (67) Google Scholar), and αB-crystallin in extracts of bovine lens can be phosphorylated in a cAMP-dependent manner (31Spector A. Chiesa R. Sredy J. Garner W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4712-4716Crossref PubMed Scopus (155) Google Scholar, 32Chiesa R. Gawinowicz-Kolks M.A. Kleiman N.J. Spector A. Biochem. Biophys. Res. Commun. 1987; 144: 1340-1347Crossref PubMed Scopus (67) Google Scholar) or by the kinase activity of the protein itself (33Kantorow M. Piatigorsky J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3112-3116Crossref PubMed Scopus (127) Google Scholar) in vitro. Recently, Wang et al. (34Wang K. Ma W. Spector A. Exp. Eye Res. 1995; 61: 115-124Crossref PubMed Scopus (40) Google Scholar) reported that H2O2 stimulated the phosphorylation of αB-crystallin in rat lens, and Bennardini et al. (35Bennardini F. Juliano C. Benetti D. Mian M. Chiesi M. Mattana A. Franconi F. Biochem. Biophys. Res. Commun. 1995; 208: 742-747Crossref PubMed Scopus (11) Google Scholar) reported that phorbol 12-myristate 13-acetate induced the phosphorylation of αB-crystallin in cultures of bovine articular chondrocytes. In this report, we show that phosphorylation of αB-crystallin in U373 MG human glioma cells and in rat tissues can be induced by various stimuli, and we also identify the sites of phosphorylation in αB-crystallin, as determined by use of antibodies that recognized each of the phosphorylation sites specifically. Phorbol 12-myristate 13-acetate (PMA), 4-α-phorbol 12-myristate 13-acetate (4α-PMA), okadaic acid, and staurosporine were obtained from Wako Pure Chemicals (Osaka, Japan). Dithiothreitol was obtained from Nacalai Tesque Inc. (Kyoto, Japan). SB202190 and PD98059 was obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). Recombinant human IL-1α and tumor necrosis factor α (with specific activities of 2 × 107 and 2.55 × 106 units/mg of protein, respectively) were obtained from Dainippon Pharmaceuticals (Osaka). Forskolin, cholera toxin, anisomycin, and protein A were obtained from Sigma. Dibutyryl cAMP was obtained from Boehringer (Mannheim, Germany). Ampholine pH 6–8, Ampholine pH 3.5–10, and CNBr-activated Sepharose 4B were obtained from Pharmacia Biotech Inc. (Tokyo, Japan). U373 MG cells (obtained from American Type Culture Collection, Rockville, MD) were grown in Eagle's minimal essential medium (Nissui Pharmaceutical Co., Tokyo) supplemented with 10% fetal calf serum (Life Technologies, Inc., Tokyo) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cells were seeded on 90-mm dishes, and the medium was changed every 2 or 3 days. When the cells had reached confluence, various chemicals, dissolved in dimethyl sulfoxide or in H2O, were added to the culture medium. After a 90-min incubation in a CO2 incubator, unless otherwise specified, cells were washed three times with phosphate-buffered saline (8 g of NaCl, 0.2 g of KCl, 1.15 g of Na2HPO4, and 0.2 g of KH2PO4 in 1000 ml of H2O) and frozen at −80 °C for a few days prior to analysis. The frozen cells on each dish were collected and suspended in 0.5 ml of 50 mm Tris-HCl, pH 7.5, containing 0.1 m NaF, 0.1 μm okadaic acid, and 5 mm EDTA. Each suspension was sonicated and centrifuged at 125,000 ×g for 20 min at 4 °C. The supernatant was used for analysis by isoelectric focusing (IEF) of αB-crystallin. In some experiments, pellets were washed once with the Tris buffer by sonication as described above and then solubilized with 8 murea for analysis by IEF. Isoelectric focusing was performed as described by O'Farrell (36O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) using the Protean II system from Bio-Rad (Tokyo). In brief, an aliquot of cell extract that contained 10–40 μg of protein was mixed with 4 volumes of sample buffer (2% Ampholine mixture (4 parts