Phosphorylation of αB-crystallin in Mitotic Cells and Identification of Enzymatic Activities Responsible for Phosphorylation*

The immunofluorescence localization of αB-crystallin in U373 MG human glioma cells with an antibody specific for αB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of αB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of αB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for αB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated αB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in αB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.

␣-Crystallin, a major structural protein of the vertebrate eye lens, is a polymeric protein with a molecular mass of about 800 kDa. The ␣-crystallin of the bovine lens is composed predominantly of two types of polypeptide, the A (␣A1 and ␣A2) and B (␣B1 and ␣B2) subunits (1). ␣A1 and ␣B1 are the phosphorylated forms of the primary gene products, ␣A2 and ␣B2 (2,3). The molecular mass of each subunit is about 20 kDa, and the similarity between the primary structures of the two subunits is greater than 50% (4). The ␣-crystallins also share sequence similarity with the small heat shock proteins (hsps) 1 of numerous species (5). Because of the striking similarities among the primary structures of the carboxyl-terminal half of each molecule (the ␣-crystallin domain), ␣A-crystallin, ␣B-crystallin, hsp27, and p20 (6) are considered to be members of the ␣-crystallin small hsp family in vertebrates (7,8).
A common feature of small hsps is their formation of large oligomeric complexes such as ␣A-crystallin and ␣B-crystallin in the lens. In the skeletal muscle, ␣B-crystallin, hsp27, and p20 seem to form a large heteropolymer, because the three proteins were copurified from the extract and coimmunoprecipitated with antibodies against each of the three proteins (6,9). The ␣-crystallin domain of each small hsp was suggested to be important for this complex formation and the chaperone activity (8). Both ␣A-crystallin (10) and ␣B-crystallin (11) are also present in nonlenticular tissues, and the expression of ␣Bcrystallin, but not ␣A-crystallin and p20, is induced in cells under various stressful conditions, as is hsp27 (12,13).
The major posttranscriptional modifications of the ␣-crystallin small hsp family are due to the phosphorylation of serine residues. The phosphorylation of hsp27 by mitogen-activated protein (MAP) kinase-activated protein (MAPKAP) kinase-2 is enhanced when cells are exposed to heat (14,15) or chemicals (16). p20 in vascular smooth muscles is phosphorylated in association with cyclic nucleotide-dependent vasorelaxation (17). In addition, p20 was phosphorylated in vitro by both cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase (17). Bovine ␣A-crystallin is phosphorylated at Ser-122 (2), and ␣B-crystallin is phosphorylated at Ser-19 or Ser-21, Ser-43 or Ser-45, and Ser-59 (18 -20). It was believed for some years that ␣B-crystallin and ␣A-crystallin in the lens were phosphorylated in a cyclic AMP-dependent manner (2,21) or by the kinase activity of the proteins themselves (22,23). However, we demonstrated recently that ␣B-crystallin in U373 MG human glioma cells is phosphorylated at three serine residues (Ser-19, -45, and -59) in response to various types of stress (24). We raised antibodies in rabbits that recognized ␣B-crystallin that had been phosphorylated at each of the three serine residues individually (24). In the present study, by using these antibodies, we found that the phosphorylation at each site in ␣B-crystallin is regulated differently during mitosis, and we obtained evidence suggesting that p44/42 MAP kinase and MAPKAP kinase-2 are responsible for phosphorylation of Ser-45 and Ser-59, respectively, in ␣B-crystallin in vivo.

EXPERIMENTAL PROCEDURES
Reagents-Affinity-purified fluorescein isothiocyanate-labeled goat antibodies against rabbit IgG were purchased from Bio Source International Inc. (Camarillo, CA). Biotin-labeled antibodies against rabbit IgG that had been absorbed with mouse serum and horseradish peroxidase-labeled streptavidin were obtained from Vector Laboratories (Burlingame, CA). Pefablock SC was obtained from Boehringer Mannheim (Tokyo, Japan). Phorbol 12-myristate 13-acetate (PMA), okadaic acid, calyculin A, taxol, thymidine, and lysyl endopeptidase were obtained from Wako Pure Chemicals (Osaka, Japan). Ampholine pH 6 -8, ampholine pH 3.5-10, and CNBr-activated Sepharose 4B were obtained from Amersham Pharmacia Biotech. Five mg of protein A (obtained from Nacalai Tesque, Kyoto) were coupled with 1 g of CNBr-activated Sepharose 4B. Recombinant human p44 MAP (Erk1, activated) kinase was obtained from StressGen Biotechnologies Co. (Victoria, Canada). MAPKAP kinase-2 purified from rabbit skeletal muscle was obtained from Upstate Biotechnology (Lake Placid, NY). Nocodazole, myelin basic protein purified from bovine brain, histone (type III-S) from calf thymus, and the catalytic subunit of protein kinase A (PKA) purified from bovine heart were obtained from Sigma-Aldrich (Tokyo). The antibody against p44/42 MAP kinase was obtained from New England Biolabs (Beverly, MA). The antibody against MAPKAP kinase-2 (C-18) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SB202190 and PD98059 were obtained from Calbiochem.
