Phosphorylation-induced Change of the Oligomerization State of alpha B-crystallin*

Hidenori ItoDagger §, Keiko KameiDagger , Ikuko IwamotoDagger , Yutaka InagumaDagger , Daisuke Nohara, and Kanefusa KatoDagger

From the Dagger  Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan and the  Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya, 467-8603, Japan

Received for publication, October 3, 2000, and in revised form, November 22, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha B-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 alpha B-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-alpha B). From results of sucrose density gradient centrifugation, we found that wild type alpha B-crystallin (wt-alpha B) and 3D-alpha B sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alpha B was significantly weaker than that of wt-alpha B. When wt-alpha B and 3D-alpha B 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, alpha B-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-alpha B or 3D-alpha B in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alpha B was less than that with wt-alpha B. From these observations, it is suggested that phosphorylation of alpha B-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1, 2). 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 (3-6) 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 (7-9), and the delta  isoform of protein kinase C (protein kinase C-delta ) (10). We previously reported that phosphorylation of HSP27 in cells leads to dissociation of large oligomers and decrease of resistance to heat (11). 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 (12, 13). HSP27 has been recognized as an actin-binding protein, with activity that modifies the polymerization state of actin (14, 15). Phosphorylation of HSP27 causes loss of its actin polymerization inhibiting activity (16).

Phosphorylation of alpha B-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 (17, 18). We first reported that alpha B-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 (19). 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 (20). It is known that alpha B-crystallin can prevent cytochalasin-induced depolymerization of actin filaments in a phosphorylation-dependent manner, although the phosphorylation does not affect its chaperone-like activity (21). alpha B-crystallin also modulates intermediate filament assembly independent of its phosphorylation state (22). Phosphorylation of alpha B-crystallin at Ser-45 occurs in mammalian mitotic cells (20) and phosphorylated forms, especially at Ser-45, increase in rat lens during postnatal development (23).

In the present study, we examined the effects of phosphorylation of alpha B-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 alpha B-crystallin and decrease in its chaperone-like activity.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Human alpha B-crystallin and Its Mutant Expression Vectors-- For expression in mammalian cells, an EcoRI fragment from human alpha B-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) (24). For site-directed mutagenesis, we used the polymerase chain reaction with oligonucleotide mutation primers and the template alpha B-crystallin expression vector. BglII and XhoI fragments from polymerase chain reaction fragments were inserted into BglII and SalI sites of pCMV5. We constructed two plasmids to express wild type alpha B-crystallin and alpha B-crystallin in which three phosphorylation sites, Ser-19, Ser-45, and Ser-59, were substituted with aspartate. We designated these plasmids as wt-alpha B-pCMV5 and 3D-alpha B-pCMV5. The mutated sites were confirmed by DNA sequencing. For expression of wt-alpha B and 3D-alpha B in Escherichia coli, their cDNAs were inserted into NdeI and XhoI sites of the vector pET30a(+) (Novagen, Madison, WI).

Purification of Recombinant wt-alpha B and 3D-alpha B-- Recombinant human alpha B-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 A600 of 0.6 and 0.5 mM isopropyl-1-thio-beta -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 alpha B-crystallin (25) and then the column was washed with 0.1 M phosphate buffer, pH 7.0. alpha B-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 7 M 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). alpha B-crystallin was eluted with a linear gradient of NaCl (0-0.4 M) in the above buffer as described previously (19).

Circular Dichroism Measurements-- Circular dichroism (CD) spectra were recorded using a Jasco J-725 spectropolarimeter. Samples of wt-alpha B and 3D-alpha B 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.

Fluorescence Studies-- 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-alpha B and 3D-alpha B 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.

Gel Filtration Chromatography-- For determination of oligomerization size of recombinant alpha B-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).

Assay of in Vitro Chaperone-like Activity-- 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 alpha B-crystallin. We also performed luciferase refolding assays in vitro in the presence of recombinant alpha B-crystallin with the method described by Lu and Cyr (26). 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, M guanidium 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 mM MgCl2, and 5 mM ATP) in the presence or absence of 1 µM recombinant alpha B-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).

