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• W2036023681 abstract "αB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934–29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346–28354). In the present study, we determined effects of phosphorylation of αB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-αB). From results of sucrose density gradient centrifugation, we found that wild type αB-crystallin (wt-αB) and 3D-αB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-αB was significantly weaker than that of wt-αB. When wt-αB and 3D-αB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, αB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-αB or 3D-αB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-αB was less than that with wt-αB. From these observations, it is suggested that phosphorylation of αB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27. αB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934–29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346–28354). In the present study, we determined effects of phosphorylation of αB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-αB). From results of sucrose density gradient centrifugation, we found that wild type αB-crystallin (wt-αB) and 3D-αB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-αB was significantly weaker than that of wt-αB. When wt-αB and 3D-αB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, αB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-αB or 3D-αB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-αB was less than that with wt-αB. From these observations, it is suggested that phosphorylation of αB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27. mitogen-activated protein small heat shock or stress proteins polyacrylamide gel electrophoresis phorbol 12-myristate 13-acetate 8-anilinonaphthalene-1-sulfonate mitogen-activated protein kinase ultraviolet circular dichroism lactate dehydrogenase Chinese hamster ovary cells Phosphorylation is one of the most important post-translational modifications and it is known that there are many protein kinase cascades in various organisms. Many of them play pivotal roles in the maintenance of cellular functions. Among them, the p44/42 MAP1 kinase and p38 MAP kinase cascades are well known for their roles, for example, in cell proliferation and stress responses (1Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (954) Google Scholar, 2Nebreda A.R. Porras A. Trends Biochem. Sci. 2000; 25: 257-260Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). These kinases phosphorylate many proteins in response to extracellular stimuli. Small heat shock or stress proteins (sHSPs) have been recognized as substrates for p44/42 MAP kinase and p38 MAP kinase cascades and it is suspected that phosphorylation of sHSPs may cause significant change in their functions. Phosphorylation of HSP27 in response to extracellular stimuli such as heat, arsenite, phorbol ester, and growth factors is well characterized (3Arrigo A.-P. Welch W.J. J. Biol. Chem. 1987; 262: 15359-15369Abstract Full Text PDF PubMed Google Scholar, 4Arrigo A.-P. Mol. Cell. Biol. 1990; 10: 1276-1280Crossref PubMed Scopus (104) Google Scholar, 5Landry J. Chretinen P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (124) Google Scholar, 6Landry 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 it has been reported to be catalyzed by MAP kinase-activated protein kinase-2 and MAP kinase-activated protein kinase-3, which are activated by p38 MAP kinase (7Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (765) Google Scholar, 8Rouse 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 (1483) Google Scholar, 9Clifton A.D. Young P.R. Cohen P. FEBS Lett. 1996; 392: 209-214Crossref PubMed Scopus (121) Google Scholar), and the δ isoform of protein kinase C (protein kinase C-δ) (10Maizels E.T. Peters C.A. Kline M. Cutler Jr., R.E. Shanmugam M. Hunzicker-Dunn M. Biochem. J. 1998; 332: 703-712Crossref PubMed Scopus (101) Google Scholar). We previously reported that phosphorylation of HSP27 in cells leads to dissociation of large oligomers and decrease of resistance to heat (11Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar). Recently, other groups documented that substitution of serine phosphorylation sites of HSP27 with aspartate or glutamate similarly results in the reduction of oligomer size, with cells expressing these mutants showing reduced tolerance to exogenous stress (12Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 13Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). HSP27 has been recognized as an actin-binding protein, with activity that modifies the polymerization state of actin (14Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar, 15Liang P. MacRae T.H. J. Cell Sci. 1997; 110: 1431-1440Crossref PubMed Google Scholar). Phosphorylation of HSP27 causes loss of its actin polymerization inhibiting activity (16Benndorf R. Hayess K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of αB-crystallin, another representative sHSP, has not been reported in response to extracellular stimuli although it was found that significant amounts of phosphorylated forms are present in mammalian lens and the phosphorylation is catalyzed by cAMP-dependent protein kinase (17Spector A. Chiesa R. Sredy J. Garner W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4712-4716Crossref PubMed Scopus (154) Google Scholar, 18Voorter C.E. de Haard-Hoekman W.A. Roersma E.S. Meyer H.E. Bloemendal H. de Jong W.W. FEBS Lett. 1989; 259: 50-52Crossref PubMed Scopus (66) Google Scholar). We first reported that αB-crystallin is also phosphorylated in response to extracellular stimuli, which also induce phosphorylation of HSP27, with three serine residues, Ser-19, Ser-45, and Ser-59, as phosphorylation sites (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We also described phosphorylation of Ser-45 and Ser-59 to be catalyzed by p44/42 MAP kinase and MAP kinase-activated protein kinase-2, respectively (20Kato K. Ito H. Kamei K. Inaguma Y. Iwamoto I. Saga S. J. Biol. Chem. 1998; 273: 28346-28354Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). It is known that αB-crystallin can prevent cytochalasin-induced depolymerization of actin filaments in a phosphorylation-dependent manner, although the phosphorylation does not affect its chaperone-like activity (21Wang K. Spector A. Eur. J. Biochem. 1996; 242: 56-66Crossref PubMed Scopus (140) Google Scholar). αB-crystallin also modulates intermediate filament assembly independent of its phosphorylation state (22Nicholl I.D. Quinlan R.A. EMBO J. 1994; 13: 945-953Crossref PubMed Scopus (393) Google Scholar). Phosphorylation of αB-crystallin at Ser-45 occurs in mammalian mitotic cells (20Kato K. Ito H. Kamei K. Inaguma Y. Iwamoto I. Saga S. J. Biol. Chem. 1998; 273: 28346-28354Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and phosphorylated forms, especially at Ser-45, increase in rat lens during postnatal development (23Ito H. Iida K. Kamei K. Iwamoto I. Inaguma Y. Kato K. FEBS Lett. 1999; 446: 269-272Crossref PubMed Scopus (19) Google Scholar). In the present study, we examined the effects of phosphorylation of αB-crystallin on its oligomerization state using site-directed mutagenesis to substitute all three phosphorylation sites with aspartate to mimic the phosphorylation state. We found this to cause changes of the oligomerization state of αB-crystallin and decrease in its chaperone-like activity. For expression in mammalian cells, an Eco RI fragment from human αB-crystallin cDNA (generously provided by Dr. A. Iwaki, Kyushu University, Japan) was inserted into the expression vector pCMV5 (generously provided by Dr. H. Itoh, National Children's Medical Research Center, Japan) (24Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). For site-directed mutagenesis, we used the polymerase chain reaction with oligonucleotide mutation primers and the template αB-crystallin expression vector.Bgl II and Xho I fragments from polymerase chain reaction fragments were inserted into Bgl II andSal I sites of pCMV5. We constructed two plasmids to express wild type αB-crystallin and αB-crystallin in which three phosphorylation sites, Ser-19, Ser-45, and Ser-59, were substituted with aspartate. We designated these plasmids as wt-αB-pCMV5 and 3D-αB-pCMV5. The mutated sites were confirmed by DNA sequencing. For expression of wt-αB and 3D-αB in Escherichia coli, their cDNAs were inserted into Nde I and Xho I sites of the vector pET30a(+) (Novagen, Madison, WI). Recombinant human αB-crystallin and its mutant were expressed in E. coli BL21(DE3). The production of recombinant protein was induced as follows; the cells were grown with shaking at 37 °C until the culture had A 600 of 0.6 and 0.5 mmisopropyl-1-thio-β-galactopyranoside was added. After 3 h, cells were harvested by centrifugation and resuspended in an extraction buffer, 25 mm Tris-HCl buffer, pH 7.5, containing 2.5 