Culture and Treatment of Cells-U373 MG human glioma 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 (ICN Biomedicals, Inc., Aurora, OH) at 37°C in a CO 2 incubator. The cells were seeded on 90-mm dishes, and the medium was changed every 2 or 3 days. HeLa S3, Swiss 3T3, 3Y-1 B1-6 (25), and NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co.) supplemented with 10% fetal calf serum. Mitotic cells were collected by double-block procedures that involved incubations with thymidine and nocodazole (26) and also by mechanical release from standard cell cultures. U373 MG cells at confluent cultures were also exposed to various chemicals for 1 h in the CO 2 incubator. Cells were then washed twice with phosphate-buffered saline (PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2 HPO 4 and 0.2 g of KH 2 PO 4 in 1000 ml of H 2 O) and frozen at Ϫ80°C for a few days prior to use. For the Western blot analysis and the assay of phosphorylation activities with the crude extracts of cells, cells were sonicated in 80 mM HEPES-NaOH buffer, pH 7.0, that contained 0.1 M NaF, 0.3 mg/ml Pefablock SC, 10 g/ml trypsin inhibitor, 0.2 M okadaic acid, and 0.2 M calyculin A. For the purification of protein kinases responsible for the phosphorylation of ␣B-crystallin, the frozen cells on each dish were collected and suspended in 20 mM Tris-HCl buffer, pH 7.5, that contained 1 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM benzamidine, and 0.03% Brij 35 (buffer A) supplemented with 0.3 mg/ml of Pefablock SC, 10 g/ml of trypsin inhibitor, 0.2 M okadaic acid, and 0.2 M calyculin A. Each suspension was sonicated and centrifuged at 125,000 ϫ g for 20 min at 4°C to obtain the soluble extracts of cells.
Immunostaining-Indirect immunofluorescence staining was performed as described previously (27). All immunostained specimens were examined under a confocal laser scanning microscope (LSM 410; Karl Zeiss, Oberkochen, Germany). Sections of mouse embryonic tissues were prepared, and the immunohistochemical staining of these sections was performed as described previously (28).
Electrophoresis and Western Blot Analysis-SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli (29) in 10 or 12.5% polyacrylamide gels. Tricine/SDS-PAGE was performed as described by Schä gger and von Jagow (30) in 16.6% polyacrylamide gels that contained 13.3% glycerol. Isoelectric focusing (IEF) was performed as described previously (24) by the method of O'Farrell (31), using the Protean II system from Bio-Rad (Tokyo). For the Western blot analysis, proteins on a gel were transferred electrophoretically to a nitrocellulose membrane, and the membrane was incubated successively for 2 h with primary antibodies and then for 1 h with peroxidase-labeled second antibodies. Antigens on the membrane were visualized on x-ray film using a chemiluminescence reagent (Renaissance; NEN Life Science Products).
Antibodies-Affinity-purified rabbit antibodies against the carboxylterminal decapeptide of ␣B-crystallin were prepared as described previously (11). Rabbit antiserum against the amino-terminal dodecapeptide of ␣B-crystallin was raised in rabbits by injecting the peptide (MDIAIHHPWIRR-Cys) conjugated with hemocyanin (Sigma) using N-(4-carboxycyclohexylmethyl)maleimide (32), and the antibodies were purified by use of a column of bovine ␣B2-crystallin-coupled Sepharose as described previously (11). Antibodies that recognize each of the three phosphorylated serine residues in human ␣B-crystallin (Ser-19, -45, and -59) were prepared as described previously (24). Because the internal sequence of the peptide (APSWFDTGLSE, residues 57-67, p59S) of human ␣B-crystallin included a residue different from that in bovine and rat ␣B-crystallin (the Phe-61 in the human sequence is Ile-61 in bovine or rat), antibodies raised with p59S of the human sequence did not react with bovine and rat ␣B1-crystallin. Therefore, the peptide of the bovine (and rat) sequence (APSWIDTGLSE-Cys; bovine p59S) was synthesized, and the antiserum was raised in rabbits as described previously (24). The antibodies that recognized the phosphorylated Ser-59S of bovine and rat ␣B-crystallin were purified with bovine p59S peptide-coupled Sepharose. The binding of anti-p19S, anti-p45S, and anti-bovine p59S to bovine ␣B1-crystallin was eliminated by preincubation of the various antibodies with bovine ␣B1-crystallin or with the FIG. 1. Specificity of antibodies that recognized each of the phosphorylated serine residues in ␣B-crystallin. Fifty-ng aliquots of ␣B1-crystallin, purified from bovine lens, were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Each membrane was then incubated for 2 h at room temperature with antibodies against p19S, p45S, or bovine p59S (0.5 g/ml) that had been preincubated overnight at 4°C with or without (None) ␣B1-crystallin purified from bovine lens (␣B1), or with the p19S, p45S, or bovine p59S peptide (10 g) of ␣B-crystallin. The membrane was then incubated for 1 h with peroxidase-labeled antibodies raised in goats against rabbit IgG, and the peroxidase activity on the membrane was visualized on x-ray film by use of a Western blotting chemiluminescence reagent (Renaissance; NEN Life Science Products). respective phosphopeptides (Fig. 1), an indication that each antibody recognized the phosphorylation of a specific serine residue in ␣B-crystallin.