Cell Culture and Preparation of Cell Extracts-- 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 M NaF, 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 alpha B-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.

Sucrose Density Gradient Centrifugation of Purified Recombinant Proteins and Cell Extracts-- Ten µg of purified recombinant alpha B-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.

Electrophoresis and Western Blot Analysis-- SDS-PAGE was performed as described by Laemmli (27) in 12.5% gels and proteins were visualized by staining with Coomassie Brilliant Blue. Isoelectric focusing was carried out as described by O'Farrell (28) using the Protean II system from Bio-Rad (19). Western blot analysis was performed as described previously using affinity purified antibodies raised in rabbits against the C-terminal decapeptide of alpha B-crystallin (25) or against phosphopeptides which correspond to the three phosphorylation sites of alpha B-crystallin (p19S, p45S, and p59S) (19). 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).

Protection of Luciferase in Transiently Transfected CHO Cells-- CHO cells were transiently co-transfected with two plasmids, 0.5 µg of wt-alpha B-pCMV5 or 3D-alpha B-pCMV5 and 0.5 µg of the luciferase expression vector, PGV-C (Toyo Ink Co.). Almost equal amounts of wt-alpha B and 3D-alpha B 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 alpha B-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.

Other Methods-- Bovine alpha B1-crystallin (phosphorylated form of alpha B-crystallin) and alpha B2-crystallin (unphosphorylated form of alpha B-crystallin) were purified from bovine lens as described previously (25). Concentrations of protein were estimated with a protein assay kit (Bio-Rad) with bovine serum albumin as the standard.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Recombinant wt-alpha B and 3D-alpha B-- To estimate the effects of phosphorylation of alpha B-crystallin on its oligomerization state, we expressed recombinant proteins in E. coli. Wild type alpha B-crystallin (wt-alpha B) and 3D-alpha B-crystallin (3D-alpha B), in which all three phosphorylation sites of alpha B-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 alpha B-crystallin and subsequent anion exchange chromatography. Purified wt-alpha B was observed at the position of the predicted molecular size in SDS-PAGE gels and 3D-alpha B was detected as a band migrating to a position pointing to a slightly greater kilodalton value (Fig. 1A). Using these recombinant proteins, far and near UV CD spectra were measured to estimate the effect of phosphorylation of alpha B-crystallin on its secondary and tertiary structure. Consistent with a previous report, wt-alpha B showed a far UV CD spectrum with a high percentage of beta -sheet/beta -turn structure in its molecule at 25 °C (Fig. 1B). The 3D-alpha B also showed a far UV CD spectrum indicating a high percentage of beta -structures at 25 °C and the pattern of the spectrum was similar to that of wt-alpha B (Fig. 1B). To estimate the effect of phosphorylation of alpha B-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-alpha B and 3D-alpha B were significantly changed and the extent of the change was more visible in the case of 3D-alpha B (Fig. 1C). 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-alpha B and 3D-alpha B were visibly different at 25 and 45 °C (Fig. 1, D and E).



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Fig. 1.   SDS-PAGE findings and far and near UV CD spectra of recombinant wt-alpha B and 3D-alpha B. A, 1-µg aliquots of recombinant wt-alpha B (lane 2) and 3D-alpha B (lane 3) were subjected to SDS-PAGE and the gel was stained with Coomassie Blue. Lane 1, molecular weight markers. B and C, far UV CD spectra of wt-alpha B (solid lines) and 3D-alpha B (dotted lines), measured at a concentration of 45 µg/ml using a path length of 10 mm in 10 mM sodium phosphate buffer, pH 7.4, at 25 °C (B) and 45 °C (C). D and E, near UV CD spectra of wt-alpha B (solid lines) and 3D-alpha B (dotted lines), measured at a concentration of 450 µg/ml using a path length of 10 mm in 10 mM sodium phosphate buffer, pH 7.4, at 25 °C (D) and 45 °C (E). Data are expressed as millidegrees. Each spectrum represents the average of 3 scans (for far UV CD spectra) or 5 scans (for near UV CD spectra).

