Phosphorylation-induced Change of the Oligomerization State
of
B-crystallin*
Hidenori
Ito
§,
Keiko
Kamei
,
Ikuko
Iwamoto
,
Yutaka
Inaguma
,
Daisuke
Nohara¶, and
Kanefusa
Kato
From the
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 |
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
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-
B). From results of sucrose density
gradient centrifugation, we found that wild type
B-crystallin
(wt-
B) and 3D-
B sedimented in fractions corresponding to apparent
molecular masses of about 500 and 300 kDa, respectively. Chaperone-like
activity of 3D-
B was significantly weaker than that of wt-
B. When
wt-
B and 3D-
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,
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-
B or 3D-
B in Chinese hamster
ovary cells caused protection of the enzyme from heat inactivation
although the degree of protection with 3D-
B was less than that with
wt-
B. From these observations, it is suggested that phosphorylation of
B-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the
case of HSP27.
 |
INTRODUCTION |
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
isoform of protein kinase C (protein kinase C-
) (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
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
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
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).
B-crystallin also modulates intermediate filament assembly independent of its phosphorylation state (22). Phosphorylation of
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
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
B-crystallin and decrease in
its chaperone-like activity.
 |
EXPERIMENTAL PROCEDURES |
Construction of Human
B-crystallin and Its Mutant Expression
Vectors--
For expression in mammalian cells, an EcoRI
fragment from human
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
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
B-crystallin and
B-crystallin in which three
phosphorylation sites, Ser-19, Ser-45, and Ser-59, were substituted
with aspartate. We designated these plasmids as wt-
B-pCMV5 and
3D-
B-pCMV5. The mutated sites were confirmed by DNA sequencing. For
expression of wt-
B and 3D-
B in Escherichia coli, their
cDNAs were inserted into NdeI and XhoI sites
of the vector pET30a(+) (Novagen, Madison, WI).
Purification of Recombinant wt-
B and 3D-
B--
Recombinant
human
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-
-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
B-crystallin (25) and then the column was
washed with 0.1 M phosphate buffer, pH 7.0.
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).
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-
B and 3D-
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-
B and 3D-
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
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
B-crystallin. We also performed luciferase
refolding assays in vitro in the presence of recombinant
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, 6 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
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
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
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
B-crystallin
(25) or against phosphopeptides which correspond to the three
phosphorylation sites of
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-
B-pCMV5 or 3D-
B-pCMV5 and 0.5 µg of
the luciferase expression vector, PGV-C (Toyo Ink Co.). Almost equal
amounts of wt-
B and 3D-
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
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
B1-crystallin
(phosphorylated form of
B-crystallin) and
B2-crystallin (unphosphorylated form of
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 |
Characterization of Recombinant wt-
B and 3D-
B--
To
estimate the effects of phosphorylation of
B-crystallin on its
oligomerization state, we expressed recombinant proteins in E. coli. Wild type
B-crystallin (wt-
B) and 3D-
B-crystallin (3D-
B), in which all three phosphorylation sites of
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
B-crystallin and subsequent anion exchange chromatography. Purified
wt-
B was observed at the position of the predicted molecular size in
SDS-PAGE gels and 3D-
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
B-crystallin on its secondary and
tertiary structure. Consistent with a previous report, wt-
B showed a
far UV CD spectrum with a high percentage of
-sheet/
-turn
structure in its molecule at 25 °C (Fig. 1B). The 3D-
B
also showed a far UV CD spectrum indicating a high percentage of
-structures at 25 °C and the pattern of the spectrum was similar
to that of wt-
B (Fig. 1B). To estimate the effect of
phosphorylation of
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-
B and
3D-
B were significantly changed and the extent of the change was
more visible in the case of 3D-
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-
B and 3D-
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- B and
3D- B. A, 1-µg aliquots of
recombinant wt- B (lane 2) and 3D- 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- B (solid lines) and
3D- 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- B (solid lines) and 3D- 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).