Preparation of ␣B-crystallin and the N-72K Peptide-Bovine and rat ␣-crystallins were purified from lenses as described previously (11). Bovine ␣B1and ␣B2-crystallins were isolated from the ␣-crystallin fraction by chromatography on a chromatofocusing column (33). The amino-terminal 72 (N-72K) peptide of ␣B2-crystallin was prepared by incubating ␣B2-crystallin with 0.5% (w/w) lysyl endopeptidase in 10 mM HEPES-NaOH buffer, pH 7.5, at 37°C for 24 -48 h. When an aliquot of the digest was subjected to reversed-phase high performance liquid chromatography on a column of TSK-ODS-80TS (0.46 ϫ 15 cm; Tosoh, Tokyo), the N-72K peptide was eluted as a major peak. The identity of N-72K was confirmed by the analysis of its amino acid sequence with a peptide sequencer (model PPSQ-10; Shimadzu, Kyoto, Japan) after the removal of acetyl methionine at the amino terminus with CNBr, as described previously (6). The lysyl endopeptidase-digested rat hsp27 was prepared with the same procedures.
Assays of Enzymatic Activities Responsible for Phosphorylation of ␣B-crystallin-The extract of cells, containing 100 g of protein, was incubated at 30°C for 20 min with 5 l of lysylendopeptidase-treated ␣B2-crystallin (3 mg/ml), 1 mM ATP, 10 mM MgCl 2 , 100 nM okadaic acid, 100 nM calyculin A, 0.05 M NaF, 0.3 mg/ml Pefablock SC, and 10 g/ml trypsin inhibitor in a reaction mixture that was brought to a final volume of 100 l with 50 mM HEPES-NaOH, pH 7.0. The reaction was stopped by the addition of 100 l of the sample buffer for Tricine/SDS-PAGE. Aliquots of 2-15 l of the mixture were subjected to Tricine/ SDS-PAGE and a Western blot analysis with antibodies that recognized each of the phosphorylated serine residues or with antibodies against the amino-terminal peptide of ␣B-crystallin, as described above. For the assays of phosphorylation with [ 32 P]ATP, we used 0.5 mM [␥-32 P]ATP (2 C/tube; ICN Biomedicals Ins., Costa Mesa, CA) and 5 mM MgCl 2 in reaction mixtures with a final volume of 50 l as described above. After 20 min at 30°C, each reaction was stopped by the addition of 50 l of the sample buffer, and 30-l aliquots were subjected to Tricine/SDS-PAGE. After drying, the gels were exposed to x-ray film.
Quantitation of Protein-The concentrations of soluble protein in extracts were estimated with a protein assay kit (Bio-Rad) with bovine serum albumin as the standard.

RESULTS
Immunocytochemical Localization of Phosphorylated Forms of ␣B-crystallin-Among the three antibodies that recognized the phosphorylated serine residues in ␣B-crystallin, anti-p45S had relatively high affinity and specificity when crude extracts of U373 MG cells that had been exposed to various types of stress were subjected to IEF and a subsequent Western blot analysis (24). Therefore, we attempted the localization of ␣Bcrystallin phosphorylated at Ser-45 by indirect immunofluorescence staining with antibodies raised against p45S. As shown in Fig. 2A, various lines of cultured cells, including U373 MG human glioma cells, HeLa S3 cells, human skin fibroblasts, Swiss 3T3 and NIH 3T3 mouse fibroblasts, and 3Y-1 B1-6 rat fibroblasts, were immunostained with antibodies against p45S. Prominent staining of phosphorylated Ser-45 in ␣B-crystallin was apparent in mitotic cells of all cell lines examined. In most cases, staining was evident as fine granular deposits, with staining throughout the cytoplasm of mitotic cells and clearly separate from the chromosomes. In some interphase cells, there was faint granular staining in the cytoplasm. The specific staining of mitotic cells was confirmed by the treatment of cells with an antimitotic agent, taxol. This treatment increased the number of cells that were immunostained with antibodies against p45S (Fig. 2B). The incubation of antibodies with p45S abolished the staining, confirming the specificity of staining (Fig. 2B, d and h). An enhanced phosphorylation of Ser-45 in ␣B-crystallin seemed to occur during mitosis. Phosphorylation began to appear during prophase and continued until telophase or cytokinesis (Fig. 2C). The phosphorylation of Ser-45 in mitotic cells was observed in vivo as well as in cultured cells, when the mouse embryonic tissues were subjected to immunohistochemical staining with the same antibody (data not shown). Samples of some human malignant tumors were also positive for immunostaining with antibodies against p45S, and only mitotic cells were immunostained in such cases (data not shown).