Intrinsic tryptophan fluorescence spectrum is thought to provide information of the environment of tryptophan residues in proteins. We also monitored fluorescence spectra of wt-alpha B and 3D-alpha B and it was revealed that the spectrum of 3D-alpha B was significantly shifted to the longer wavelength and the intensity of fluorescence increased (Fig. 2A), indicating a difference of the environment of tryptophan residues in wt-alpha B and 3D-alpha B and a decrease in the extent of hydrophobicity of 3D-alpha B. To estimate the extent of hydrophobicity of wt-alpha B and 3D-alpha B, 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 (29). As shown in Fig. 2B, the fluorescence intensity of ANS bound to 3D-alpha B was lower than that bound to wt-alpha B, indicating a smaller extent of hydrophobicity of 3D-alpha B than that of wt-alpha B and the results were consistent with the results of intrinsic tryptophan fluorescence spectra (Fig. 2A).



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Fig. 2.   Intrinsic tryptophan fluorescence spectra of wt-alpha B and 3D-alpha B (A) and fluorescence spectra of ANS bound to wt-alpha B and 3D-alpha B (B). Intrinsic tryptophan fluorescence spectra of wt-alpha B (solid line) and 3D-alpha B (dotted line) and fluorescence spectra of ANS bound to wt-alpha B (solid line) and 3D-alpha B (dotted line) at room temperature were monitored as described under "Experimental Procedures."

Oligomelization States of Recombinant wt-alpha B and 3D-alpha B-- 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 (11). To estimate oligomerization states of recombinant wt-alpha B and 3D-alpha B, we fractionated each preparation of alpha B-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 alpha B-crystallin. As shown in Fig. 3A, oligomerization states of wt-alpha B and 3D-alpha B were quite different. Quantitation of the intensity of bands of Western blot analysis revealed that wt-alpha B mainly sedimented at a position which corresponded to an apparent molecular mass of about 500 kDa, while 3D-alpha B mainly sedimented at a position which corresponded to apparent molecular masses of about 300 kDa, with a broader range (Fig. 3B). We also estimated oligomerization sizes of wt-alpha B and 3D-alpha B using gel filtration chromatography and it revealed that the oligomerization sizes of wt-alpha B and 3D-alpha B were about 550 and 390 kDa, respectively (data not shown).



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Fig. 3.   Oligomerization states of recombinant wt-alpha B and 3D-alpha B. A, 10 µg each of recombinant wt-alpha B and 3D-alpha B were subjected to sucrose density gradient centrifugation and subsequent fractionation from the bottom. Ten-µl aliquots of each fraction were subjected to SDS-PAGE and subsequent Western blot analysis. B, visualized bands were quantitated and graphed. Arrowheads indicate the positions at which beta -D-galactosidase from E. coli (Gal, 540 kDa) and bovine serum albumin (BSA, 67 kDa) sedimented. Open circles, wt-alpha B; closed circles, 3D-alpha B.

In Vitro Chaperone-like Activity of wt-alpha B and 3D-alpha B-- Stress proteins are known to have a chaperone activity and there are several reports describing such activity for alpha B-crystallin (30, 31). To determine the effects of phosphorylation on this parameter, we assessed thermal aggregation of LDH in the presence or absence of recombinant wt-alpha B or 3D-alpha B. As shown in Fig. 4, A and B, the degree of prevention from thermal aggregation of LDH by 3D-alpha B was slightly weaker than that of wt-alpha B and this tendency was more obvious when the ratio of LDH to alpha B-crystallin was higher. Moreover, we estimated the ability of wt-alpha B and 3D-alpha B to refold denatured firefly luciferase. In the absence of alpha B-crystallin, the activity of luciferase remained to be lowered (Fig. 4C). In the presence of wt-alpha B or 3D-alpha B, refolding of luciferase was clearly observed, but the refolding activity of 3D-alpha B was weaker than that of wt-alpha B (Fig. 4C).



