From the Department of Biochemistry, University of
Mississippi Medical Center, Jackson, Mississippi 39216 and
§ New England Biolabs, Beverly, Massachusetts 01915
Received for publication, May 6, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Protein B23 is a multifunctional nucleolar
protein whose molecular chaperone activity is proposed to play role in
ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that
protein B23 has several characteristics typical of molecular
chaperones, including anti-aggregation activity, promoting the
renaturation of denatured proteins, and preferential binding to
denatured substrates. However, until now there has been no proposed
mechanism for release of a bound substrate. Protein B23 can be
phosphorylated by protein kinase CK2 (CK2) in a segment required for
chaperone activity. The presence of bound substrate enhanced the rate
of CK2 phosphorylation of protein B23 by 2-3-fold, and this
enhancement was dependent on a nonpolar region in its N-terminal end.
Formation of a complex between B23 and chaperone test substrates
(rhodanese or citrate synthase) was inhibited by CK2 phosphorylation.
Furthermore, CK2 phosphorylation of a previously formed B23-substrate
complex promoted its dissociation. The dissociation of complexes
between B23 and the human immunodeficiency virus-Rev protein required
both CK2 phosphorylation and competition with a Rev nuclear
localization signal peptide, suggesting that Rev binds B23 at two
separate sites. These studies suggest that unlike many molecular
chaperones, which directly hydrolyze ATP, substrate release by protein
B23 is dependent on its phosphorylation by CK2.
The nucleolus is the subcellular domain for the assembly of
ribosomal subunits in which the initial building blocks are
preribosomal RNA, 5 S rRNA, and ribosomal and nonribosomal proteins
(1). This is a complex, multistep process that takes place in an
environment that is likely to contain high concentrations of
macromolecules. Although the concentrations in the various compartments
of the nucleolus are not known, they are likely to be at least as high as and probably much higher than the estimated overall cellular macromolecular concentration of 200-300 mg/ml (2). At these concentrations proteins are faced with the potential problems of
denaturation, aggregation, and immobilization as they participate in
ribosome biogenesis. In addition to the macromolecular crowding effect,
the process of incorporation of proteins into preribosomal complexes
could expose hydrophobic surfaces and so further increase the
probability for aggregation. Such a tendency to aggregate is usually
relieved by the action of a variety of molecular chaperones (3), and it
seems likely that specialized molecular chaperones exist in the
nucleolus to aid in ribosome assembly. In fact, recent proteomic
analyses of nucleoli identified 14 potential molecular chaperones in
HeLa cell nucleoli (4). These could be important in preventing
aggregation of proteins in the nucleolus.
To date, one nucleolar protein has been shown to have a general
molecular chaperone activity by several criteria. Szebeni and
Olson (5) tested a nucleolar nonribosomal protein called B23 (also
called NO38, nucleophosmin/NPM, or numatrin) for chaperone activity
using several protein substrates typically used to test proteins for
chaperone activity. Protein B23 was found to prevent aggregation of the
HIV-11 Rev protein and also
to suppress the aggregation of several other proteins during thermal
denaturation. In addition, B23 preserves enzyme activities under high
temperature conditions and actually promotes the renaturation and
restoration of activities of enzymes previously denatured with
guanidine-HCl. Finally, protein B23 preferentially binds denatured
substrates; this is accompanied by an increase in exposure of the
hydrophobic regions of the proteins in the B23-substrate complexes. A
histone chaperone activity has also been proposed for protein B23,
based on the observation that it binds to histones and mediates the
formation of nucleosomes (6).
Because protein B23 is predominantly localized in nucleolus, it is
believed to serve as a factor in ribosome biogenesis. Protein B23 binds
RNA and DNA (7) and interacts with other nucleolar proteins, including
nucleolin (8), protein P120 (9), and the HIV-1 Rev protein (10, 11). It
has intrinsic endoribonuclease activity (12), and its capacity to
preferentially cleave in the ITS2 region of pre-rRNA suggests that it
could participate in late stages of ribosome biogenesis (13). In
addition, protein B23 seems to have multiple functions outside of the
nucleolus. The ability of the protein to shuttle between the nucleus
and cytoplasm (14) and to bind nuclear localization signal
(NLS)-containing peptides (15) suggests a role in nuclear import. In
support of this latter notion, protein B23 has been shown to stimulate the import of NLS-containing proteins into isolated nuclei (16), and it
has been proposed that this ability is related to its molecular chaperone activity (5). The protein also stimulates the activity of DNA
polymerase Molecular chaperones are a diverse group of proteins that share the
common general function of modulating protein conformation (20). They
appear to operate through repeated cycles of binding partially unfolded
substrates, release of the bound substrate, and folding after release
until the correct conformation is achieved (21). Typically, substrate
release by chaperones is coupled to ATP hydrolysis (20). Like the
molecular chaperones in general, protein B23 preferentially binds
denatured substrates (5). However, protein B23 does not directly
hydrolyze ATP and, until now, a mechanism for release of substrate had
not been found.