mm EDTA, 0.3 mg/ml Pefablock SC (Roche Molecular Biochemicals), and 100 μg/ml trypsin inhibitor. Each suspension was sonicated at 0 °C and centrifuged at 12,000 × g at 4 °C for 30 min. The supernatant was applied to a column of Sepharose beads which coupled with affinity purified antibodies against C-terminal peptides of αB-crystallin (25Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (188) Google Scholar) and then the column was washed with 0.1 m phosphate buffer, pH 7.0. αB-crystallin trapped on the column was eluted with ActiSep Elution Medium (Sterogen, Arcadia, CA). The eluate was desalted and mixed with equal volume of 0.1 m sodium acetate buffer, pH 4.5, containing 7m urea, 1 mm EDTA and then applied to a column (0.8 cm, inner diameter × 7.5 cm) of TSK-SP-5PW (Tosoh, Tokyo, Japan). αB-crystallin was eluted with a linear gradient of NaCl (0–0.4 m) in the above buffer as described previously (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Circular dichroism (CD) spectra were recorded using a Jasco J-725 spectropolarimeter. Samples of wt-αB and 3D-αB purified from bacteria were dialyzed overnight against 10 mm sodium phosphate buffer, pH 7.4, and used at concentrations of 450 and 45 μg/ml for near and far UV CD spectrometry, respectively. The path length of the cell was 10 mm for both spectrometry. Near and far UV CD spectra were accumulated 5 and 3 times, respectively. Intrinsic tryptophan fluorescence spectra were recorded using a JASCO FP-770 fluorescence spectrophotometer with the excitation wavelength of 295 nm. Samples containing 50 μg/ml wt-αB and 3D-αB in 10 mm sodium phosphate buffer, pH 7.4, were used in 10-mm path length cuvettes. The excitation and emission band passes were set at 3 nm. Spectra were monitored from 300 to 400 nm at room temperature. For the 8-anilinonaphthalene-1-sulfonate (ANS) binding studies, 10 μl of 10 mm ANS (Wako Pure Chemical, Osaka, Japan) was added to 3 ml of 50 μg/ml protein solution in 10 mm sodium phosphate buffer, pH 7.4, containing 0.1 m NaCl and incubated at room temperature for 2 h. Samples were transferred to 10-mm path length cuvettes and fluorescence spectra were monitored. The excitation and emission band passes were set at 5 nm. The excitation wavelength was set at 350 nm and spectra were monitored from 400 to 600 nm at room temperature. For determination of oligomerization size of recombinant αB-crystallin, we performed gel filtration chromatography with a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated in 50 mm sodium phosphate buffer, pH 7.4, containing 0.1 m NaCl. A calibration curve was generated by using a high molecular weight protein standard (Amersham Pharmacia Biotech). Chaperone-like activity was measured as the ability to protect against heat-induced aggregation of lactate dehydrogenase (LDH, 100 μg/ml, purified from rabbit muscle, Roche Molecular Biochemicals), monitored by measuring the turbidity at 360 nm at 50 °C in 25 mm sodium phosphate buffer, pH 7.0, containing 100 mm NaCl and 2 mm EDTA for 60 min in the presence or absence of 5 or 10 μg/ml recombinant αB-crystallin. We also performed luciferase refolding assays in vitro in the presence of recombinant αB-crystallin with the method described by Lu and Cyr (26Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Firefly luciferase (14 mg/ml) (Promega, Madison, WI) was diluted 42-fold into denaturation buffer (25 mm HEPES, pH 7.4, containing 50 mm KCl, 5 mm MgCl2, 6 mguanidium HCl, and 5 mm dithiothreitol). The denaturation reaction was allowed to proceed for 40 min at room temperature. Two-microliter aliquots were removed from the denaturation mixture and mixed with 100 μl of refolding buffer (25 mm HEPES, pH 7.4, containing 50 mm KCl, 5 mmMgCl2, and 5 mm ATP) in the presence or absence of 1 μm recombinant αB-crystallin and the mixtures were incubated at 30 °C. Two-microliter aliquots were removed at various times and mixed with 60 μl of luciferase assay reagents (PicaGene Luminescence Kit; Toyo Ink Co., Japan). Relative light units were counted using a TD-20/20 luminometer (Tuner Designs, CA). COS-m6 cells and CHO cells were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., Tokyo, Japan), supplemented with 10% fetal calf serum (Equitech-Bio, Inc., Ingram, TX). Cells were seeded in 35-mm dishes and transfected with 1 μg of plasmids by using LipofectAMINE Plus reagent (Life Technologies, Inc., Gaithersburg, MD). Cells were harvested at 48 h after transfection and suspended in 