Western Blot Analysis of Extracts of Mitotic Cells-To test the results obtained in our immunocytochemical studies, we collected U373 MG cells at the mitotic phase after successive treatments of cells with thymidine and nocodazole. We subjected extracts of untreated control cells and of mitotic cells to IEF and a subsequent Western blot analysis with antibodies against the carboxyl-terminal peptide of ␣B-crystallin (Fig. 3A, ␣B-C) or with antibodies against p45S (Fig. 3A, p45S). The extracts were also subjected to SDS-PAGE and a subsequent Western blot analysis with antibodies against p19S, p45S, p59S, and the carboxyl-terminal peptide of ␣B-crystallin (Fig.  3B). After IEF, the two bands of the acidic form of ␣B-crystallin, corresponding to those of ␣B1-crystallin purified from bovine lens, were clearly detected in the case of mitotic cells after immunostaining with antibodies both against the carboxylterminal peptide of ␣B-crystallin and against p45S. The results of the SDS-PAGE and Western blot analysis revealed that the increases in phosphorylated serine residues in ␣B-crystallin in mitotic cells corresponded to phosphorylation at Ser-19 and -45, while the levels of phosphorylated Ser-59 tended to be reduced in mitotic cells. These results suggest that the phosphorylation of these three serine residues in ␣B-crystallin is catalyzed by multiple protein kinases.
The increases in the levels of ␣B-crystallin that had been phosphorylated at Ser-45 in mitotic 3Y-1 B1-6 cells and in mitotic BRL 3A rat liver cells were confirmed by IEF and Western blot analysis, as shown in Fig. 3C. Phosphorylated ␣B-crystallin in U373 MG cells was detected after 6 h of exposure to nocodazole, and two bands of phosphorylated ␣B-crystallin were detected in cells that had been exposed to nocodazole for 12-18 h. However, when cells that had been exposed to nocodazole for 12 h were returned to the standard medium, the phosphorylated ␣B-crystallin disappeared completely within 6 h (data not shown).
These results confirmed our immunohistochemical finding that the level of ␣B-crystallin phosphorylated at Ser-45 was elevated in mitotic cells. In addition, the SDS-PAGE and Western blot analyses revealed that the level of ␣B-crystallin phosphorylated at Ser-19 was also elevated, while that of ␣B-crystallin phosphorylated at Ser-59 was depressed in mitotic cells.
Activities of Protein Kinases That Phosphorylate ␣B-crystallin in Mitotic Cells-When bovine ␣B2-crystallin was incubated with extracts of U373 MG cells in the presence of [␥-32 P]ATP and Mg 2ϩ , the 32 P label was barely incorporated into ␣B2-crystallin, even when we used extracts of cells that had been exposed to PMA and okadaic acid (Fig. 4A). However, when ␣B2-crystallin that had been treated with lysyl endopeptidase was used as a substrate, the N-72K peptide was phosphorylated significantly even with extracts of control cells, and the phosphorylation was markedly enhanced with extracts of cells that had been exposed to PMA and okadaic acid (Fig. 4A). These results suggest that the aggregation properties of the purified ␣B-crystallin might be different from the native form in cells and that protein kinases might be unable to gain access to sites of potential phosphorylation. The treatment of ␣B2crystallin with lysyl endopeptidase resulted in the destruction of the ␣-crystallin domain and the formation of N-72K peptide, which contained each of the three phosphorylation sites in ␣B-crystallin accessible to protein kinases.
To clarify whether the changes in the levels of ␣B-crystallin phosphorylated at the various serine residues were the result of changes in the activities of protein kinases that are specific for the respective residues, we assayed the phosphorylation activities in extracts of control and mitotic U373 MG cells using the N-72K peptide of ␣B2-crystallin as the substrate. After incubation at 30°C for 20 min in the presence of ATP and Mg 2ϩ , aliquots of each reaction mixture were subjected to Tricine/SDS-PAGE and subsequent autoradiography (Fig. 4C) or a Western blot analysis (Fig. 4B) with the antibodies that recognized each of the three phosphorylated serine residues. As shown in Fig. 4B, the levels of N-72K phosphorylated at Ser-19 and Ser-45 that had been produced during incubations with extracts of mitotic cells were much higher than the levels after incubations with extracts of control cells, while the levels of N-72K phosphorylated at Ser-59 after incubations with ex-tracts of mitotic cells were lower than those after incubations with extracts of control cells. Thus, the extent of incorporation of 32 P into N-72K was similar for the extracts of control and mitotic cells, as shown in the autoradiogram in Fig. 4C.