Protein B23 has been shown to be phosphorylated in vitro
(22) and in vivo (23) by protein kinase CK2 (CK2).
Furthermore, the CK2 sites are located in a segment of the molecule
that is essential for chaperone activity (24). Therefore, we considered the possibility that phosphorylation of protein B23 by CK2 could promote the release of bound substrates. Here we report a possible mechanism for the dissociation of a substrate from protein B23. Phosphorylation of the B23-substrate complex by CK2 alters the interaction of B23 with denatured proteins and promotes their release
from the complex. In this mechanism, the release of the chaperone
substrate appears to be indirectly dependent on hydrolysis of ATP.
Because CK2 and protein phosphatases are present in the nucleus and
nucleolus, cycles of phosphorylation and dephosphorylation could
control the binding and release of substrate proteins in vivo. This mechanism has not previously been observed in other molecular chaperones.
Proteins and Peptides--
Recombinant rat proteins B23.1 and
B23.2 used in these studies were produced in Escherichia
coli and purified essentially as previously described (11) except
that the respective cDNAs were inserted into the pET 11c vector
(Novagen) for expression. Recombinant deletion mutants ( Assays and Other Procedures--
Protein B23 was labeled with
125I as previously described by Szebeni et al.
(16). Recombinant protein B23 or its mutants were phosphorylated
in vitro with CK2 from Roche Molecular Biochemicals or
recombinant CK2 Phosphopeptide Mapping--
Phosphorylated mixtures of B23 and
rhodanese were digested with 20 units of mouse submaxillary gland
endoproteinase Arg-C (Calbiochem) for 8 h at 37 °C in 1%
NaHCO3 (pH 8.0). Aliquots of the digestion products were
run on SDS-PAGE gels containing 20% polyacrylamide, and the
Coomassie-stained gels were subjected to phosphorimaging. The samples
were also digested for 24 h with 0.03 mg/ml tosylphenylalanyl
chloromethyl ketone-treated trypsin (Sigma) using the same buffer and
temperature conditions as above. The digested mixtures were subjected
to reverse phase HPLC using a C18 column (Vydac 218MS54) and a gradient
of 0-60% acetonitrile containing 0.1% trifluoroacetic acid.
Fractions (0.5 ml) were collected and subjected to scintillation counting.
Binding of Denatured Substrate Enhances the Rate of Phosphorylation
of Protein B23 by CK2--
The major phosphorylation site of rat
protein B23 by CK2 (serine 125) is located in the first highly acidic
segment of the protein (22). Another potential phosphorylation site
(threonine 185) is found in the second highly acidic segment. These
sites are part of the region essential for maximal chaperone activity (24) and their phosphorylation could be altered by the presence of
bound substrate. Therefore, we examined the effect of bound, heat-denatured proteins to protein B23 on its phosphorylation by
CK2.
Rhodanese, which was previously used as a substrate to test chaperone
activity, was incubated at an equimolar ratio with protein B23 for 20 min at 65 °C to form a complex. Incorporation of 32P
from [
Similar experiments were performed using two other proteins, citrate
synthase (heated at 50 °C) and the HIV-1 Rev protein (heated to
30 °C). The results were generally very similar to those obtained
with rhodanese, with 2-3-fold stimulation of the rate of
phosphorylation (Fig. 1, B and C).
To ensure that the enhanced rate of phosphorylation was not because of
incorporation of phosphate into nonspecific sites, phosphopeptide
mapping studies were done on the phosphorylated protein mixtures.
Protein B23 was phosphorylated with CK2 and [
Protein B23 has also been shown to be phosphorylated in the C-terminal
region by a cdc2-type protein kinase (23). To determine whether the
effect seen with CK2 in the presence of denatured proteins was specific
or was due to phosphorylation in general, protein B23 was also
incubated with a cdc2-type protein kinase. When the complex was
phosphorylated by the cdc2-type kinase there was no significant
increase in the rate of incorporation of radioactivity into protein B23
in the presence of any of the three denatured chaperone substrates
mentioned above (data not shown). Thus, the effect of complex formation
on the rate of phosphorylation is specific to the CK2 sites.
The N-terminal Region of Protein B23 Is Required for Stimulation of
CK2 Phosphorylation by Denatured Chaperone Substrates--
Protein B23
has several distinctive segments and functional domains in its primary
structure including a nonpolar N-terminal domain, a basic C-terminal
region, and two highly acidic segments in the middle. Previous studies
(24) showed that maximal molecular chaperone activity requires nonpolar
and acidic segments in the N-terminal half of the polypeptide chain
(see the schematic in Fig.