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Fig. 4.   Protection against LDH aggregation at 50 °C by recombinant wt-alpha B or 3D-alpha B (A and B) and refolding of luciferase by recombinant wt-alpha B or 3D-alpha B (C). A and B, LDH was aggregated at 50 °C in the presence (closed symbols) or absence (open circles) of wt-alpha B (closed triangles) and 3D-alpha B (closed squares) for 60 min and the absorbance at 360 nm was monitored. The ratios of LDH:alpha B-crystallin were 10:1 (100 µg:10 µg) (A) and 20:1 (100 µg:5 µg) (B). C, refolding of luciferase at 30 °C in the presence (closed symbols) or absence (opened circles) of recombinant wt-alpha B (closed squares) and 3D-alpha B (closed triangles) was measured as described under "Experimental Procedures."

Oligomerization States of wt-alpha B and 3D-alpha B in Transiently Transfected Mammalian Cells-- To estimate the effects of phosphorylation on the oligomerization state of alpha B-crystallin in mammalian cells, we performed a transient expression of wt-alpha B and 3D-alpha B in COS-m6 cells. Consistent with the results obtained with the purified recombinant proteins, the band for 3D-alpha B was observed at a slightly different position from wt-alpha B after the SDS-PAGE and subsequent immunostaining with antibodies against C-terminal peptide of alpha B-crystallin (Fig. 5A). Extracts of cells transiently expressing wt-alpha B or 3D-alpha B were subjected to sucrose density gradient centrifugation with subsequent fractionation and Western blot analysis. The sedimentation profiles of wt-alpha B and 3D-alpha B were quite different (Fig. 5B). Quantitation of the intensity of each band revealed that wt-alpha B mainly sedimented at a position corresponding to an apparent molecular mass of about 500 kDa while 3D-alpha B 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 alpha B-crystallin in transiently-transfected COS-m6 cells fractionated within narrow ranges.



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Fig. 5.   SDS-PAGE and subsequent Western blot analysis of extracts of cells transiently expressing wt-alpha B or 3D-alpha B and their fractions obtained after sucrose density gradient centrifugation. A, COS-m6 cells were transiently transfected with plasmids to express wt-alpha B (lanes 1 and 2) or 3D-alpha B (lanes 3 and 4) as described under "Experimental Procedures" and 10-µg aliquots of cell extracts were subjected to SDS-PAGE and subsequent Western blot analysis with antibodies against the C-terminal peptide of alpha B-crystallin. B, cell extracts (0.2 ml) were fractionated by sucrose density gradient centrifugation. Ten-µl aliquots of each fraction were subjected to SDS-PAGE with subsequent Western blot analysis. C, densities of visualized bands in B were quantitated and graphed. Arrowheads indicate the positions at which beta -D-galactosidase from E. coli (Gal, 540 kDa) and bovine serum albumin (BSA, 67 kDa) sedimented. Open circles, cells expressing wt-alpha B; closed circles, cells expressing 3D-alpha B.

Effects of Phosphorylation of alpha B-crystallin on Its Oligomerization State in U373 MG Cells-- We previously reported that various stresses induce phosphorylation of alpha B-crystallin in U373 MG cells, a combination of PMA and okadaic acid treatment markedly increasing phosphorylated forms (19). We therefore estimated whether phosphorylation of endogenous alpha B-crystallin causes change in its oligomerization state. Endogenous alpha B-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 alpha B-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 alpha B-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 alpha B-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 alpha B-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 alpha B-crystallin being shifted to a smaller molecular size as compared with that of untreated control cells (Fig. 7). These results indicate phosphorylation of endogenous alpha B-crystallin to cause a change in oligomerization state also in cells in vivo.