Our results indicate that the increased levels of ␣B-crystallin phosphorylated at Ser-19 and -45 and the decreased levels of ␣B-crystallin phosphorylated at Ser-59 resulted from changes in the activities of different protein kinases.
Partial Purification of Protein Kinases Responsible for Phosphorylation of N-72K Peptide from U373 MG Cells-To identify the enzymatic activities responsible for the phosphorylation of ␣B-crystallin, we partially purified the phosphorylation activities from U373 MG cells. Frozen cells from about 30 dishes After fixation with 4% paraformaldehyde, pH 7, the cells were immunostained with antibodies against p45S (a, b, c, e, f, and g) or with the same antibodies that had been preincubated with p45S (50 g/ml; d and h). Bar, 100 m. C, immunostaining of cells at various phases of the cell cycle with antibodies against p45S. 3Y1 B1-6 rat fibroblasts, cultured on a coverslip, were immunostained with antibodies against p45S. Staining at various phases of the cell cycle was monitored by confocal laser scanning microscopy, as follows: at interphase (a); at prometaphase (b); at metaphase (c); at anaphase (d); at telophase (e); during cytokinesis (f); in postmitotic daughter cells (g); and at postmitotic interphase (h). Bar, 20 m. that had been exposed to 4 mM H 2 O 2 and 2.5 nM calyculin A at 37°C for 1 h were collected at 0°C in 10 ml of buffer A supplemented with 0.3 mg/ml of Pefablock SC, 10 g/ml of trypsin inhibitor, 0.2 M okadaic acid, and 0.2 M calyculin A. After sonication, the homogenate was centrifuged at 4°C for 20 min at 125,000 ϫ g. The supernatant was passed through a filter (W-25-2; Tosoh) and then incubated at 30°C for 10 min with 0.5 mM ATP and 5 mM MgCl 2 . This procedure increased the protein kinase activity for Ser-45 by about 2-fold. The extract containing about 10 mg of protein was then applied to a column of DEAE-5PW (0.8 ϫ 7.5 cm; Tosoh) that had been equilibrated with buffer A on a fast protein liquid chromatography system (Amersham Pharmacia Biotech). After washing the column with 10 ml of buffer A, protein kinase activities for the three serine residues in N-72K peptide were eluted with 30 ml of buffer A that contained a linear gradient of NaCl (0 -0.5 M) in the same buffer at a flow rate of 1 ml/min. Protein kinase activities that phosphorylated Ser-19 (Ser-19 kinase) and Ser-59 (Ser-59 kinase) were eluted in similar fractions, and Ser-45 kinase was eluted at fractions with the buffer containing a slightly higher concentration of salt (not shown). The fractions (5 ml) containing the kinase activities were pooled and concentrated and applied to a column of Superdex 200 were subjected to IEF (A) or SDS-PAGE (B) and a subsequent Western blot analysis with antibodies against the carboxyl-terminal peptide of ␣B-crystallin (␣B-C) or against the p19S, p45S, or p59S peptide. C, extracts of control and of mitotic 3Y-1 B1-6 cells containing 30 g of protein or 100 ng of ␣-crystallin purified from rat lens (␣-C) were subjected to IEF and a subsequent Western blot analysis with antibodies against the carboxyl-terminal peptide of ␣B-crystallin. D, extracts of control and of mitotic BRL 3A cells containing 0.5 mg of protein were incubated at 4°C overnight with 5 g of the antibodies against the carboxyl-terminal peptide of ␣B-crystallin together with 100 l of protein A-coupled Sepharose beads. After washing, the Sepharose beads were heated in the sample buffer for IEF. After centrifugation to remove the beads, the supernatant was subjected to IEF, together with 100 ng of ␣-crystallin purified from rat lens (␣-C), and subsequent immunostaining with antibodies against the carboxyl-terminal peptide of ␣B-crystallin, as described above. p0, unphosphorylated ␣B-crystallin; p1 and p2, phosphorylated ␣B-crystallin.

FIG. 4. Examination of the protein kinases in extracts of U373 MG cells that phosphorylate the three serine residues in ␣Bcrystallin.