3A). To determine whether
specific segments are also important for stimulation of phosphorylation
of protein B23 by CK2, we tested this effect using the B23.2 isoform
and the
The CK2 phosphorylation experiments were performed on the B23.2 isoform
and on the deletion mutants in the presence and absence of the
denatured rhodanese substrate as above. In these experiments the rates
of phosphorylation of proteins B23.1, B23.2, the C-terminal deletion
mutant Phosphorylation of Protein B23 by CK2 Promotes Dissociation of the
B23-Substrate Complex--
One of the characteristics of protein B23
that is typical of molecular chaperones is its preference for binding
denatured substrates (5). The formation of the complex between
denatured substrate and protein B23 seems to be largely dependent on
hydrophobic interactions between the proteins. In this complex, the
substrate is seemingly irreversibly bound to protein B23. To determine
whether CK2 phosphorylation of protein B23 could facilitate
dissociation of the substrate, we examined the unmodified and
phosphorylated complexes by sedimentation analyses. Equimolar mixtures
of 125I-labeled control or CK2-phosphorylated protein B23
and rhodanese were heated and layered onto sucrose gradients and
subjected to ultracentrifugation. The gradients were fractionated, and
enzyme activity and radioactivity were measured in each fraction (Fig. 4). When rhodanese and B23 were mixed at
room temperature they sedimented as separate peaks (Fig.
4A), indicating a lack of interaction, as previously shown
(5). Heating the mixture at 60 °C caused the proteins to sediment
essentially as a single peak (Fig. 4B). However, when
phosphorylated protein B23 and rhodanese were heated together at
60 °C the two proteins again sedimented as separate peaks (Fig.
4C). Similar results were obtained with a CK2-phosphorylated B23-citrate synthase complex analyzed under similar conditions (Fig. 4,
D-F). In control experiments, mock phosphorylations of B23
were performed in the absence of CK2, ATP, or MgCl2 before heating with the substrate; under these conditions B23 and the substrate protein sedimented together (Fig. 4, G-I). This
indicates that the phosphorylation reaction was essential for
dissociation of the B23-substrate complex, which was not promoted
simply by the presence of CK2, ATP, or MgCl2.
To confirm phosphorylation at the CK2 site (Ser-125) experiments
similar to the ones above were performed using a mutant protein in
which Ser-125 was converted to alanine (S125A). In these experiments there was no dissociation of the B23-substrate complex after treatment with CK2 either with rhodanese (Fig.
5C) or with citrate synthase (Fig. 5F). This result strengthens the case that
phosphorylation of Ser-125 of protein B23 is essential for dissociation
of the bound substrate. Taken together, the results of this series of experiments indicate that CK2-phosphorylated protein B23 has greatly reduced affinity for the denatured substrates.
Because the presence of a bound substrate enhances B23 phosphorylation
and phosphorylated B23 has reduced affinity for the substrate, we
considered the possibility that phosphorylation of a preformed complex
could also promote its dissociation. To answer this question,
experiments were performed in which an equimolar mixture of
125I-labeled protein B23 and rhodanese was heated as above,
and the resulting complex was phosphorylated by CK2. Aliquots were
taken for sedimentation analysis at various times after initiation of the phosphorylation reaction (Fig. 6).
With each time point after the addition of CK2, there was an increase
in the level of phosphorylation and a corresponding increase in the
proportion of the two components dissociated from the complex. At the
5- and 15-min time points, ~0.1 and 0.7 mol of phosphate were
incorporated per mol of B23, respectively. About 9 and 65% of the
rhodanese was dissociated from the complex at the 5- and 15-min time
points, respectively. When the complex was phosphorylated for 60 min,
the molar ratio of phosphate to B23 was ~1.2, with greater than 95%
dissociation of the complex (data not shown). Thus, phosphorylation of
protein B23 by CK2 not only inhibits its ability to form a complex with a denatured substrate, but it also is able to promote the
dissociation of a preformed complex in a manner that is roughly
proportional to the level of phosphorylation.
Studies using B23 mutants (Fig. 3) indicated that the nonpolar
N-terminal end of the protein is required for chaperone activity and
suggested that this region binds denatured protein substrates (24). To
determine whether this segment is required for substrate binding, we
performed the sedimentation analyses with the Additional Requirements for Dissociation of the HIV-1 Rev-B23
Complex--
Earlier experiments (10, 11) showed that there is a
strong affinity between nonphosphorylated protein B23 and the HIV Rev
protein and that protein B23 also acts as a molecular chaperone toward
Rev (5). To determine the effect of CK2 phosphorylation of protein B23
on its Rev binding properties we performed experiments similar to the
ones above using phosphorylated and nonphosphorylated protein B23.