Ampholine pH 6–8 and 1 part Ampholine pH 3.5–10), 9.5 m urea, 2% Nonidet P-40, and 5% 2-mercaptoethanol). Fifty-microliter aliquots of the mixture were then applied to a gel composed of 9.2 m urea, 2% Ampholine mixture, 4% acrylamide, and 2% Nonidet P-40, and electrophoresis was performed at 400 V for 16 h at 16.5 °C. For Western blot analysis, proteins on a gel were transferred electrophoretically to a polyvinylidene difluoride membrane (type GV; Nihon Millipore Ltd., Yonezawa, Japan), and the membrane was immunostained, as described previously (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar), with antibodies against the carboxyl-terminal decapeptide (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar) or against specifically phosphorylated forms of αB-crystallin that had been prepared as described below. Cells were labeled with [32P]orthophosphate as described previously (27Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar), and then they were incubated for 90 min in a CO2 incubator with 1 μm PMA and 0.2 μm okadaic acid. The cells were washed three times with phosphate-buffered saline and then frozen at −80 °C for 2 h. The frozen cells were scraped off the plates, disrupted by passage through a 24-gauge needle, and then centrifuged as described above. The soluble fraction of the cells (100 μg of protein) was incubated at 4 °C for 5 h with 5 μg of affinity-purified rabbit IgG against αB-crystallin (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar), and then 50 μl of a suspension of protein A-Sepharose were added to the mixture with further incubation at 4 °C overnight. The Sepharose beads were washed three times with 0.5 ml of 50 mm Tris-HCl, pH 8.0, containing 1 m NaCl and 0.1% Nonidet P-40 and then once with 50 mm Tris-HCl, pH 7.0. The beads were subjected to rapid agitation on a mixer for 5 min with 50 μl of the sample buffer for IEF, and then the mixture was centrifuged at 10,000 ×g for 5 min. The supernatants were subjected to IEF or analysis of phosphoamino acids. The immunopurified extract was subjected to SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically from the gel to a polyvinylidene difluoride membrane. The membrane was subjected to autoradiography. Each band containing phosphorylated αB-crystallin was cut out and subjected to hydrolysis in 6 n HCl at 110 °C for 1 h. The hydrolysate was then evaporated in dryness under nitrogen gas. The residue was dissolved in 5 μl of formate/acetate buffer (formic acid, acetic acid, and water (26:78:900, v/v)), pH 1.9, supplemented with 67 μg each of phosphoserine, phosphothreonine, and phosphotyrosine (Sigma) and applied to a silica gel plate (Merck, Darmstadt, Germany). Electrophoresis was performed in the above buffer at pH 1.9 and 30 mA for 40 min. Electrophoresis in the second dimension was performed in a different buffer (acetic acid, pyridine, and water (10:1:200, v/v)) at pH 3.5 and 35 mA for 30 min. The phosphoamino acids on the dried plate were visualized by staining with ninhydrin, and then the plate was subjected to autoradiography at −80 °C. The sites of phosphorylation in αB-crystallin were determined by high-performance liquid chromatography-electrospray ionization/triple stage quadrupole mass spectrometry (HPLC-ESI/TSQMS) (37Nakayama H. Uchida K. Shinkai F. Shinoda T. Okuyama T. Seta K. Isobe T. J. Chromatogr. 