A, extracts of control cells (Cn) or extracts of cells that had been exposed for 60 min to 1 M PMA plus 0.2 M okadaic acid (PMA & OA), each containing 100 g of protein, were incubated at 30°C for 20 min with 5 l of bovine ␣B2-crystallin (1 mg/ml) or 5 l of lysyl endopeptidase-digested ␣B2-crystallin (1 mg/ml) in the presence of 0.5 mM [␥-32 P]ATP (2 Ci/tube), 5 mM MgCl 2 , 100 nM okadaic acid, 100 nM calyculin A, 0.05 M NaF, 200 g/ml Pefablock SC, and 10 g/ml trypsin inhibitor in a final volume of 50 l with 50 mM HEPES-NaOH, pH 7.0. The reaction was stopped by the addition of 50 l of the sample buffer for Tricine/SDS-PAGE. Thirty-l aliquots of the mixture were subjected to Tricine/SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue (protein staining) and exposed to x-ray film (autoradiogram). The locations of ␣B-crystallin (␣B-cry) and the N-72K peptide (N-72K) are indicated by arrowheads. B, extracts of control cells (Cont) and of mitotic cells (Mc) that contained 100 g of protein were incubated at 30°C for 20 min with 5 l of lysyl endopeptidase-digested ␣B2-crystallin (3 mg/ml) in the presence of 1 mM ATP, 10 mM MgCl 2 , 100 nM okadaic acid, 100 nM calyculin A, 0.05 M NaF, 200 g/ml Pefablock SC, and 10 g/ml trypsin inhibitor in a final volume of 100 l with 50 mM HEPES-NaOH, pH 7.0. The reaction was stopped by the addition of 100 l of the sample buffer for Tricine/SDS-PAGE. Two-l aliquots (for detection of N-72K) or 10-l aliquots (for the detection of phosphorylated serine residues) of the mixture were subjected to Tricine/SDS-PAGE and a Western blot analysis with antibodies that recognized each of the phosphorylated serine residues (p19S, p45S, and p59S) or antibodies against the amino-terminal dodecapeptide of ␣B-crystallin (␣B-N). Bl, incubated without a cell extract. C, extracts of control and mitotic cells that contained 100 g of protein were incubated at 30°C for 20 min with 5 l of the peptidase-treated ␣B2-crystallin in the presence of 0.5 mM [␥-32 P]ATP and 5 mM MgCl 2 in reaction mixture with a final volume of 50 l, and after terminating the reaction by the addition of the sample buffer for electrophoresis, 30-l aliquots of the mixture were subjected to Tricine/SDS-PAGE, and the gel was exposed to x-ray film ( 32 P) as described above.
after the chromatography of Superdex 200. These results suggest that each of the three serine residues is phosphorylated by the three different protein kinases.
Characterization of the Protein Kinases That Phosphorylate Ser-45 and Ser-59 -We previously observed (24) that the phosphorylation of Ser-45 was preferentially stimulated in U373 MG cells that had been exposed to PMA and that the phosphorylation of Ser-59 was stimulated in cells exposed to arsenite. The phosphorylation induced by PMA was selectively suppressed by PD98059 (34), an inhibitor of MAP kinase kinase, while that induced by arsenite or other stimuli, except for PMA and okadaic acid, was strongly suppressed in the presence of SB202190 (35), an inhibitor of p38 MAP kinase (24). To confirm whether the changes in the levels of ␣B-crystallin phosphorylated at each site were due to increased activities responsible for the phosphorylation of the respective serine residue, we assayed the protein kinase activities in extracts of cells that had been exposed to 200 M arsenite or 1 M PMA in the presence or absence of SB202190 and PD98059. As shown in Fig. 6, the arsenite-induced stimulation of the phosphorylation activity for Ser-59 was strongly inhibited in the presence of SB202190, and the PMA-induced activation of Ser-45 and Ser-19 kinases (but not that of Ser-59 kinase) was suppressed by PD98059. These results suggested that Ser-45 kinase (and Ser-19 kinase) is a member of the cascade of p44/42 MAP kinase and that Ser-59 kinase is involved downstream of the cascade of p38 MAP kinase. The apparent molecular masses of the two protein kinases estimated from the elution profiles from the column of Superdex 200 suggested that Ser-45 kinase and Ser-59 kinase might be p44/42 MAP kinase and MAPKAP kinase-2, respectively. In fact, the Western blot analyses of each fraction from the column of Superdex 200 with antibodies against p44/42 MAP kinase and MAPKAP kinase-2 revealed that p44/42 MAP kinase and MAPKAP kinase-2 were eluted from the column with profiles almost identical to those of Ser-45 kinase and Ser-59 kinase, respectively (Fig. 5B). The results of the SDS-PAGE and Western blot analysis of partially purified preparations of Ser-45 kinase and Ser-59 kinase with antibodies against p44/42 MAP kinase and against MAPKAP kinase-2 are shown in Fig. 7. Both p44/42 MAP kinase and MAPKAP kinase-2 were clearly detected in the Ser-45 kinase preparation and Ser-59 kinase preparation, respectively, after the protein staining of gels or Western blot analysis of the nitrocellulose membrane (Fig. 7). Therefore, we characterized the preparations of Ser-45 kinase and Ser-59 kinase that had been obtained after chromatography on a column of Superdex 200 by comparing them with recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscles. As shown in Fig. 8A, Ser-45 kinase and p44 MAP kinase selectively phosphorylated Ser-45, whereas Ser-59 kinase and MAPKAP kinase-2 phosphorylated Ser-59 in the N-72K peptide. The bands weakly stained at Ser-19 and Ser-45 by the Ser-59 kinase preparation were