Sedimentation analyses were performed with equimolar mixtures of Rev
and protein B23 where Rev was labeled with 125I, and the
concentrations of protein B23 were determined by an enzyme-linked
immunosorbent assay using a monoclonal antibody to protein B23. The
experiments showed that protein B23 binds the Rev protein both at
4 °C and at 30 °C (Fig. 8,
B and C), indicating that the complex can be
formed without heating. Furthermore, phosphorylation of protein B23 was
not capable of dissociating the complex formed at either temperature
(Fig. 8, D and E). This observation suggests that
protein B23 has an additional binding site for the Rev protein. One of
them could be a general binding site for denatured proteins as above
and the other could be a site that interacts more or less specifically
with the Rev protein. To test this hypothesis, additional experiments
were performed in which a synthetic peptide based on the NLS of the Rev
protein (Rev37-47; ARRNRRRRWREYC) was used as a competitor. It was
reported earlier that the Rev37-47 sequence is necessary for nucleolar
localization of the Rev protein (27), and protein B23 binds the
Rev37-47 NLS peptide with high affinity (15). In another series of
experiments, equimolar amounts of the Rev protein were incubated at 4 and 30 °C with phosphorylated samples of protein B23 and
nonphosphorylated controls. All of the incubation mixtures included a
10-fold molar excess of the Rev37-47 peptide. Fig. 6F shows
that the Rev37-47 peptide was able to prevent formation of the Rev-B23
complex at 4 °C. However, competition with the Rev peptide was not
able to prevent formation of the complex at 30 °C (Fig.
8G). When phosphorylated protein B23 was incubated with Rev
in the presence of the Rev37-47 peptide either at 4 or at 30 °C the
two proteins sedimented as separate peaks (Fig. 8, H and
I). This indicated that both phosphorylation of protein B23
and the presence of the competing peptide were required to dissociate
the complex formed at 30 °C. To determine whether the competition
was due to a specific sequence and not simply caused by the charge of
the peptide, another peptide with a sequence that was the reverse of
that of the Rev NLS peptide was used in a similar experiment. In this
case, the reverse NLS peptide was not capable of dissociating the
phosphorylated complex (Fig. 8J), indicating that the
interaction has a relatively high degree of specificity. Thus, complete
dissociation of the Rev-B23 complex requires phosphorylation of protein
B23 as well as competing peptide. This supports the idea that there are
two sites of interaction between Rev and protein B23, with two
different modes of binding. One mode is through a generic, nonpolar
substrate-binding site whose affinity is controlled by phosphorylation,
and the other operates via a negatively charged site that interacts
with the basic Rev NLS.
Protein B23 is a multifunctional protein that has many
characteristics typically exhibited by molecular chaperones using
in vitro assays (5). The current studies not only identify
additional chaperone-like properties, but they also suggest that the
protein B23-substrate interactions are regulated by a novel mechanism. First, protein B23 seems to detect the binding of a denatured substrate
and respond by making sites for CK2 phosphorylation more accessible.
Second, phosphorylation of B23 by CK2 promotes the dissociation of
B23-substrate complexes. Finally, there seem to be two sites for
binding at least one substrate, the HIV-1 Rev protein; that is, one
site that recognizes nonpolar regions and another that interacts with
basic segments of proteins. The former is regulated by phosphorylation,
but what controls affinity for the latter has not been determined.
A key feature of molecular chaperones is that they are able to detect
bound substrates and regulate their release through allosteric
interactions. For example, in Hsp70 (28) and the Hsp100-Clp family
(29), the binding of unfolded peptide increases the rate of ATP
hydrolysis. In the case of the GroE molecular chaperones (3, 30),
binding of a compact folding intermediate triggers a complex series of
events including ATP binding, attachment of GroES, hydrolysis of the
ATP, and eventual release of the substrate. With protein B23, the
binding of substrate does not affect ATP hydrolysis directly, but it
appears to enhance the accessibility of the CK2 phosphorylation sites
of protein B23. The stimulation of CK2 phosphorylation by substrate
binding has at least one other precedent in the nucleosome assembly
factors NAP-1 and NAP-2; the addition of histones enhances CK2
phosphorylation of these proteins (31). In protein B23, the increased
rate of phosphorylation is dependent on the presence of the nonpolar
region in the N-terminal end, since the effect is not seen when that
segment is deleted. Thus, the nonpolar region seems to communicate with
the adjacent highly acidic segment, which contains the major CK2
phosphorylation site.
As indicated above, in most molecular chaperones the release of
substrate is coupled with ATP hydrolysis, which is directly carried out
by ATPase activity in the chaperone itself. Although protein B23 is
capable of binding ATP (32), extensive studies in our laboratory have
never detected any significant ATPase activity in the protein itself
under any conditions.2 Thus,
it is unlikely that the affinity of B23 for a substrate is regulated by
direct nucleoside triphosphate hydrolysis. This observation prompted us
to search for an alternative mechanism for dissociation of the complex.
Because protein B23 is phosphorylated by CK2 (22, 23) and has been
shown to be an interacting partner of the enzyme (33), it seemed
reasonable to examine the possible effect of phosphorylation by this
protein kinase on chaperone substrate binding. It was found that
phosphorylation of protein B23 with CK2 before the addition of
denatured substrate inhibits the formation of a stable complex.
In the sedimentation assay, the interaction between CK2-phosphorylated
B23 and the heat-denatured substrate protein was not detectable.
