(Received for publication, October 16, 1996)
From the Department of Pharmacology, University of
Washington, Seattle, Washington 98195 and the § Department
of Anatomy and Structural Biology, Albert Einstein College of Medicine,
Bronx, New York 10461
Casein kinase 2 (CK2) is a multifunctional second
messenger-independent protein serine/threonine kinase that
phosphorylates many different proteins. To understand the function and
regulation of this enzyme, biochemical methods were used to search for
CK2-interacting proteins. Using immobilized glutathione
S-transferase fusion proteins of CK2, the nucleolar protein
Nopp140 was identified as a CK2-associated protein. It was found that
Nopp140 binds primarily to the CK2 regulatory subunit, . The
possible in vivo association of Nopp140 with CK2 was also
suggested from a coimmunoprecipitation experiment in which Nopp140 was
detected in immunoprecipitates of CK2 prepared from cell extracts.
Further studies using an overlay technique with radiolabeled CK2 as a
probe revealed a direct CK2-Nopp140 interaction. Using deletion mutants
of CK2
subunits, the binding region of the CK2
subunit to Nopp140
has been mapped. It was found that the NH2-terminal 20 amino acids of CK2
are involved. Since Nopp140 has been identified
as a nuclear localization sequence-binding protein and has been shown
to shuttle between the cytoplasm and the nucleus, the finding of a
CK2-Nopp140 interaction could shed light on our understanding of the
function and regulation of CK2 and Nopp140.
Protein phosphorylation is known to be a very important means of cellular regulation, and in recent years much information about protein kinases and phosphatases, especially those involved in the mitogen-activated protein kinase pathway, has been obtained (for review, see Refs. 1-3). Little is known, however, about the function and regulation of one particular protein kinase, casein kinase 2 (CK2),1 although increasing data suggest that it may be an important mediator in mitogenic signal transduction (for review, see Ref. 4).
CK2 is a multifunctional, second messenger-independent eukaryotic
protein serine/threonine kinase present in the nucleus and cytoplasm of
all eukaryotic cells. The holoenzyme form is generally composed of
catalytic subunits ,
, and a regulatory subunit
combined so
as to form
2
2,
2,
or
2
2 heterotetramers. The
and
subunits are catalytically active, whereas the
subunit is inactive
but can stimulate the catalytic activity of
and
under certain
circumstances. Furthermore, the
subunit stabilizes the
and
subunits and can facilitate substrate recognition (4). CK2 is widely
expressed, and its sequence is highly conserved throughout evolution,
indicating that the enzyme may have a critical role in cell function.
In fact, it has been shown in Saccharomyces cerevisiae that
the simultaneous disruption of genes encoding CK2
and
subunits
is lethal (5). CK2 phosphorylates a large number of proteins including
enzymes involved in nucleic acid synthesis, transcription and protein synthesis factors, structural proteins, and signal transduction proteins, suggesting a global role in the regulation of cellular processes (6). Several recent studies demonstrate that CK2 is important
for cell growth and division. First, it has been shown that CK2
activity is required for progression of the cell cycle (7, 8) and that
enzyme levels are elevated in many rapidly proliferating cells and
tumor cells (4). Second, CK2 reportedly associates with several
growth-related proteins including p53 (9), c-Raf (10), and
c-Mos.2 Thus it may modulate cellular
responses to growth factor stimulation.2 Most strikingly,
overexpression of CK2
in transgenic mice caused a high
predisposition for lymphoma development, and coexpression of the CK2
transgene with c-myc resulted in the rapid development of
murine perinatal leukemia associated with disruption of lymphoid cell
functions (12).
In spite of the above mentioned studies, it is still not entirely clear exactly what physiological roles CK2 may have and how the activity of this enzyme is regulated in vivo. CK2 has a vast number of the potential substrates. It is generally considered constitutively active (13) and is not known to respond to any second messenger molecules. These features makes it difficult to study the function and regulation of this enzyme (for review, see Refs. 4, 6, 14, and 15). Therefore, efforts have been made to search for proteins that interact with CK2 for a greater understanding of its role. For example, the nucleolar protein nucleolin was identified as a CK2-associating protein in previous work (16-18).