50 mm Tris-HCl buffer, pH 7.5, containing 0.1 mNaF, 5 mm EDTA, 0.3 mg/ml Pefablock SC, 0.2 μm okadaic acid, and 0.2 μm calyculin A. Each suspension was sonicated at 0 °C and centrifuged at 125,000 × g for 20 min at 4 °C to obtain soluble extracts of cells. For phosphorylation of αB-crystallin by extracellular stimuli, we used U373 MG human glioma cells grown in Eagle's minimal essential medium (Nissui Pharmaceutical Co.). Cells were seeded in 90-mm dishes and when they reached confluence, they were treated for 90 min at 37 °C with 0.1 μm phorbol 12-myristate 13-acetate (PMA) and 0.1 μm okadaic acid (Wako Pure Chemical), and then cell extracts were obtained as described above. Ten μg of purified recombinant αB-crystallin in 0.2 ml of 50 mm Tris-HCl, pH 7.5, containing 0.1 m NaF and 5 mm EDTA or extracts of cells (0.2-ml aliquots) were layered over 3.6-ml linear gradient of sucrose (10–40%) in 50 mm Tris-HCl, pH 7.5, containing 0.1 m NaF and 5 mm EDTA and centrifuged at 130,000 × g for 16 h at 4 °C in a swinging bucket rotor (RPS56T; Hitachi, Tokyo, Japan). Each sample was then fractionated into 15 test tubes from the bottom. SDS-PAGE was performed as described by Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (204264) Google Scholar) in 12.5% gels and proteins were visualized by staining with Coomassie Brilliant Blue. Isoelectric focusing was carried out as described by O'Farrell (28O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) using the Protean II system from Bio-Rad (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Western blot analysis was performed as described previously using affinity purified antibodies raised in rabbits against the C-terminal decapeptide of αB-crystallin (25Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (188) Google Scholar) or against phosphopeptides which correspond to the three phosphorylation sites of αB-crystallin (p19S, p45S, and p59S) (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Peroxidase-labeled antibodies raised in goat against rabbit IgG was employed as the second antibodies. Peroxidase activity on nitrocellulose sheets was visualized on x-ray films using a Western blot chemiluminescence reagent (Renaissance, PerkinElmer Life Sciences, Boston, MA). In some experiments, peroxidase activity was also detected with the aid of a luminoimage analyzer LAS-1000 (Fuji Film, Tokyo, Japan) and the relative densities of protein bands were quantified with relevant software. We also quantified relative densities of bands on x-ray films using an NIH image program (National Institute of Health, Bethesda, MD). CHO cells were transiently co-transfected with two plasmids, 0.5 μg of wt-αB-pCMV5 or 3D-αB-pCMV5 and 0.5 μg of the luciferase expression vector, PGV-C (Toyo Ink Co.). Almost equal amounts of wt-αB and 3D-αB were expressed in CHO cells at 24 to 48 h after transfection as detected by Western blot analysis of cell extracts with antibodies against the C-terminal peptide of αB-crystallin under our experimental conditions (data not shown). When cells reached confluence, they were subjected to heat treatment at 45 °C for 70 min and then cultured at 37 °C for 16 h. Cells were harvested and the luciferase activity in cell extracts was measured using a PicaGene Luminescence Kit (Toyo Inc.) according to the manufacturer's protocol. Bovine αB1-crystallin (phosphorylated form of αB-crystallin) and αB2-crystallin (unphosphorylated form of αB-crystallin) were purified from bovine lens as described previously (25Kato K. Shinohara H. Kurobe N. Inaguma Y. Shimizu K. Ohshima K. Biochim. Biophys. Acta. 1991; 1074: 201-208Crossref PubMed Scopus (188) Google Scholar). Concentrations of protein were estimated with a protein assay kit (Bio-Rad) with bovine serum albumin as the standard. To estimate the effects of phosphorylation of αB-crystallin on its oligomerization state, we expressed recombinant proteins in E. coli. Wild type αB-crystallin (wt-αB) and 3D-αB-crystallin (3D-αB), in which all three phosphorylation sites of αB-crystallin, Ser-19, Ser-45, and Ser-59, were substituted with aspartate to mimic the phosphorylated state, were purified using a column of Sepharose beads coupled with antibodies against the C-terminal peptide of αB-crystallin and subsequent anion exchange chromatography. Purified wt-αB was observed at the position of the predicted molecular size in SDS-PAGE gels and 3D-αB was detected as a band migrating to a position pointing to a slightly greater kilodalton