1996; 730: 279-287Crossref PubMed Scopus (19) Google Scholar). The system consisted of a Model 140A liquid chromatograph (Perkin-Elmer Applied Biosystems, Foster City, CA) that was connected to a Model TSQ-700 mass spectrometer equipped with an ESI interface (Finnigan MAT, San Jose, CA). αB-Crystallin (2 μg), purified from U373 MG cells that had been exposed to 1 μm PMA and 0.2 μm okadaic acid for 90 min as described below, was digested with tosylphenylalanyl chloromethyl ketone-treated trypsin at 37 °C for 5 h in 25 mm Tris-HCl, pH 8.0, at a enzyme/substrate ratio of 1:50 (w/w). The digest was fractionated by reversed-phase chromatography on a capillary column (0.5 mm, inner diameter, × 150 mm) packed with Aquapore C18 (particle size, 5 mm; Perkin-Elmer Applied Biosystems) with a gradient of 0–80% acetonitrile in 0.1% formic acid over 20 min at a flow rate of 8.5 ml/min. The eluate was introduced directly to the ESI interface of the mass spectrometer. After mapping of tryptic peptides, the phosphopeptides were analyzed by tandem mass spectrometry (MS/MS) for identification of the sites of phosphorylation. The spectrometer was operated under the following conditions: electrospray voltage, 4.5 kV; temperature, 200 °C; and electron multiplier voltage, 1000 V for peptide mapping and 1600 V for sequencing. For analysis of phosphorylation sites by mass spectrometry, αB-crystallin was purified from extracts of U373 MG cells that had been exposed to 1 μm PMA plus 0.2 μmokadaic acid for 90 min. The cells were scraped from ∼100 dishes (90 mm in diameter), sonicated, and centrifuged as described above. The supernatant (∼80 ml) was incubated at room temperature with 0.5 mg of affinity-purified antibodies against the carboxyl-terminal decapeptide of αB-crystallin. After 2 h of a gentle shaking, 1 ml of a 50% (v/v) suspension of protein A-coupled Sepharose beads (Sigma) was added to the mixture with additional incubation at 4 °C overnight. The Sepharose beads were washed with 50 mm Tris-HCl, pH 8.0, containing 1 m NaCl and 0.1% Nonidet P-40 and then with 50 mm Tris-HCl, pH 7.0. αB-Crystallin trapped on the beads was eluted with 0.1 m sodium acetate buffer, pH 4.5, containing 7 m urea, 1 mm glycol ether diaminetetraacetate, and 1 mm dithiothreitol. The eluate was then applied to a column (0.8 cm, inner diameter, × 7.5 cm) of TSK-SP-5PW (Tosoh, Tokyo), and αB-crystallin was eluted with a linear gradient of NaCl (0–0.4 m) in the above buffer as described previously (38Kato K. Goto S. Inaguma Y. Hasegawa K. Morishita R. Asano T. J. Biol. Chem. 1994; 269: 15302-15309Abstract Full Text PDF PubMed Google Scholar). Three peptides corresponding to internal sequences of human αB-crystallin, containing phosphorylated Ser-19 (residues 17–27, FHSPSRLFDQF+C; p19S), Ser-45 (residues 44–54, LSPFYLRPPSF+C; p45S), and Ser-59 (residues 57–67, APSWFDTGLSE+C; p59S), respectively, were synthesized. Each peptide was conjugated with hemocyanin (Sigma) usingN-(4-carboxycyclohexylmethyl) maleimide (Zieben Chemicals Co., Ltd., Tokyo) (39Yoshitake S. Yamada Y. Ishikawa E. Masseyeff R. Eur. J. Biochem. 1979; 101: 395-399Crossref PubMed Scopus (169) Google Scholar). Antisera were raised in rabbits by injection of each conjugate (0.5 mg of peptide/animal), and antibodies were purified with bovine lens αB1-crystallin-coupled Sepharose in the case of antibodies against p19S and p45S and with p59S peptide-coupled Sepharose in the case of antibodies against p59S by the previously described procedure (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar). Male Wistar rats (body weight, ∼250 g) were treated in accordance with the guidelines of the Animal Care and Use Committee of the Institute for Developmental Research. The rats were subjected to heat stress as described previously (40Inaguma Y. Hasegawa K. Goto S. Ito H. Kato K. J. Biochem. (Tokyo). 