In this report, we provide evidence that CK2 and the nucleolar protein
Nopp140 associate as a molecular complex in vitro and probably in vivo. These studies were performed using
immobilized GST fusion proteins of CK2 subunits and a
32P-radiolabeled CK2 overlay technique and by
coimmunoprecipitation of the two proteins from cell extracts. The
region of the subunit of CK2 which binds to Nopp140 was also
mapped. The possible roles of CK2 in the regulation of Nopp140 as well
as in rRNA synthesis and ribosomal protein transport are supported by
the results of this study.
Nucleotide oligomers used as polymerase chain
reaction primers were synthesized by Drs. Y. F. Lee and P. S. H.
Chou (Biopolymer Facility, Department of Immunology, University of
Washington) and by Integrated DNA Technologies, Inc. BL-21 (DE3) pLysS
competent cells were purchased from Novogen. [-32P]ATP
was obtained from Amersham. The PD-10 gel filtration column and
glutathione-Sepharose 4B beads were purchased from Pharmacia Biotech
Inc. All other chemical reagents were purchased from Sigma.
Recombinant CK2 subunits and
and holoenzymes
2
2 and
2
2 were expressed and purified from
baculovirus-infected Sf-9 cells as described
elsewhere.3 Recombinant Nopp140 was
expressed in Escherichia coli BL-21 (DE3) cells transformed
with pET8c/Nopp140 (Nopp140 bacterial expression vector). Since the
overexpressed Nopp140 protein was predominantly insoluble and
segregated into inclusion bodies, it was denatured and renatured by
solubilization in 6 M urea followed by extensive dilution
and then purified using a hydroxylapatite column (19).
Polyclonal antibodies of Nopp140 were raised in
rabbits against a synthetic peptide of Nopp140 (20). Polyclonal
antibodies against CK2 subunits ,
, and
were prepared in
this laboratory (21).
3T3 L1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum until confluence. The cells were washed by phosphate-buffered saline, harvested in lysis buffer (50 mM Tris-Cl, pH 7.5, 50 mM NaF, 0.25 M NaCl, 0.1% Triton X-100, 0.25 M sucrose, 2 mM EDTA, 10 µg/ml leupeptin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and sonicated twice for 10 s. After centrifugation for 30 min at 13,000 rpm, the supernatants were used as cell lysates.
Construction of CK2 Deletion MutantsDeletion mutants of
GST-CK2 were constructed by truncating the NH2-terminal
(
1-20,
1-40,
1-80,
1-120,
1-141) and COOH-terminal (
161-215) amino acids of CK2
to give mutants
GST-
21-215, GST-
41-215,
GST-
81-215, GST-
121-215,
GST-
142-215, and GST-
1-160. The
cDNA for each deletion mutant was obtained by polymerase chain
reaction amplification of human CK2
in Bluescript KS plasmid (22).
For NH2-terminal deletion mutations, the sense polymerase
chain reaction primers were: 5
-GCGGATCCTTCTTCTGTGAAGTGGATG-3
(GST-
21-215); 5
-GCGGATCCGAGCAGGTCCCTCACTATC-3
(GST-
41-215); 5
-GCGGATCCGGATTGATCCACGCCCGCT-3
(GST-
81-215); 5
-GCGGATCCCCCATTGGCCTTTCAGACA-3
(GST-
121-215); and 5
-GCGGATCCGATGTGTACACACCCAAGT-3
(GST-
142-215). The T7 24-mer primer was used as
the antisense primers for all of the NH2-terminal deletion
mutants of CK2
. For the COOH-terminal deletion mutant,
GST-
1-160, the sense primer was
5
-GCGGATCCAGCAGCTCAGAGGAGGTGT-3
, and the antisense primer was
5
-GCGGATCCTCAGCCGAAGTAGGCGCCATCC-3
. After polymerase chain reaction,
the DNA fragments were purified, digested with BamHI, and
ligated into pGEX-2T vector (Pharmacia).
GST-CK2, GST-CK2
, and GST-CK2
and its deletion
mutants were expressed in and purified from E. coli and
immobilized on glutathione-Sepharose resin (18). The immobilized
protein were eluted using an elution buffer containing 10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0. To remove the GST tag from GST-CK2
, a thrombin cleavage
procedure developed by Pharmacia was employed. GST fusion proteins of
CK2 holoenzyme
2
2 and
2
2 were generated by mixing an equal
amount of the immobilized GST-CK2
with CK2
, or GST-CK2
with
CK2
.