probably due to the contaminated activities of Ser-19 kinase and Ser-45 kinase in the Ser-59 kinase preparation. The activity for Ser-19 kinase was barely detected in the Ser-59 kinase preparation, and p44 MAP kinase and MAPKAP kinase-2 did not phosphorylate Ser-19. Next, we tested the activities of phos- After 1 h, the cells were collected, sonicated, and centrifuged to obtain the soluble extracts as described under "Experimental Procedures." Each extract containing 100 g of protein was incubated with the lysyl endopeptidase-digested ␣B2-crystallin at 30°C for 20 min, and the phosphorylated serine residues in N-72K peptide were analyzed using antibodies against p19S, p45S, and p59S, as described in the legend to Fig. 5. ␣B-N, the N-72K peptide detected by antibodies against the amino-terminal peptide of ␣B-crystallin; Lane 1, untreated control cells. phorylation of each protein kinase for hsp27 and myelin basic protein. As shown in Fig. 8B, Ser-45 kinase and p44 MAP kinase phosphorylated myelin basic protein and the N-72K peptide but barely phosphorylated ␣B2-crystallin, hsp27, and its lysyl endopeptidase-digest. In contrast, Ser-59 kinase and MAPKAP kinase-2 phosphorylated, to a similar extent, hsp27 and the peptides derived from ␣B2-crystallin and hsp27 but barely phosphorylated ␣B2-crystallin.
These results suggest that the phosphorylation of ␣B-crystallin is catalyzed by three different protein kinases; that Ser-45 and -59 in the molecule were phosphorylated by p44/42 MAP kinase and MAPKAP kinase-2, respectively; and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division. However, it remains to be clarified which protein kinase that is enhanced in mitotic cells phosphorylates Ser-19 in ␣B-crystallin.

DISCUSSION
The phosphorylation of ␣B-crystallin in U373 MG human glioma cells is induced by exposure of the cells to various types of stress, such as arsenite, PMA, okadaic acid, anisomycin, H 2 O 2 , sorbitol, NaCl, and heat (24). All three serine residues (Ser-19, -45 and -59) are phosphorylated to some extent in response to each stress. Immunofluorescence localization of ␣B-crystallin that had been phosphorylated at Ser-45 in U373 MG cells upon exposure to PMA plus okadaic acid revealed no characteristic differences from that in control cells (data not shown), but it did reveal that all mitotic cells were intensely immunostained with antibodies against p45S, independently of the treatment of cells with chemicals. The intense staining of mitotic cells with antibodies against p45S was observed in all cell lines examined, as well as in sections of mouse embryos. The increases in the levels of ␣B-crystallin phosphorylated at Ser-45 in mitotic cells were confirmed biochemically by IEF or the SDS-PAGE of cell extracts and subsequent Western blot analysis with antibodies against p45S or against the carboxylterminal peptide of ␣B-crystallin. The SDS-PAGE and Western blot analysis of extracts of mitotic cells revealed that ␣B-crystallin phosphorylated at Ser-19 was also elevated. By contrast, the levels of ␣B-crystallin phosphorylated at Ser-59 were considerably lower in mitotic cells than in control cells. These results suggest that the phosphorylation of the three serine residues in ␣B-crystallin in mitotic cells is regulated independently by different protein kinases.
As we reported previously that the synthesis and accumulation of ␣B-crystallin in cells is induced by agents that promote the disassembly of microtubules (27), the total amount of ␣Bcrystallin, estimated with a specific immunoassay (11), in-  creased significantly in mitotic U373 MG cells and 3Y-1 cells that had been collected after exposure to nocodazole (data not shown). However, it remains to be clarified whether the induced synthesis of ␣B-crystallin in cells exposed to nocodazole is related to the phosphorylation states of ␣B-crystallin in the same cells.
When bovine ␣B2-crystallin was incubated with extracts of U373 MG cells in the presence of [␥-32 P]ATP and Mg 2ϩ , the radiolabel was barely incorporated into ␣B2-crystallin, even when we used extracts of cells that had been extensively exposed to stress (Fig. 3A). These results suggest that the aggregation properties of ␣B2-crystallin, separated from ␣A-crystallins and ␣B1-crystallin in the presence of 6 M urea (33), might be different from the native form that is found in cells and that protein kinases might be unable to gain access to sites of potential phosphorylation. Therefore, we treated ␣B2-crystallin with lysyl endopeptidase to destroy the ␣-crystallin domain and to prepare the N-72K peptide, which contained each of the three phosphorylation sites in ␣B-crystallin. Using N-72K peptide as the substrate, we were able to detect the activities of protein kinases that phosphorylated ␣B-crystallin in a cell-free system. The activities of protein kinase that phosphorylated Ser-19 and -45 were enhanced, and the activity of the kinase that phosphorylated Ser-59 was depressed in mitotic cells, as expected from the results of the Western blot analyses of phosphorylated forms of ␣B-crystallin. We obtained results similar to those presented above with naturally mitotic cells that had been prepared without chemical treatment (data not shown), an indication that the observed phenomena were not due to direct effects of thymidine and nocodazole on cells.