value (Fig.1 A). Using these recombinant proteins, far and near UV CD spectra were measured to estimate the effect of phosphorylation of αB-crystallin on its secondary and tertiary structure. Consistent with a previous report, wt-αB showed a far UV CD spectrum with a high percentage of β-sheet/β-turn structure in its molecule at 25 °C (Fig. 1 B). The 3D-αB also showed a far UV CD spectrum indicating a high percentage of β-structures at 25 °C and the pattern of the spectrum was similar to that of wt-αB (Fig. 1 B). To estimate the effect of phosphorylation of αB-crystallin on its conformational stabilities against temperature increasing, we monitored far UV CD spectrum at a higher temperature. At 45 °C, far UV CD spectra for wt-αB and 3D-αB were significantly changed and the extent of the change was more visible in the case of 3D-αB (Fig. 1 C). The near UV CD spectra of proteins are thought to reflect the contribution of aromatic amino acid to protein tertiary structure. The pattern of near UV CD spectra of wt-αB and 3D-αB were visibly different at 25 and 45 °C (Fig. 1, D and E). Intrinsic tryptophan fluorescence spectrum is thought to provide information of the environment of tryptophan residues in proteins. We also monitored fluorescence spectra of wt-αB and 3D-αB and it was revealed that the spectrum of 3D-αB was significantly shifted to the longer wavelength and the intensity of fluorescence increased (Fig.2 A), indicating a difference of the environment of tryptophan residues in wt-αB and 3D-αB and a decrease in the extent of hydrophobicity of 3D-αB. To estimate the extent of hydrophobicity of wt-αB and 3D-αB, we carried out ANS binding assays. ANS fluorescence is weak in aqueous solutions and its fluorescence quantum yield increases in a hydrophobic environment. This property of ANS has been exploited to monitor the hydrophobic surface of proteins (29Cardamone M. Puri N.K. Biochem. J. 1992; 282: 589-593Crossref PubMed Scopus (401) Google Scholar). As shown in Fig. 2 B, the fluorescence intensity of ANS bound to 3D-αB was lower than that bound to wt-αB, indicating a smaller extent of hydrophobicity of 3D-αB than that of wt-αB and the results were consistent with the results of intrinsic tryptophan fluorescence spectra (Fig. 2 A). We previously reported one representative small heat shock protein, HSP27, to be phosphorylated by various stresses, with a resultant decrease in oligomerization size and increase in the dissociated form (11Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar). To estimate oligomerization states of recombinant wt-αB and 3D-αB, we fractionated each preparation of αB-crystallin by sucrose density gradient centrifugation. Aliquots of each fraction were subjected to SDS-PAGE followed by immunostaining with antibodies against the C-terminal peptide of αB-crystallin. As shown in Fig.3 A, oligomerization states of wt-αB and 3D-αB were quite different. Quantitation of the intensity of bands of Western blot analysis revealed that wt-αB mainly sedimented at a position which corresponded to an apparent molecular mass of about 500 kDa, while 3D-αB mainly sedimented at a position which corresponded to apparent molecular masses of about 300 kDa, with a broader range (Fig. 3 B). We also estimated oligomerization sizes of wt-αB and 3D-αB using gel filtration chromatography and it revealed that the oligomerization sizes of wt-αB and 3D-αB were about 550 and 390 kDa, respectively (data not shown). Stress proteins are known to have a chaperone activity and there are several reports describing such activity for αB-crystallin (30Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1701) Google Scholar, 31Rao P.V. Horwitz J. Zigler Jr., J.S. Biochem. Biophys. Res. Commun. 1993; 190: 786-793Crossref PubMed Scopus (109) Google Scholar). To determine the effects of phosphorylation on this parameter, we assessed thermal aggregation of LDH in the presence or absence of recombinant wt-αB or 3D-αB. As shown in Fig.4, A and B, the degree of prevention from thermal aggregation of LDH by 3D-αB was slightly weaker than that of wt-αB and this tendency was more obvious when the ratio of LDH to αB-crystallin was higher. Moreover, we estimated the ability of wt-αB and 3D-αB to refold denatured firefly luciferase. In the absence of αB-crystallin, the activity of luciferase remained to be lowered (Fig. 4 C). In the presence of wt-αB or 3D-αB, refolding of luciferase was clearly observed, but the refolding activity of 3D-αB was