3T3 L1 cell lysates (200 µl) were incubated at 4 °C either with 35 µl (50%; v/v) of glutathione beads immobilized with GST-CK2 fusion proteins (approximately 2 µg) for 4 h or with protein A-Sepharose conjugated with a mixture of antiserum against each CK2 subunit (18) for 2 h. The beads were then washed four times with an immunoprecipitation buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 mM dithiothreitol, 0.025% Triton X-100) by centrifugation. Bound proteins were extracted using Laemmli sample buffer and analyzed by SDS-PAGE, followed by immunoblotting with antibodies. Alternatively, the proteins bound to the immobilized CK2 were phosphorylated by GST-CK2 fusion proteins using a procedure described previously (18).
Immunoprecipitation of Nopp140Approximately 10,000,000 BRL cells were washed twice and scraped into ice-cold phosphate-buffered saline. After pelleting, the cells were lysed in 0.5 ml of lysis buffer (50 mM Tris, pH 7.4; 1% Triton X-100; 0.2% SDS; 150 mM NaCl; 2 mM phenylmethylsulfonyl fluoride; and 1 µg/ml each leupeptin, antipain, chymostatin, and pepstatin A) by tip sonication (five times for 30 s on ice). The cell lysate was clarified for 10 min in a microcentrifuge and incubated with 5 µg of anti-Nopp140 peptide IgGs in the absence and presence of 5 µM free competing peptide for 1 h at room temperature. The antibody-antigen complexes were adsorbed to 5 µl of packed protein A-Sepharose beads (Pharmacia) for an additional 1-h incubation at room temperature. The beads were washed four times with 1 ml of wash buffer (50 mM Tris, pH 7.4, 0.1% Triton X-100, 0.02% SDS, 150 mM NaCl), and the antibody-antigen complexes were eluted with Laemmli sample buffer and analyzed by SDS-PAGE (19).
32P-Labeled CK2 Overlay MethodRecombinant CK2
holoenzyme 2
2 (obtained from Sf-9 cells)
was radiolabeled by autophosphorylation and then subjected to a PD-10
column (Pharmacia) to remove [
-32P]ATP and other
inorganic chemicals (18). Bacterially expressed pure Nopp140 was
subjected to 8% SDS-PAGE and transferred onto a polyvinylidene
difluoride membrane. The membrane was preincubated in Blotto blocking
buffer (5% milk in 20 mM phosphate, pH 7.4, 0.15 mM NaCl) overnight and then overlaid with
32P-labeled CK2 probe (in fresh Blotto) for 4 h at
room temperature. After extensive washing, the associated CK2 band was
detected by autoradiography. Bovine serum albumin (1-2 µg) was also
loaded on the same SDS-polyacrylamide gel as a negative control.
Each of the recombinant CK2s:
CK2, CK2
,
2
2, and
2
2 (30-120 ng), was incubated at
30 °C with 0.5 µg of recombinant Nopp140 in 25 µl of
phosphorylation buffer (20 mM Tris-Cl, pH 7.5, 20 mM MgCl2, and 0.1 mM
[
-32P]ATP (2,000 cpm/pmol ATP)). After 30 min, the
reaction was stopped by adding 8 µl of 4 × sample buffer, and
the proteins were resolved by SDS-PAGE. The protein phosphorylation was
detected by autoradiography.
To test whether CK2-Nopp140 association
would affect the catalytic activity of CK2, CK2 activity was assayed by
a routine as well as a modified procedure. In the routine procedure,
the assay was carried out in the same way as described previously (23,
24) in the presence and absence of Nopp140 in the reaction solution. In
the modified procedure, CK2 holoenzyme was preincubated with
[-32P]ATP in a reaction buffer (50 mM
Tris-Cl, pH 7.5, 10 mM MgCl2, 0.1 mM [
-32P]ATP (2,000 cpm/pmol)), with and
without Nopp140 protein (0.075 mg/ml), for 20 min at 30 °C. Then,
CK2 substrate peptide RRRDDDSDDD (DSD; final concentration 0.1 mM) was added to the reaction mixture and incubated for
another 10 min. Aliquots of reaction mixture was spotted onto P81
paper, washed, and assayed as described previously (23, 24).