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Intrinsic tryptophan fluorescence spectrum is thought to provide
information of the environment of tryptophan residues in proteins. We
also monitored fluorescence spectra of wt-
B and 3D-
B and it was
revealed that the spectrum of 3D-
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-
B and 3D-
B and a
decrease in the extent of hydrophobicity of 3D-
B. To estimate the
extent of hydrophobicity of wt-
B and 3D-
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-
B was lower than that bound to wt-
B,
indicating a smaller extent of hydrophobicity of 3D-
B than that of
wt-
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- B and 3D- B
(A) and fluorescence spectra of ANS bound to
wt- B and 3D- B
(B). Intrinsic tryptophan fluorescence spectra of
wt- B (solid line) and 3D- B (dotted line)
and fluorescence spectra of ANS bound to wt- B (solid
line) and 3D- B (dotted line) at room temperature
were monitored as described under "Experimental Procedures."
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Oligomelization States of Recombinant wt-
B and 3D-
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-
B and 3D-
B, we
fractionated each preparation of
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
B-crystallin. As shown in Fig.
3A, oligomerization states of
wt-
B and 3D-
B were quite different. Quantitation of the intensity
of bands of Western blot analysis revealed that wt-
B mainly
sedimented at a position which corresponded to an apparent molecular
mass of about 500 kDa, while 3D-
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-
B and 3D-
B using gel filtration chromatography and it revealed that the oligomerization sizes of wt-
B and 3D-
B were about 550 and 390 kDa, respectively (data not shown).

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Fig. 3.
Oligomerization states of recombinant
wt- B and 3D- B.
A, 10 µg each of recombinant wt- B and 3D- 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
-D-galactosidase from E. coli (Gal, 540 kDa)
and bovine serum albumin (BSA, 67 kDa) sedimented.
Open circles, wt- B; closed circles,
3D- B.
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In Vitro Chaperone-like Activity of wt-
B and
3D-
B--
Stress proteins are known to have a chaperone activity
and there are several reports describing such activity for
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-
B or 3D-
B. As shown in Fig.
4, A and B, the
degree of prevention from thermal aggregation of LDH by 3D-
B was
slightly weaker than that of wt-
B and this tendency was more obvious
when the ratio of LDH to
B-crystallin was higher. Moreover, we
estimated the ability of wt-
B and 3D-
B to refold denatured
firefly luciferase. In the absence of
B-crystallin, the activity of
luciferase remained to be lowered (Fig. 4C). In the presence
of wt-
B or 3D-
B, refolding of luciferase was clearly observed,
but the refolding activity of 3D-
B was weaker than that of wt-
B
(Fig. 4C).

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Fig. 4.
Protection against LDH aggregation at
50 °C by recombinant wt- B or
3D- B (A and
B) and refolding of luciferase by recombinant
wt- B or 3D- B
(C). A and B, LDH was
aggregated at 50 °C in the presence (closed symbols) or
absence (open circles) of wt- B (closed
triangles) and 3D- B (closed squares) for 60 min and
the absorbance at 360 nm was monitored. The ratios of
LDH: 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- B
(closed squares) and 3D- B (closed triangles)
was measured as described under "Experimental Procedures."
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Oligomerization States of wt-
B and 3D-
B in Transiently
Transfected Mammalian Cells--
To estimate the effects of
phosphorylation on the oligomerization state of
B-crystallin in
mammalian cells, we performed a transient expression of wt-
B and
3D-
B in COS-m6 cells. Consistent with the results obtained with the
purified recombinant proteins, the band for 3D-
B was observed at a
slightly different position from wt-
B after the SDS-PAGE and
subsequent immunostaining with antibodies against C-terminal peptide of
B-crystallin (Fig. 5A). Extracts of cells transiently expressing wt-
B or 3D-
B were
subjected to sucrose density gradient centrifugation with subsequent
fractionation and Western blot analysis. The sedimentation profiles of
wt-
B and 3D-
B were quite different (Fig. 5B).
Quantitation of the intensity of each band revealed that wt-
B mainly
sedimented at a position corresponding to an apparent molecular mass of
about 500 kDa while 3D-
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
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- B or 3D- B and
their fractions obtained after sucrose density gradient
centrifugation. A, COS-m6 cells were transiently
transfected with plasmids to express wt- B (lanes 1 and
2) or 3D- 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
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 -D-galactosidase from E. coli (Gal, 540 kDa) and bovine serum albumin (BSA, 67 kDa) sedimented. Open circles, cells expressing wt- B;
closed circles, cells expressing 3D- B.