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Fig. 6.   Phosphorylation of alpha B-crystallin induced by PMA and okadaic acid in U373 MG cells results in change of the oligomerization state of alpha B-crystallin. A, cells were treated with 0.1 µM PMA and 0.1 µM okadaic acid for 90 min and 200-µl extracts were fractionated by centrifugation on a sucrose density gradient (10-40%). Aliquots (15 µl) of each fraction were subjected to SDS-PAGE and Western blot analysis with antibodies against C-terminal peptides of alpha B-crystallin. Upper panel, unstimulated control cells; lower panel, cells treated with PMA and okadaic acid. B, densities of visualized bands shown in A were quantitated and graphed. Arrowheads indicate the positions at which beta -D-galactosidase from E. coli (Gal, 540 kDa) and bovine serum albumin (BSA, 67 kDa) sedimented. Open circles, control cells; closed circles, cells treated with PMA and okadaic acid. C, aliquots (15 µl) of each fraction from sucrose density gradient centrifugation of extracts of cells treated with PMA and okadaic acid were subjected to SDS-PAGE and Western blot analysis with antibodies recognizing each of the three phosphorylation sites in alpha B-crystallin (p19S, p45S, and p59S, respectively). D, densities of the bands shown in C were quantitated and graphed. Arrowheads indicate the positions at which beta -D-galactosidase from E. coli (Gal, 540 kDa) and bovine serum albumin (BSA, 67 kDa) sedimented. Closed circles, phosphorylated Ser-59; open circles, phosphorylated Ser-45; closed squares, phosphorylated Ser-19.



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Fig. 7.   Isoelectric focusing and Western blot analysis of fractions of U373 MG cells treated with PMA and okadaic acid obtained by sucrose density gradient centrifugation. Aliquots (20 µl) of each fraction from sucrose density gradient centrifugation of extracts of cells untreated (A) or treated with PMA and okadaic acid (B) were subjected to isoelectric focusing and Western blot analysis with antibodies recognizing the C-terminal peptide of alpha B-crystallin. p0, unphosphorylated alpha B-crystallin; p1, p2, and p3, alpha B-crystallin phosphorylated at one, two, or three sites, respectively.

Protection of Luciferase by alpha B-crystallin against Heat Treatment in CHO Cells-- To compare chaperone-like activity of wt-alpha B or 3D-alpha B 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-alpha B was not decreased but rather increased after heat treatment (Fig. 8). In cells expressing 3D-alpha B, 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-alpha B (Fig. 8).



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Fig. 8.   Protection of luciferase against heat by wt-alpha B and 3D-alpha B in CHO cells. Cells transiently coexpressing wt-alpha B or 3D-alpha B, with luciferase, on reaching confluence, were subjected to heat treatment at 45 °C for 70 min, cultured at 37 °C for 16 h, harvested, and quantitated for luciferase activity. The data shown are percentages relative to luciferase activity of untreated cells, each bar showing mean ± S.D. of results for three dishes. The data are representative of three separate experiments. Open bars, cells transfected with empty vector; closed bars, cells expressing wt-alpha B; shaded bars, cells expressing 3D-alpha B. The bar indicated with "*" is statistically different from wt-alpha B with p < 0.005 (Student's t test). Controls, cells without heat treatment; heat shock, cells treated with heat.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report here for the first time that phosphorylation of alpha B-crystallin results in change in its oligomerization state. Oligomerization states of wt-alpha B and 3D-alpha B 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 and 5). Moreover, the oligomerization size of alpha B-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 alpha B-crystallin were detected in fractions corresponding to a smaller oligomerization size (Figs. 6 and 7). These results suggest that phosphorylation of alpha B-crystallin causes a reduction of its oligomerization. We and other groups previously reported that the phosphorylation of HSP27 caused similar dissociation of large oligomers (11, 32). It has also been described that HSP20, another sHSP (33), is phosphorylated by a cyclic nucleotide-dependent pathway which results in reduction of its oligomer size (34). From these data, we conclude that alpha B-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 alpha B-crystallin, but in fact, both oligomerized and dissociated forms of HSP27 and HSP20 are detectable in extracts of normal tissues and cells, while alpha B-crystallin is only present as an oligomer about 500 kDa under the same conditions (33).