1995; 117: 1238-1243Crossref PubMed Scopus (58) Google Scholar). After 20 min of heat stress at 42 °C, rats were killed under ether anesthesia, and tissues were dissected out on dry ice and kept frozen at −80 °C for a few days. The frozen tissues were homogenized in 10 volumes of 50 mm Tris-HCl, pH 7.5, containing 0.1 m NaF, 5 mm EDTA, and 0.1 μm okadaic acid, and each suspension was sonicated and centrifuged at 125,000 × g for 20 min at 4 °C. Supernatants were subjected to IEF followed by Western blot analysis. The insoluble fraction was also analyzed as described above. Levels of cyclic AMP in cells were determined using a cAMP enzyme immunoassay system (EIA, Amersham International, Buckinghamshire, United Kingdom). Concentrations of protein in soluble fractions of cells and tissues and in fractions solubilized with urea from pellets after centrifugation were determined with a protein assay kit (Bio-Rad), with bovine serum albumin as the standard (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar). Bovine αB1-crystallin and αB2-crystallin and rat α-crystallin, used as standards for IEF, were purified from lenses as described previously (8Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (189) Google Scholar). Human αB-crystallin was purified from skeletal muscle (14Kato K. Shinohara H. Goto S. Inaguma Y. Morishita R. Asano T. J. Biol. Chem. 1992; 267: 7718-7725Abstract Full Text PDF PubMed Google Scholar). U373 MG human glioma cells were exposed to NaAsO2 at various concentrations for 60 min, and then cell extracts were subjected to IEF with subsequent Western blot analysis. As shown in Fig. 1 A, arsenite acted in a dose-dependent manner to induce the generation of a form of αB-crystallin that had a lower isoelectric point than the control. Since the newly induced form of αB-crystallin had an isoelectric point similar to that of αB1-crystallin, a phosphorylated form of αB-crystallin, it appeared that the observed modification of αB-crystallin under the above conditions was due to phosphorylation. The arsenite-induced phosphorylated form of αB-crystallin was detected after 20 min of exposure, and its level increased in a time-dependent manner for 120 min (Fig. 1 B). When cells that had been exposed to arsenite for 120 min were incubated for 2 h in the standard medium, the phosphorylated form of αB-crystallin disappeared (Fig. 1 B). These results suggest that considerable dephosphorylating activity was present in the cells. We next examined whether or not okadaic acid, an inhibitor of phosphoserine/phosphothreonine protein phosphatases, could induce the accumulation of the phosphorylated form of αB-crystallin. As shown in Fig. 2 A, exposure of cells to 0.2 μm okadaic acid stimulated the accumulation of αB-crystallin in a time-dependent manner. These results suggest that the phosphorylation and dephosphorylation of αB-crystallin in cells are under dynamic equilibrium. The band that migrated between the unphosphorylated (p0) and phosphorylated (p1) forms, which was also detected in the preparation of α-crystallin purified from lens, seemed to be αB-crystallin that had received other modification. Generation of the phosphorylated form of αB-crystallin was also observed when cells were exposed to IL-1α and PMA, a potent activator of protein kinase C, although the extent of the phosphorylation induced by IL-1α was smaller than that induced by arsenite or PMA (Fig. 2 B). 4α-PMA, an inactive analog of PMA, and tumor necrosis factor α each barely induced any phosphorylation of αB-crystallin (Fig.2 B). It has been reported that αB-crystallin in extracts of bovine lens can be phosphorylated in a cAMP-dependent manner in vitro (31Spector A. Chiesa R. Sredy J. Garner W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4712-4716Crossref PubMed Scopus (155) Google Scholar, 32Chiesa R. Gawinowicz-Kolks M.A. Kleiman N.J. Spector A. Biochem. Biophys. Res. Commun. 1987; 144: 1340-1347Crossref PubMed Scopus (67) Google Scholar). However, activators of protein kinase A, such as dibutyryl cAMP, forskolin, and cholera toxin, barely induced any phosphorylation of αB-crystallin in U373 MG cells (Fig.2 C). Other compounds, such as H2O2 (oxidative stress), sorbitol and NaCl (hypertonic stress), and anisomycin (an activator of p38 MAP kinase) (41Meier R. Rouse J. Cuenda A. Nebreda A.R. Cohen P. Eur. J. Biochem. 