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Effects of Phosphorylation of
B-crystallin on Its
Oligomerization State in U373 MG Cells--
We previously reported
that various stresses induce phosphorylation of
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
B-crystallin causes change in its
oligomerization state. Endogenous
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
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
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
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
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
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
B-crystallin to cause a
change in oligomerization state also in cells in vivo.

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Fig. 6.
Phosphorylation of
B-crystallin induced by PMA and okadaic acid in
U373 MG cells results in change of the oligomerization state of
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 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
-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 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
-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 B-crystallin. p0, unphosphorylated
B-crystallin; p1, p2, and p3, B-crystallin
phosphorylated at one, two, or three sites, respectively.
|
|
Protection of Luciferase by
B-crystallin against Heat Treatment
in CHO Cells--
To compare chaperone-like activity of wt-
B or
3D-
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-
B was not decreased but
rather increased after heat treatment (Fig. 8). In cells expressing
3D-
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-
B (Fig. 8).

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Fig. 8.
Protection of luciferase against heat by
wt- B and 3D- B in CHO
cells. Cells transiently coexpressing wt- B or 3D- 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- B; shaded bars, cells expressing 3D- B.
The bar indicated with "*" is statistically different
from wt- B with p < 0.005 (Student's t
test). Controls, cells without heat treatment; heat
shock, cells treated with heat.
|
|
 |
DISCUSSION |
We report here for the first time that phosphorylation of
B-crystallin results in change in its oligomerization state.
Oligomerization states of wt-
B and 3D-
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
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
B-crystallin were detected in fractions
corresponding to a smaller oligomerization size (Figs. 6 and 7). These
results suggest that phosphorylation of
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
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
B-crystallin, but
in fact, both oligomerized and dissociated forms of HSP27 and HSP20 are
detectable in extracts of normal tissues and cells, while
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
B-crystallin. Nicoll and Quinlan (22)
found both unphosphorylated and phosphorylated forms of
B-crystallin
to be equally modulating intermediate filament assembly. Wang et
al. (35) further showed both forms of
B-crystallin purified
from rat lens exhibited similar chaperone-like activity. While it has
also been reported that
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-
B showed a less chaperone-like activity
as compared with wt-
B (Fig. 4). Most of the phosphorylated forms of
B-crystallin in rat lens are phosphorylated at one or two sites and
the amount of
B-crystallin phosphorylated at all three sites is
limited (19). Therefore the chaperone-like activity of the
phosphorylated form of
B-crystallin purified from lens may not be
much different from that of unphosphorylated
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-
B
existing as large oligomers exhibited more powerful chaperone-like activity than 3D-
B forming smaller oligomers (Fig. 4). However, it
has been reported that mutation of R120G in
B-crystallin causes a
larger oligomer than wt-
B which demonstrate less chaperone-like activity (36, 37). These observations suggest that the oligomerization state of
B-crystallin may not be determining in this respect.
We also examined the ability of
B-crystallin to protect luciferase
against heat in CHO cells (Fig. 8). In cells expressing wt-
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-
B, the
ability of 3D-
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-
B and 3D-
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-
B was more prominent than that of
wt-
B (Fig. 1C), indicating that the secondary structure
of 3D-
B sensitively changed in response to an increasing temperature.
The biological significance of phosphorylation and changes in the
oligomerization size of
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
B-crystallin, with small oligomers perhaps
preferentially interacting with other proteins. In our previous study,
we detected in vitro phosphorylation of
B-crystallin
using an N-terminal 72-amino acid (N72-K) peptide as a substrate
because bovine
B2-crystallin was not phosphorylated in vitro under any conditions which caused phosphorylation
of
B-crystallin in vivo (20). The N72-K peptide can be
obtained by digestion of bovine unphosphorylated
B2-crystallin by lysyl endopeptidase and this peptide
contains all three phosphorylation sites of
B-crystallin without the
-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
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
B-crystallin. Elucidation of the possibility should help to clarify
the functions of
B-crystallin.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Akiko Iwaki and Dr. Hiroshi Itoh
for generous gifts of cDNAs for human
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.
 |
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