There have been several reports concerning the phosphorylation state and chaperone-like activity of alpha B-crystallin. Nicoll and Quinlan (22) found both unphosphorylated and phosphorylated forms of alpha B-crystallin to be equally modulating intermediate filament assembly. Wang et al. (35) further showed both forms of alpha B-crystallin purified from rat lens exhibited similar chaperone-like activity. While it has also been reported that alpha B-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 (21). We carried out thermal aggregation assays of LDH and luciferase refolding assays using recombinant proteins and found that 3D-alpha B showed a less chaperone-like activity as compared with wt-alpha B (Fig. 4). Most of the phosphorylated forms of alpha B-crystallin in rat lens are phosphorylated at one or two sites and the amount of alpha B-crystallin phosphorylated at all three sites is limited (19). Therefore the chaperone-like activity of the phosphorylated form of alpha B-crystallin purified from lens may not be much different from that of unphosphorylated alpha B-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-alpha B existing as large oligomers exhibited more powerful chaperone-like activity than 3D-alpha B forming smaller oligomers (Fig. 4). However, it has been reported that mutation of R120G in alpha B-crystallin causes a larger oligomer than wt-alpha B which demonstrate less chaperone-like activity (36, 37). These observations suggest that the oligomerization state of alpha B-crystallin may not be determining in this respect.

We also examined the ability of alpha B-crystallin to protect luciferase against heat in CHO cells (Fig. 8). In cells expressing wt-alpha B, 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 (38). As compared with wt-alpha B, the ability of 3D-alpha B to protect luciferase activity against heat in CHO cells was significantly reduced (Fig. 8), consistent with the results in vitro as shown in Fig. 4.

Far UV CD spectra of recombinant wt-alpha B and 3D-alpha B, purified from E. coli, were similar at 25 °C (Fig. 1B), indicating mutation does not cause significant change of secondary structure and it is in agreement with results for HSP27 (13). 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. 1D 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-alpha B was more prominent than that of wt-alpha B (Fig. 1C), indicating that the secondary structure of 3D-alpha B sensitively changed in response to an increasing temperature.

The biological significance of phosphorylation and changes in the oligomerization size of alpha B-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 alpha B-crystallin, with small oligomers perhaps preferentially interacting with other proteins. In our previous study, we detected in vitro phosphorylation of alpha B-crystallin using an N-terminal 72-amino acid (N72-K) peptide as a substrate because bovine alpha B2-crystallin was not phosphorylated in vitro under any conditions which caused phosphorylation of alpha B-crystallin in vivo (20). The N72-K peptide can be obtained by digestion of bovine unphosphorylated alpha B2-crystallin by lysyl endopeptidase and this peptide contains all three phosphorylation sites of alpha B-crystallin without the alpha -crystallin domain, which is thought to play an important role in oligomerization of the molecule (39). These experiments suggested that the protein kinase(s) responsible for phosphorylation of alpha B-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 alpha B-crystallin. Elucidation of the possibility should help to clarify the functions of alpha B-crystallin.


    ACKNOWLEDGEMENTS

We thank Dr. Akiko Iwaki and Dr. Hiroshi Itoh for generous gifts of cDNAs for human alpha B-crystallin and expression vector, pCMV5, respectively.


    FOOTNOTES

* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan. Tel.: 81-568-88-0811 (ext. 3582); Fax: 81-568-88-0829; E-mail: itohide@inst-hsc.pref.aichi.jp.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009004200


    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; sHSPs, small heat shock or stress proteins; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; ANS, 8-anilinonaphthalene-1-sulfonate; MAPK, mitogen-activated protein kinase; UV CD, ultraviolet circular dichroism; LDH, lactate dehydrogenase; CHO cells, Chinese hamster ovary cells.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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