This is a curious result since phosphorylated B23 maintained its
ability to prevent thermal aggregation of the substrate and protect the
enzyme from inactivation under elevated temperature conditions despite
the undetectable interaction with the substrate. Because the chaperone
properties would have to involve some kind of contact between
phosphorylated B23 and the substrate, it is likely that the affinity
between the two components is reduced but is sufficient to mediate the
chaperone activity. However, because of the length of time required,
the sedimentation assay is probably not capable of identifying low
affinity interactions. Thus, in the phosphorylated protein, the
chaperone activities may be mediated through transient interactions
between protein B23 and the substrate; i.e. B23 might
rapidly and repeatedly associate with and dissociate from the
substrate, and this could be sufficient to sustain the chaperone activity.
The more important observation is that phosphorylation of protein B23
in a pre-formed B23-substrate complex can promote the dissociation of
the complex. This could be physiologically relevant since it was also
shown that complex formation enhances the rate of phosphorylation.
Thus, the bound form of protein B23 would tend to be preferentially
targeted for phosphorylation compared with the unbound form; this could
be the basis of a mechanism for regulating the cycle of binding and release.
Although the proposed release mechanism seems applicable to many
substrates, there are added considerations for the B23 interaction with
the HIV-1 Rev protein. The Rev-B23 complex is not dissociable by CK2
phosphorylation alone. This complex seems to be held together by a
combination of nonpolar and electrostatic interactions, and the latter
set of interactions are not responsive to phosphorylation. Complete
dissociation requires a combination of phosphorylation and competition
with a peptide with the sequence of the Rev NLS. It is possible that
specificity of the B23 chaperone resides in these electrostatic
interactions. However, this raises the question of how the dissociation
of these sites is achieved. Whether additional co-chaperones are
required or whether competition by other proteins could displace the
bound protein remain unanswered.
How might this system operate in living organisms? Because of the
abundance of protein B23 and the high concentration of proteins in the
nucleolus, one could imagine that it would interact with a variety of
nucleolar proteins, either ribosomal or nonribosomal proteins or both.
To remove a partially unfolded protein from an aggregation pathway,
protein B23 might recognize and bind to exposed surfaces on the protein
(Fig. 9). The substrate protein would
remain bound to B23 until it is phosphorylated by CK2. The latter
enzyme is abundant in the nucleolus (34) and migrates to the nucleolus
under heat shock conditions (35). Furthermore, CK2 is up-regulated
during cell growth (36), in which nucleolar activity and protein
concentration are also elevated. The latter conditions would increase
the probability for proteins to be partially unfolded and to be bound
by protein B23. The binding event could allow time for the substrate
protein to properly fold either while associated with protein B23 or
after its release. CK2 might then preferentially phosphorylate protein
B23-substrate complexes to promote their dissociation. Once the
substrate is released, protein B23 could then be dephosphorylated by
phosphoprotein phosphatases, which are also present in the nucleolus
(4, 37). The latter event would restore the potential for B23 to bind a
partially unfolded substrate. This cycle could be repeated over and
over to facilitate the ribosome assembly process. Whether protein B23 is a general chaperone as suggested by the current studies using generic chaperone substrates or whether it recognizes specific substrates in the nucleolus is currently under investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(17), and it may serve a factor in DNA recombination through its capacity to promote annealing of complementary strands of
DNA (18). Finally, protein B23 seems to control the duplication of
centrosomes in mammalian cells (19). Taken together, these observations
have led researchers to conclude that B23 is a multifunctional protein
with roles both in and outside of the nucleolus.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C35,
N35,
N90, and
N119) were expressed using the Qiagen pQE-30 vector and
purified as described by Hingorani et al. (24). The mutant
protein B23, where the CK2 phosphorylation site was changed to alanine
(S125A), was prepared using the SOEing technique as previously
described (15) and expressed in the pQE-31 vector as above. The
cDNA for the HIV-1 Rev protein was cloned into the pET11c vector
(Novagen), and the recombinant protein was expressed in E. coli, and it was purified from bacterial extracts essentially as
reported by Karn et al. (25). The purified protein was
refolded as described by Wingfield et al. (26). Peptides
based on the NLS sequence of the HIV-1 Rev protein were synthesized by
Coast Scientific: NLS (residues 37-47); rNLS (residues 37-47, reverse
order). The monoclonal antibody to protein B23 was a gift from Dr.
P. K. Chan of Baylor College of Medicine.
subunit, generously provided by Dr. Khalil Ahmed
(Veterans Affairs Medical Center, Minneapolis MN); there were no
significant differences observed between the two enzyme preparations.
For CK2 phosphorylation samples of protein B23 or mutants (0.3 mg/ml)
were incubated in a reaction mixture containing ~1 milliunits/ml CK2,
20 mM MES buffer (pH 7.2), 130 mM KCl, 0.4 mM MgCl2, 48 mM dithiothreitol, 100 µM ATP in a total volume of 1 ml at 30 or 37 °C. The
enzyme reaction was linear for at least 30 min. The number of
phosphoryl groups incorporated into the protein was calculated from the
radioisotope incorporation, the specific activity of the
[
-32P]ATP, and the amount of protein as determined by
the Bio-Rad protein assay (15). For the preparations of B23 used for
the studies examining the effect of phosphorylation on B23-substrate complex formation, the incubations were carried out for 60 min; under
these conditions there was incorporation of an average of ~1.2
phosphoryl groups per molecule (15). Protein B23 was phosphorylated by
a cdc2-type kinase (Upstate Biotechnology) as described by Szebeni
et al. (15). Enzyme activities of citrate synthase and rhodanese were measured as indicated in a previous study (5). Sucrose
density gradient centrifugation of protein B23-substrate complexes was
performed according to a previously published procedure (5).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP into the complex catalyzed by CK2 was
monitored in the linear phase of the reaction (Fig.