weaker than that of wt-αB (Fig. 4 C). To estimate the effects of phosphorylation on the oligomerization state of αB-crystallin in mammalian cells, we performed a transient expression of wt-αB and 3D-αB in COS-m6 cells. Consistent with the results obtained with the purified recombinant proteins, the band for 3D-αB was observed at a slightly different position from wt-αB after the SDS-PAGE and subsequent immunostaining with antibodies against C-terminal peptide of αB-crystallin (Fig. 5 A). Extracts of cells transiently expressing wt-αB or 3D-αB were subjected to sucrose density gradient centrifugation with subsequent fractionation and Western blot analysis. The sedimentation profiles of wt-αB and 3D-αB were quite different (Fig. 5 B). Quantitation of the intensity of each band revealed that wt-αB mainly sedimented at a position corresponding to an apparent molecular mass of about 500 kDa while 3D-αB sedimented at ∼100 kDa. These results were also similar to the case with purified recombinant proteins, as shown in Fig. 3, although both forms of αB-crystallin in transiently-transfected COS-m6 cells fractionated within narrow ranges. We previously reported that various stresses induce phosphorylation of αB-crystallin in U373 MG cells, a combination of PMA and okadaic acid treatment markedly increasing phosphorylated forms (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We therefore estimated whether phosphorylation of endogenous αB-crystallin causes change in its oligomerization state. Endogenous αB-crystallin in control U373 MG cells formed a large oligomer with an apparent molecular mass of about 500 kDa. After treatment of cells with PMA and okadaic acid, the sedimentation profile of αB-crystallin was clearly shifted to a smaller molecular size (Fig. 6,A and B). Using antibodies specifically recognizing each of the three phosphorylation sites of αB-crystallin, we examined whether phosphorylated forms were present in fractions containing smaller oligomers in U373 MG cells treated with PMA and okadaic acid. The phosphorylated form of αB-crystallin, detected by each of the three specific antibodies, was observed in fractions corresponding to the relatively smaller molecular masses (Fig. 6,C and D). The same fractions used in the experiment for Fig. 6 were also subjected to isoelectric focusing with subsequent Western blot analysis using antibodies against C-terminal peptide of αB-crystallin. It was confirmed that treatment of cells with PMA and okadaic acid at 37 °C for 90 min resulted in the induction of multiple phosphorylated bands which had more acidic isoelectric points, the sedimentation profile of αB-crystallin being shifted to a smaller molecular size as compared with that of untreated control cells (Fig. 7). These results indicate phosphorylation of endogenous αB-crystallin to cause a change in oligomerization state also in cells in vivo. To compare chaperone-like activity of wt-αB or 3D-αB in cells in vivo, we estimated the protective activity of each protein against heat inactivation of luciferase in transiently-transfected CHO cells. When cells reached confluence after transfection, they were subjected to heat treatment at 45 °C for 70 min and then cultured at 37 °C for 16 h. In mock-transfected cells, luciferase activity was almost completely inactivated after heat treatment (Fig. 8). In contrast, luciferase activity in cells expressing wt-αB was not decreased but rather increased after heat treatment (Fig. 8). In cells expressing 3D-αB, luciferase activity was protected as compared with mock-transfected cells but the degree of the protection was significantly less than that of cells expressing wt-αB (Fig. 8). We report here for the first time that phosphorylation of αB-crystallin results in change in its oligomerization state. Oligomerization states of wt-αB and 3D-αB were found to be quite different on analysis by sucrose density gradient centrifugation with subsequent Western blot analysis of both purified recombinant proteins or extracts of cells transiently expressing these proteins (Figs. 3 and5). Moreover, the oligomerization size of αB-crystallin in U373 MG cells, which had been treated with PMA and okadaic acid to induce extensive phosphorylation, became smaller and the majority of phosphorylated forms of αB-crystallin were detected in fractions corresponding to a smaller oligomerization size (Figs. 6 and 7). These results suggest that phosphorylation of αB-crystallin causes a reduction of its