To examine the activation of CK2 by deletion mutants of CK2
, CK2
assays were conducted using baculovirus-expressed CK2
(approximately
5 ng for each reaction) and bacterially expressed and purified
GST-CK2
mutants. CK2
was premixed with GST-CK2
mutants for 10 min at room temperature prior to the assay to allow proper folding of
the holoenzyme. An excess amount of CK2
compared with CK2
was
used in this experiment to give maximal stimulation (23, 24).
GST-CK2
holoenzymes were reconstituted in vitro by incubating
bacterially expressed, immobilized GST-CK2 or GST-CK2
with CK2
(obtained by thrombin cleavage of GST tag from GST-CK2
) and
immobilized on glutathione-Sepharose resin. These forms were then
incubated with 3T3 L1 cell lysates for 4 h at 4 °C. After extensive washing, phosphorylation reactions were initiated by adding a
buffer containing [
-32P]ATP and MgCl2 to
the beads (18) and stopped using Laemmli sample buffer. The
phosphorylated CK2-interacting proteins were analyzed by SDS-PAGE and
autoradiography (Fig. 1). Several CK2-associated phosphoproteins were detected from the GST-CK2 resin, which had been
incubated with cell lysates (Fig. 1, lanes 1 and
2) compared with the control experiment (lane 3),
which shows only the autophosphorylation of the CK2 holoenzyme. Among
them, one protein of 140 kDa was the most highly phosphorylated and
bound to both forms of the CK2 holoenzyme.
One possible candidate for the highly phosphorylated protein p140 was a
nucleolar protein, Nopp140, known to be a very good CK2 substrate and
known to migrate on SDS gels with a similar molecular weight (20). A
polyclonal antibody against protein Nopp140 was used to test this
possibility. A binding experiment using GST-CK2 fusion proteins as
described above was conducted, except that the phosphorylation step was
omitted, and the CK2 interacting proteins were transferred
electrophoretically to a polyvinylidene difluoride membrane and
examined by Nopp140 immunoblotting. As illustrated in Fig.
2A, anti-Nopp140 antiserum recognized a protein of 140 kDa which bound to both GST-CK2 holoenzymes, GST + CK2
and GST
+ CK2
(Fig. 2A, left and
center lanes), but not to GST (right lane),
suggesting a specific interaction between CK2 and Nopp140.
To investigate which subunits of CK2 bind to Nopp140, immobilized GST
fusion proteins of each CK2 subunit, GST-CK2, GST-CK2
, and
GST-CK2
, were used for the type of binding experiment described above. A strong CK2
-Nopp140 interaction was detected when less than
2 µg of GST-CK2
was used (Fig. 2B, left
lane), whereas no interaction was observed between Nopp140 and 2 µg of GST-CK2
, or Nopp140 and 2 µg of GST-CK2
(center and right lanes), suggesting that the CK2
holoenzyme-Nopp140 association most probably occurred through the
subunit of CK2. However, with a higher amount (between 5 and 10 µg)
of GST-CK2
, detectable affinity toward Nopp140 was seen; GST-CK2
was associated very weakly with Nopp140 (data not shown).
To clarify further whether the
CK2-Nopp140 interaction is a direct association, a radioactive CK2
overlay experiment was carried out using 32P-labeled
purified CK2 protein as a probe (18). Bacterially expressed pure
Nopp140 protein was immobilized on the membrane as described (see
"Experimental Procedures"). As demonstrated in Fig.
3, lane 1, Nopp140 did bind to the
radiolabeled CK2 probe, whereas a control protein, bovine serum albumin
(lane 2, 1 µg of bovine serum albumin), did not bind. This
indicates that the CK2-Nopp140 interaction is specific and direct. It
is noteworthy that in this experiment, a dephosphorylated form of
Nopp140 was used because the purified Nopp140 was expressed in
bacteria, giving a band of 100 kDa on the gel, whereas in intact cells,
most of the Nopp140 is in a highly phosphorylated form (20). Taken
together, the experiments of Figs. 2 and 3 make it seem very likely
that the CK2-Nopp140 association is not dependent on the
phosphorylation state of Nopp140.