1A). The presence of denatured
rhodanese in the complex accelerated the rate of incorporation
~2-fold compared with the heated control without rhodanese. Because
heat treatment of protein B23 alone did not enhance the rate of
incorporation of 32P, the binding of the substrate must be
responsible for the increased rate of phosphorylation. In control
samples, in which MgCl2 was omitted from the otherwise
complete reaction mixture, there was no significant incorporation of
32P in either the unheated or heated sample. This indicated
that nonspecific adsorption of labeled ATP was not responsible for the
enhanced rate of phosphorylation.
View larger version (13K):
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Fig. 1.
Denatured chaperone substrate enhances the
rate of phosphorylation of protein B23 by protein kinase CK2. The
substrates were preincubated at 1:1 molar ratios with protein B23 for
20 min at the elevated temperatures indicated. A, rhodanese
(65 °C). B, citrate synthase (50 °C). C,
HIV-1 Rev protein (30 °C). The resulting complexes were then
incubated with CK2 and [ -32P]ATP for 12 min, and
incorporation of 32P was monitored by scintillation
counting. The temperatures for the CK2 reactions were 37 °C for
rhodanese and citrate synthase and 30 °C for HIV-1 Rev. Each curve
represents an average of the results from three separate experiments
with the following pretreatment conditions before incubation of the
samples with CK2.
, B23 untreated;
, B23 plus untreated
substrate;
, B23 treated at elevated temperature;
, B23 plus
substrate treated at elevated temperature;
, B23 untreated with no
MgCl2 in the reaction mixture;
, B23 plus substrate
treated at elevated temperature with no MgCl2 in the
reaction mixture.
-32P]ATP
for 60 min in the presence of rhodanese either before or after heating
the mixture at 65 °C. Under the conditions of phosphorylation, CK2
alone did not incorporate significant label (Fig.
2A, lane 1) due to
autophosphorylation. The labeling patterns of the heated and unheated,
undigested B23-rhodanese mixtures treated with CK2 were virtually
identical, with incorporation predominantly into the protein B23 band,
with traces of label in degradation products or minor contaminants
(Fig. 2A, lanes 2 and 4). There was no
significant incorporation into rhodanese (location indicated), and
there were no differences in labeling patterns between heated and
unheated samples. When the samples were subjected to digestion with
endoproteinase Arg-C only one major band was obtained in either case
(lanes 3 and 5). The molecular mass of the
phosphopeptide cleavage product of endoproteinase Arg-C was estimated
to be ~5 kDa; this fits with the molecular mass of the fragment
between residues 102 and 142. Because this segment contains only one
serine residue, the presence of this cleavage product supports the
notion that only one serine residue is phosphorylated by CK2. To
confirm that there were no additional sites labeled when the
heat-treated B23-substrate complex was phosphorylated with CK2, similar
experiments were done with tryptic digestion. In this case, the reverse
phase HPLC elution profiles of the untreated and heat-treated samples
were virtually identical, with only one peak of radioactivity (Fig. 2B). These studies indicate that the presence of the
denatured substrate did not expose additional sites to nonspecific
phosphorylation by CK2, and only serine 125 is phosphorylated by CK2
under these conditions (see Ref. 22).
View larger version (42K):
[in a new window]
Fig. 2.
Denatured chaperone substrate does not alter
the sites phosphorylated in protein B23 by protein kinase CK2.
Protein B23 was phosphorylated with CK2 and [ -32P]ATP
for 60 min in the presence of rhodanese either before or after heating
the mixture at 65 °C. The mixtures were then digested with either
endoproteinase Arg-C or trypsin as described under "Experimental
Procedures." A, the Arg-C digests and controls were run on
20% polyacrylamide SDS gels, which were subsequently subjected to
phosphorimaging. Lane 1, reaction run with CK2 alone (not
heated and not digested); lanes 2 and 3, B23 and
rhodanese not heated before CK2 treatment; lanes 4 and
5, B23/rhodanese mixture heated before CK2 treatment;
lanes 2 and 4, undigested samples; lanes
3 and 5, digested samples. The bars on the
right indicate the positions of molecular mass standards:
from top to bottom, 209, 124, 80, 49.1, 34.8, 28.9, 20.6, and 7.1 kDa. The positions of protein B23 (B23),
rhodanese (Rh), and the major phosphopeptide (P)
are indicated on the right. B, the tryptic digests were
separated by reverse phase HPLC, and radioactivity in the fractions was
determined by scintillation counting. Solid line, B23 and
rhodanese not heated before CK2 treatment; dashed line, B23
and rhodanese heated before CK2 treatment.