1996; 236: 796-805Crossref PubMed Scopus (112) Google Scholar), also stimulated the phosphorylation of αB-crystallin in the soluble fraction of U373 MG cells (Fig.3 A). The phosphorylated form of αB-crystallin was also detected in the insoluble fraction of cells that had been treated with H2O2 (Fig.3 A) or with arsenite or PMA (data not shown). However, except in the case of heat stress, most of the phosphorylated form of αB-crystallin was detected in the soluble fraction of the cells. The phosphorylated form of αB-crystallin was not detected in the soluble fraction of cells that had been heated at 43–47 °C for 20 min (Fig. 3 B). αB-Crystallin in cells is converted from a soluble form to an insoluble form soon after heat treatment (13Inaguma Y. Shinohara H. Goto S. Kato K. Biochem. Biophys. Res. Commun. 1992; 182: 844-850Crossref PubMed Scopus (50) Google Scholar). Therefore, we solubilized the insoluble fractions of cell extracts using 8 m urea and then subjected the resultant mixtures to IEF and subsequent Western blot analysis. As shown in Fig.3 B, we detected the heat stress-induced and temperature-dependent phosphorylation of αB-crystallin in the insoluble fraction of U373 MG cells. The arsenite-induced phosphorylation of αB-crystallin was suppressed in the presence of 2 mm dithiothreitol, whereas the PMA-induced phosphorylation was selectively inhibited by 100 nm staurosporine, an inhibitor of protein kinase C (Fig.4 A). By contrast, the phosphorylation of αB-crystallin that was stimulated by anisomycin (Fig. 4 B) or by other chemicals (data not shown) was unaffected by the presence of dithiothreitol or staurosporine. However, an inhibitor of p38 MAP kinase, SB202190 (42Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kemar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar), inhibited the phosphorylation that was induced by arsenite, anisomycin, H2O2, sorbitol, NaCl, and heat, but not the phosphorylation that was induced by PMA and okadaic acid (Fig.5). On the other hand, an inhibitor of p44 MAP kinase kinase, PD98059 (43Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar), selectively suppressed the phosphorylation of αB-crystallin inducible by PMA (Fig. 5). The phosphorylation that was induced by anisomycin (Fig. 5) or by each of the other stimuli described above (data not shown) was barely inhibited by the presence of PD98059. These results indicate that the phosphorylation of αB-crystallin by the various agents involves different signal transduction cascades.Figure 5Effects of SB202190, an inhibitor of p38 MAP kinase, and PD98059, an inhibitor of p44 MAP kinase kinase, on the phosphorylation of αB-crystallin. Cells were exposed, in the presence (+) or absence (−) of 10 μm SB202190 (SB) or 50 μm PD98059 (PD), to 200 μm arsenite, 1 μm PMA, 10 μg/ml anisomycin (AMC), 0.2 μm okadaic acid (OA), 4 mm H2O2, 0.4m sorbitol, 0.15 m NaCl, or heat (45 °C for 30 min) in the standard medium, and soluble extracts of cells (the urea-solubilized extract of pellets in the case of heat-treated cells) were analyzed by IEF and subsequent Western blotting.Control, untreated cells. Polyvinylidene difluoride membranes were stained as described in the legend to Fig. 1.p0, unphosphorylated αB-crystallin; p1 andp2, phosphorylated αB-crystallin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When U373 MG cells were exposed to PMA plus okadaic acid, generation of three acidic forms of αB-crystallin was induced in a time-dependent manner (Fig.6, A and B,p1, p2, and p3). To confirm that the modifica" @default.
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