oligomerization. We and other groups previously reported that the phosphorylation of HSP27 caused similar dissociation of large oligomers (11Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar, 32Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (559) Google Scholar). It has also been described that HSP20, another sHSP (33Kato K. Goto S. Inaguma Y. Hasegawa K. Morishita R. Asano T. J. Biol. Chem. 1994; 269: 15302-15309Abstract Full Text PDF PubMed Google Scholar), is phosphorylated by a cyclic nucleotide-dependent pathway which results in reduction of its oligomer size (34Brophy C.M. Dickinson M. Woodrum D. J. Biol. Chem. 1999; 274: 6324-6329Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). From these data, we conclude that αB-crystallin shares a characteristic feature of sHSPs,i.e. phosphorylation-dependent reduction of oligomerization. The changes in HSP27 and HSP20 in response to phosphorylation were more drastic than those with αB-crystallin, but in fact, both oligomerized and dissociated forms of HSP27 and HSP20 are detectable in extracts of normal tissues and cells, while αB-crystallin is only present as an oligomer about 500 kDa under the same conditions (33Kato K. Goto S. Inaguma Y. Hasegawa K. Morishita R. Asano T. J. Biol. Chem. 1994; 269: 15302-15309Abstract Full Text PDF PubMed Google Scholar). There have been several reports concerning the phosphorylation state and chaperone-like activity of αB-crystallin. Nicoll and Quinlan (22Nicholl I.D. Quinlan R.A. EMBO J. 1994; 13: 945-953Crossref PubMed Scopus (393) Google Scholar) found both unphosphorylated and phosphorylated forms of αB-crystallin to be equally modulating intermediate filament assembly. Wang et al. (35Wang K. Ma W. Spector A. Exp. Eye. Res. 1995; 61: 115-124Crossref PubMed Scopus (40) Google Scholar) further showed both forms of αB-crystallin purified from rat lens exhibited similar chaperone-like activity. While it has also been reported that αB-crystallin prevents cytochalasin-induced depolymerization of actin filaments in a phosphorylation-dependent manner, in the absence of cytochalasin, its effects on the actin polymerization state are phosphorylation-independent (21Wang K. Spector A. Eur. J. Biochem. 1996; 242: 56-66Crossref PubMed Scopus (140) Google Scholar). We carried out thermal aggregation assays of LDH and luciferase refolding assays using recombinant proteins and found that 3D-αB showed a less chaperone-like activity as compared with wt-αB (Fig. 4). Most of the phosphorylated forms of αB-crystallin in rat lens are phosphorylated at one or two sites and the amount of αB-crystallin phosphorylated at all three sites is limited (19Ito H. Okamoto K. Nakayama H. Isobe T. Kato K. J. Biol. Chem. 1997; 272: 29934-29941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Therefore the chaperone-like activity of the phosphorylated form of αB-crystallin purified from lens may not be much different from that of unphosphorylated αB-crystallin and major effects can only be observed by using 3D mutants as models. The relationship between oligomerization state and chaperone-like activity is also not clear. Under our experimental conditions, wt-αB existing as large oligomers exhibited more powerful chaperone-like activity than 3D-αB forming smaller oligomers (Fig. 4). However, it has been reported that mutation of R120G in αB-crystallin causes a larger oligomer than wt-αB which demonstrate less chaperone-like activity (36Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (334) Google Scholar, 37Perng M.D. Muchowski P.J. van Den I.P. Wu G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These observations suggest that the oligomerization state of αB-crystallin may not be determining in this respect. We also examined the ability of αB-crystallin to protect luciferase against heat in CHO cells (Fig. 8). In cells expressing wt-αB, luciferase activity after heat treatment did not decrease but rather increased (Fig. 8). The reason for this result is not clear although it is in line with a previous report (38van de Klundert F. van den IJssel P. Stege G.J.J. de Jong W.W. Biochem. Biophys. Res. Commun. 