C35,
N35,
N90,
N119 deletion mutants (Fig.
3A).
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Fig. 3.
The N-terminal region of protein B23 is
required for stimulation of CK2 phosphorylation by denatured chaperone
substrates. A, schematic representation of rat protein
B23.1 and N- and C-terminal deletion mutants. Light
gray, nonpolar region; black, acidic regions, the first
of which contains the major CK2 phosphorylation site; dark
gray, short segments containing cdc2-type phosphorylation sites;
gray with horizontal lines, C-terminal segment
unique to B23.1. B, the CK2 phosphorylation experiments as
described in Fig. 1A were performed using the B23.1 and
B23.2 isoforms and the C35,
N35,
N90, and
N119 deletion
mutants in the presence and absence of the denatured rhodanese
chaperone substrate. The incorporation of 32P is expressed
as % of control (B23.1 phosphorylated in the absence of denatured
substrate) after 12 min of incubation with CK2. Data are shown as
means ± S.E. (error bars) of three independent
experiments.
C35, or the N-terminal deletion mutant
N35 were enhanced
almost 3-fold by the presence of denatured rhodanese, with no
significant differences among the four forms of the protein (Fig.
3B). In contrast, rates of labeling of the
N90 and
N119 deletion mutants by CK2 in the presence of the denatured
substrate were similar to that of the control, i.e. there
was no enhancement of the rate of phosphorylation. Because the nonpolar
region in the N-terminal half of protein B23 is absent in the latter
two mutants, these experiments confirm that this segment is essential not only for optimal chaperone activity but also for the
substrate-induced enhancement of CK2 phosphorylation of the protein.
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Fig. 4.
Effect of phosphorylation of protein B23 on
formation of the B23-substrate complex. Protein B23 was labeled
with 125I and phosphorylated with CK2. The phosphorylated
and nonphosphorylated B23 proteins (250 µg/ml) were incubated with
equimolar concentrations of rhodanese or citrate synthase either at
room temperature or at 60 °C for 30 min in 50 mM
Tris-HCl buffer (pH 7.8). After incubation the mixtures were applied to
5-30% linear sucrose gradients and centrifuged in a Beckman SW60
rotor for 17 h at 35,000 rpm. Fractions (0.35 ml) were collected,
and the radioactivity of protein B23 and the enzyme activity of
rhodanese or citrate synthase were measured. A, B23 and
rhodanese incubated at room temperature. B, B23 and
rhodanese incubated at 60 °C. C, phosphorylated B23 and
rhodanese incubated at 60 °C. D, B23 and citrate synthase
incubated at room temperature. E, B23 and citrate synthase
incubated at 60 °C. F, phosphorylated B23 and citrate
synthase incubated at 60 °C. G, B23 and rhodanese
incubation conditions were the same as in panel C except
that CK2 was omitted. H, incubation as in panel C
done in the absence of ATP. I, incubation as in panel
C was done in the absence of MgCl2. , enzyme
activity of substrate protein;
, B23 relative concentrations
determined by 125I radioactivity.
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Fig. 5.
Mutation of serine 125 of protein B23
abolishes the ability of CK2 phosphorylation to prevent formation of
the B23-denatured substrate complex. The S125A mutant of protein
B23 was labeled with 125I and treated with CK2 as in Fig.
4. The treated and untreated mutant B23 proteins (250 µg/ml) were
incubated with equimolar concentrations of rhodanese or citrate
synthase either at room temperature or at 60 °C for 30 min in 50 mM Tris-HCl buffer (pH 7.8). The samples were subjected to
sucrose gradient ultracentrifugation analyses as in Fig. 4.
A, B23 mutant S125A and rhodanese incubated at room
temperature. B, S125A and rhodanese incubated at 60 °C.
C, CK2-treated S125A and rhodanese incubated at 60 °C.
D, S125A and citrate synthase incubated at room temperature.
E, S125A and citrate synthase incubated at 60 °C.
F, CK2 treated S125A and citrate synthase incubated at
60 °C. , enzyme activity of substrate protein;
, B23 relative
concentrations determined by 125I radioactivity.
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Fig. 6.
Effect of phosphorylation of protein B23 on
the denatured rhodanese-B23 complex. Protein B23 was labeled with
125I and incubated with an equimolar concentration of
rhodanese either at room temperature or at 60 °C for 30 min in 50 mM Tris-HCl buffer (pH 7.8). The samples treated at the
elevated temperature were incubated with CK2 at 37 °C for the times
indicated, layered onto 5-30% linear sucrose gradients, and
centrifuged in Beckman SW41 rotor for 18 h at 38,000 rpm.