Coimmunoprecipitation of CK2 and Nopp140
The association of
CK2 and Nopp140 was also studied by coimmunoprecipitation of the two
proteins from cell lysates. Immunoprecipitation of endogenous Nopp140
was performed in 3T3 L1 cell lysates (data not shown) and BRL cell
lysates (Fig. 4A), and in both cases CK2 was
detected in the immunoprecipitates of Nopp140 when anti-CK2 antiserum was used for immunoblotting analysis. In addition to CK2,
NAP57, a Nopp140-binding protein (25), was also coprecipitated with
Nopp140 (Fig. 4A). The specific precipitation of Nopp140 and
coprecipitation of NAP57 and CK2
are present only in the absence of
competing peptide (
lanes). It was found that a significant amount of CK2 was complexed with Nopp140 in the coprecipitation experiment; as determined by immunoblotting, it was estimated that more
than half of the total cellular CK2 was removed from the supernatants
by precipitation with anti-Nopp140 antibodies (data not shown).
In a parallel experiment, immunoprecipitation of endogenous CK2 from
3T3 L1 cell lysates was performed using a mixture of anti-CK2,
anti-CK2
, and anti-CK2
antiserum. The presence of protein
Nopp140 was examined by immunoblotting of the CK2 immunoprecipitate with a polyclonal anti-Nopp140 antiserum (20). As shown in Fig. 4B, Nopp140 was detected in the immunoprecipitate of CK2
(lane 2) but was not detected in the preimmune sera
immunoprecipitate (lane 1), indicating a specific
interaction of these two proteins. Together, coimmunoprecipitation of
CK2 and Nopp140 suggested an in vivo association of these
two proteins.
GST-CK2 deletion mutants were prepared for mapping
the region of CK2
subunit which associates with Nopp140. The
constructs used are shown in Fig. 5A.
Expression of these deletion constructs produced
NH2-terminal and COOH-terminal truncated GST-CK2
proteins. To characterize whether these deletion mutants could
stimulate the activity of CK2
as the wild type
does, the
enzymatic activity of the recombinant
subunit (from Sf-9 cells) was
measured in the presence of an excess amount (five times more) of
either wild type
subunit or the deletion mutants of
. As
illustrated in Fig. 5B, at 30 °C under our assay
condition (with 0.1 M NaCl in final reaction mixture),
CK2
showed very low activity because of the inhibition of NaCl (Fig.
5B, far left lane). Addition of the wild type
GST-
protein greatly stimulated the catalytic activity of the
subunit (second lane). However, among all of the deletion mutants of
, only the mutant with deletion of the 20 NH2-terminal amino acids, GST-
21-215, could
stimulate the activity of CK2
to the same extent as the wild type
(third lane). Deletion of amino acids 1-40
(GST-
41-215) greatly decreased the ability of
to
activate CK2
(fourth lane), and further deletion up to
first 80 amino acids (GST-
81-215) almost totally abolished the stimulation to
(fifth lane). This suggests
that deletion of amino acids 21-80 causes a major structural change in
the subunit. According to Kusk et al. (26), from their study in the yeast two-hybrid system, amino acids 20-60 are needed for strong
-
interaction; so it is very possible that a tetrameric structure is needed to obtain full CK2 activity. Consistent with the
data reported previously (27, 28), the COOH terminus of the
subunit
was important for activating the
subunit because it is responsible
for the
-
interaction; deletion of the COOH-terminal amino acids
160-215 caused a big decrease in the activation of the
subunit by
. Further investigation is under way to make a more thorough
analysis of structure-function relationships in CK2
.
Mapping of the Binding Region of CK2
The
deletion mutants of CK2 were used in the in vitro
CK2-Nopp140 binding studies. After incubating the immobilized
GST-CK2
deletion mutants with 3T3 L1 cell lysates, the bound
proteins were eluted and analyzed by SDS-PAGE and Nopp140
immunoblotting. Nopp140 was not detected in any of the eluates of the
NH2-terminal deletion mutants (Fig. 5C,
lanes 2-6) but was detected in the eluate from the
COOH-terminal deletion mutant GST-CK2
1-160 (Fig.
5C, lane 7). Since GST-CK2
21-215
is the smallest NH2-terminal deletion, the lack of its
binding ability to Nopp140 indicated that the first 20 NH2-terminal amino acids are most probably involved in the
CK2-Nopp140 interaction.