1999; 254: 164-168Crossref PubMed Scopus (23) Google Scholar). As compared with wt-αB, the ability of 3D-αB to protect luciferase activity against heat in CHO cells was significantly reduced (Fig. 8), consistent with the resultsin vitro as shown in Fig. 4. Far UV CD spectra of recombinant wt-αB and 3D-αB, purified fromE. coli, were similar at 25 °C (Fig. 1 B), indicating mutation does not cause significant change of secondary structure and it is in agreement with results for HSP27 (13Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). From the results of near UV CD spectra, intrinsic fluorescence spectra and ANS binding assay, it is suggested that mutation causes significant change of the environment of aromatic amino acid and the extent of hydrophobicity (Figs. 1 D and 2). At a higher temperature, the pattern of far UV CD spectra of both proteins were changed and the change in the pattern of 3D-αB was more prominent than that of wt-αB (Fig. 1 C), indicating that the secondary structure of 3D-αB sensitively changed in response to an increasing temperature. The biological significance of phosphorylation and changes in the oligomerization size of αB-crystallin is still unclear. Since the effects on oligomerization state and chaperone-like activity were less than those found for HSP27, phosphorylation may confer another biological activity on αB-crystallin, with small oligomers perhaps preferentially interacting with other proteins. In our previous study, we detected in vitro phosphorylation of αB-crystallin using an N-terminal 72-amino acid (N72-K) peptide as a substrate because bovine αB2-crystallin was not phosphorylatedin vitro under any conditions which caused phosphorylation of αB-crystallin in vivo (20Kato K. Ito H. Kamei K. Inaguma Y. Iwamoto I. Saga S. J. Biol. Chem. 1998; 273: 28346-28354Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The N72-K peptide can be obtained by digestion of bovine unphosphorylated αB2-crystallin by lysyl endopeptidase and this peptide contains all three phosphorylation sites of αB-crystallin without the α-crystallin domain, which is thought to play an important role in oligomerization of the molecule (39Boelens W.C. Croes Y. de Ruwe M. de Reu L. de Jong W.W. J. Biol. Chem. 1998; 273: 28085-28090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These experiments suggested that the protein kinase(s) responsible for phosphorylation of αB-crystallin may not be able to access the potential phosphorylation sites in the large oligomer under our experimental conditions in vitro, whereas in vivo cellular machinery which folds proteins in adequate forms might make them accessible. There could be proteins which specifically interact with the phosphorylated form of αB-crystallin. Elucidation of the possibility should help to clarify the functions of αB-crystallin. We thank Dr. Akiko Iwaki and Dr. Hiroshi Itoh for generous gifts of cDNAs for human αB-crystallin and expression vector, pCMV5, respectively." @default.
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• W2036023681 title "Phosphorylation-induced Change of the Oligomerization State of αB-crystallin" @default.
• W2036023681 cites W1481540262 @default.
• W2036023681 cites W1482432683 @default.
• W2036023681 cites W1513598783 @default.
• W2036023681 cites W1531522492 @default.
• W2036023681 cites W1552489964 @default.
• W2036023681 cites W1557531431 @default.
• W2036023681 cites W1562088891 @default.
• W2036023681 cites W1581180757 @default.
• W2036023681 cites W1606423922 @default.
• W2036023681 cites W1755868117 @default.
• W2036023681 cites W1913069793 @default.
• W2036023681 cites W1967167898 @default.
• W2036023681 cites W1968802798 @default.
• W2036023681 cites W1973766879 @default.
• W2036023681 cites W1976462601 @default.
• W2036023681 cites W1988603521 @default.
• W2036023681 cites W1994861776 @default.
• W2036023681 cites W1999145875 @default.
• W2036023681 cites W1999852323 @default.
• W2036023681 cites W2008359273 @default.
• W2036023681 cites W2009556803 @default.
• W2036023681 cites W2011574538 @default.
• W2036023681 cites W2019012009 @default.
• W2036023681 cites W2023610686 @default.
• W2036023681 cites W2025198517 @default.
• W2036023681 cites W2033395773 @default.
• W2036023681 cites W2044031095 @default.
• W2036023681 cites W2044122905 @default.
• W2036023681 cites W2071962920 @default.
• W2036023681 cites W2082925886 @default.
• W2036023681 cites W2085054634 @default.
• W2036023681 cites W2100837269 @default.
• W2036023681 cites W2130224280 @default.
• W2036023681 cites W2142181775 @default.
• W2036023681 cites W2154904733 @default.
• W2036023681 cites W2156759248 @default.
• W2036023681 cites W2166500974 @default.
• W2036023681 cites W2443634062 @default.
• W2036023681 cites W4232192299 @default.
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