Fractions (0.5 ml) were collected, and the radioactivity of protein B23
and the enzyme activity of rhodanese were measured. A,
rhodanese and B23 mixed at room temperature. B, heat-treated
rhodanese and B23. C, heat-treated rhodanese and B23 applied
to gradient 5 min after the addition of CK2. D, heat-treated
rhodanese and B23 applied to gradient 15 min after the addition of CK2.
The data are expressed as arbitrary units of either cpm or enzyme
activity. Solid line, rhodanese activity; dotted
line, B23 radioactivity.
N35,
N90, and
C35 deletion mutants. These experiments showed that removal of a
short segment from the N-terminal end of the protein (
N35) had no
effect on the binding of denatured substrate (Fig. 7B). As with the wild type
protein, phosphorylation by CK2 promoted dissociation of the complex
(Fig. 7C). Essentially the same set of results was obtained
with the
C35 deletion mutant (data not shown). In contrast, the
N90 deletion mutant was unable to bind denatured rhodanese, which
aggregated and lost its enzyme activity during thermal denaturation
(Fig. 7E). As expected, the same result was seen with
phosphorylated
N90 (Fig. 7F). This observation confirms
that the N-terminal region protein B23 is essential for substrate
binding as well as for the phosphorylation enhancement effect.
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Fig. 7.
Effect of phosphorylation of protein B23
deletion mutants on B23-substrate complex formation. Protein B23
deletion mutants N35 (A-C) and
N90 (D-F)
were labeled with 125I and phosphorylated with CK2. The
phosphorylated and nonphosphorylated protein samples were incubated at
60 °C with equimolar concentrations of rhodanese and subjected to
linear sucrose gradients as described in Fig. 4. For sedimentation
analysis 0.65-ml fractions were collected. A and
D, rhodanese and the B23 mutant mixed at room temperature.
B and E, heat-treated rhodanese and B23 mutants.
C and F, heat-treated mixture of phosphorylated
B23 mutants and rhodanese.
, rhodanese activity;
, B23
radioactivity.
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Fig. 8.
Sedimentation analyses of phosphorylated
protein B23-Rev protein complex. An equimolar mixture of
CK2-phosphorylated or nonphosphorylated protein B23 and
125I-labeled Rev protein were incubated (185 µg/ml) for
60 min at 4 or 30 °C in 10 mM sodium phosphate (pH 7.4),
140 mM NaCl, 200 mM KCl, and 0.05% (w/v)
digitonin, applied to 5-30% linear sucrose gradients, and centrifuged
in Beckman SW60 rotor for 18 h at 35,000 rpm. Fractions (0.35 ml)
were collected, and the radioactivity of Rev protein was measured.
Protein B23 concentrations were determined by an enzyme-linked
immunosorbent assay using a monoclonal antibody. A, protein
B23 and Rev protein subjected to sedimentation in two separate tubes.
B, B23 and Rev incubated at 4 °C. C, B23 and
Rev incubated at 30 °C. D, phosphorylated protein B23 and
Rev incubated at 4 °C. E, phosphorylated protein B23 and
Rev incubated at 30 °C. F, protein B23 and Rev incubated
at 4 °C in the presence of the NLS peptide. G, protein
B23 and Rev incubated at 30 °C in the presence of the NLS peptide.
H, phosphorylated protein B23 and Rev incubated at 4 °C
in the presence of the NLS peptide. I, phosphorylated
protein B23 and Rev incubated at 30 °C in the presence of the NLS
peptide. J, phosphorylated protein B23 and Rev incubated at
30 °C in the presence of the reverse NLS peptide. , Rev
radioactivity;
, relative B23 concentration determined by
enzyme-linked immunosorbent assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
A model for the plausible involvement of
phosphorylation of protein B23 in its molecular chaperone
activity. Under the macromolecular crowding conditions of the
nucleolus proteins would have a tendency to form aggregates when
partially unfolded. Protein B23 could recognize and bind to an exposed
surface of the protein and temporarily remove it from the aggregation
pathway. Refolding of the substrate protein could be accomplished
either in the complex or after its release. Phosphorylation of the
complex by casein kinase II (abundant in the nucleolus) would reduce
the affinity of B23 for a substrate and promote dissociation of the
complex. After release of substrate, a phosphoprotein phosphatase
(PPase) could dephosphorylate protein B23. The latter step
would restore the affinity of B23 for partially unfolded substrates and
allow it to participate in additional cycles of binding and release of
substrates.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. P. K. Chan for providing the anti-protein B23 monoclonal antibody and Dr. Khalil Ahmed for supplying recombinant CK2. We gratefully acknowledge Michael Wallace for protein chemistry expertise, Szilvia Szebeni for preparing protein B23 and the HIV-1 Rev protein, and Dr. Mona Norcum for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health, by the Medical Guardian Society of the University of Mississippi, and by funds from the National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR) Protein Structure and Localization Group Project.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, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216. Tel.: 601-984-1500; E-mail: molson@biochem.umsmed.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M204411200
2 A. Szebeni and M. O. J. Olson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; CK2, protein kinase CK2 (also known as casein kinase II); NLS, nuclear localization signal; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography.
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