By using the N-72K peptide, we found that the phosphorylation of each of the three serine residues in ␣B-crystallin is catalyzed by three different protein kinases. The enzymatic activity that phosphorylates Ser-45 was eluted from a column of Superdex 200 at fractions corresponding to about 40 kDa with a elution profile similar to that of p44/42 MAP kinase, and the activities that phosphorylate Ser-19 and -59 were located in fractions corresponding to about 60 kDa with an elution profile similar to that of MAPKAP kinase-2. However, the phosphorylation activity for Ser-19 was low and unstable. Human recombinant p44 MAP kinase and the Ser-45 kinase fraction selectively phosphorylated Ser-45 in N-72 K peptide, and MAP-KAP kinase-2 purified from rabbit muscle and the Ser-59 kinase fraction selectively phosphorylated Ser-59 in N-72K peptide. The Ser-45 kinase fraction was able to phosphorylate myelin basic protein, as was recombinant p44 MAP kinase, and the Ser-59 kinase fraction was able to phosphorylate hsp27, as was MAPKAP kinase-2. The antibodies against p44/42 MAP kinase and against MAPKAP kinase-2 immunoprecipitated only some portions (20 -30%) of enzymatic activities, under our conditions, for phosphorylation of Ser-45 and Ser-59, respectively, in extracts of cells as well as in solutions of recombinant p44 MAP kinase and MAPKAP kinase-2 purified from rabbit skeletal muscle, respectively (data not shown). However, the present results, together with the inhibitory effects of PD98059 and SB202190 on the activation of Ser-45 kinase and Ser-59 kinase, respectively, in cells, strongly suggest that the protein kinase responsible for the phosphorylation of Ser-45 is p44/42 MAP kinase and that responsible for the phosphorylation of Ser-59 is MAPKAP kinase-2.
The phosphorylation of ␣B-crystallin in U373 MG cells was induced in response to various stimuli with a cyclic AMPindependent process (24), However, it was reported that ␣Band ␣A-crystallins in the bovine lens were phosphorylated in a cyclic AMP-dependent manner in vitro (2,21) or by their own autokinase activity (22,23). Therefore, we tested whether PKA would phosphorylate ␣B-crystallin and hsp27 or their fragments prepared by digestion with lysyl endopeptidase under the present conditions. As shown in Fig. 9A, the catalytic subunit of PKA purified from bovine heart phosphorylated the protease-treated ␣B2-crystallin and hsp27, but it barely phosphorylated ␣B2-crystallin and hsp27, although hsp27 was phosphorylated by the Ser-59 kinase fraction and MAPKAP kinase-2 (Fig. 8B). The serine residue in N-72K peptide that had been phosphorylated by PKA was limited to Ser-59 (Fig.  9B), as were the Ser-59 kinase fraction and MAPKAP kinase-2.
These results indicate that PKA can catalyze the phosphorylation of Ser-59 in N-72K peptide as can MAPKAP kinase-2. Since the reaction medium for the assay of the phosphorylation with N-72K peptide did not include cyclic AMP, PKA in extracts of cells seemed to be inactive and was unable to phosphorylate N-72K peptide under the present conditions. However, these results suggest that the previous in vitro finding of the phosphorylation of ␣B-crystallin by PKA in the presence of cyclic AMP were the phosphorylation at Ser-59 in ␣B-crystallin.
Although the biological significance of the enhanced phosphorylations of Ser-19 and -45 and the suppressed phosphorylation of Ser-59 in ␣B-crystallin during cell division remains to be clarified, the present results suggest that ␣B-crystallin might be involved in nuclear functions. were incubated with 10 milliunits of PKA purified from bovine heart for 20 min, and the reaction mixture was subjected to Tricine/SDS-PAGE and subsequent autoradiography, as described in the legend to Fig. 4. Molecular mass markers (in kDa) are indicated with arrowheads. B, the lysyl endopeptidase-digested ␣B2-crystallin was incubated for 10 or 20 min at 30°C with 10 milliunits of PKA or 5 g of the Ser-59 kinase fraction (Ser-59 K), and the reaction mixture was subjected to Tricine/SDS-PAGE and a Western blot analysis with antibodies against p45S and p59S and against the amino-terminal peptide of ␣B-crystallin (␣B-N), as described in the legend to Fig. 5.