To understand how the
CK2 subunit might affect the specificity of CK2 toward Nopp140 as a
substrate, bacterially expressed Nopp140 was subjected to
phosphorylation by baculovirus-expressed CK2 catalytic subunits
and
and by holoenzymes
2
2 and
2
2. The phosphorylation reactions were
carried out in a buffer without NaCl in order to have maximal
or
catalytic activity (
and
are inhibited by NaCl). When a
high concentration of CK2 (120 ng) was used, Nopp140 was found to be
phosphorylated efficiently by all of the forms of CK2 after incubation
in a phosphorylation buffer for 10 min at 30 °C; phosphorylation
caused a gel mobility shift from 100 to 140 kDa (data not shown).
However, when a lower amount of CK2 was used (approximately 30 ng),
each form of holoenzyme,
2
2 and
2
2, showed much higher specific activity
toward Nopp140 than the monomeric active subunits,
and
,
although their relative activities toward a CK2 substrate peptide, DSD,
had already been deliberately adjusted to the same level by dilution.
Phosphorylation of Nopp140 by
2
2 and
2
2 for 20 min at 30 °C resulted in a massive incorporation of 32P and a significant gel mobility
shift (Fig. 6A, lanes 1 and
2), whereas phosphorylation by
and
under the same
condition was much weaker and did not shift the band significantly
(Fig. 6A, lanes 3 and 4). From the
densitometric reading, phosphorylation of Nopp140 by the holoenzymes
CK2 was 4-fold higher than by the monomeric enzymes (Fig.
6B), indicating that Nopp140 is a much better substrate for
the holoenzyme CK2 than for the monomeric
and
subunit.
Effect of CK2-Nopp140 Association on CK2 Activity Using a Different Substrate
To examine the impact of the CK2-Nopp140 association on the enzymatic activity of CK2, CK2 was assayed in the presence or absence of Nopp140 using purified Sf-9 cell-expressed CK2 and bacterially expressed Nopp140. CK2 peptide substrate DSD was used for the assay. Besides the routine assay method (23, 24), a modified method in which Nopp140 was first phosphorylated for 20 min by CK2 followed by adding the DSD peptide to start the reaction, was also used to decrease the competition that might be caused by Nopp140 as an alternative substrate. In each case, no significant change of CK2 activity was detected when Nopp140 was present in the reaction mixture. This occurred even though it was possible that in the experiment using the alternative method Nopp140 phosphorylation may not have been absolutely complete at the point at which DSD was added. Also, a slightly lower [32P]ATP concentration would be present due to its utilization for Nopp140 phosphorylation. These factors would have been expected to decrease activity in the DSD phosphorylation reaction.
In this study we have identified a nucleolar protein, Nopp140, as a CK2-associated protein. The interaction of the two proteins was shown to be direct and not dependent on the phosphorylation state of Nopp140. Furthermore, a possible in vivo interaction of CK2 and Nopp140 was suggested by the coimmunoprecipitation of the two proteins from cell lysates.
Nopp140 was first isolated as a nuclear localization sequence-binding protein (29). Immunostaining and immunoelectron microscopy revealed that Nopp140 is a nucleolar protein that shuttles between the cytoplasm and the nucleolus. A possible role of Nopp140 as a chaperone for import into or export from the nucleolus was suggested (20). Having 49 phosphorylation consensus sites for CK2, and upon their phosphorylation an additional 33, Nopp140 can be highly phosphorylated by CK2 in intact cells (20), giving an apparent molecular mass of 140 kDa on SDS-PAGE. Only phosphorylated Nopp140 binds to the nuclear localization sequence-containing peptide (20). However, a precise understanding of the function of Nopp140 and its phosphorylation by CK2 is not available.
Our data showed that CK2 most probably interacts with Nopp140 through
its subunit, although some binding of the protein with the
subunit of CK2 was seen. One conceivable role of the CK2-Nopp140
interaction could be increasing the substrate specificity for Nopp140
phosphorylation. To address this point, monomeric forms of the enzyme,
and
, and the holoenzymes,
2
2 and
2
2, were used for the phosphorylation of
Nopp140. It was found that Nopp140 was a much better substrate for the
holoenzyme form of CK2. Until now, with nearly all substrates including
the routinely used CK2 substrate peptide DSD, the holoenzyme form of
CK2 always exhibits a higher activity than the monomeric
or
subunit. An approximately 3-5-fold stimulation of CK2
activity by
CK2
has normally been observed when DSD peptide is used in the assay (18, 30). In our experiment in which we compare the ability of the
and
subunits to phosphorylate Nopp140 with that of the holoenzyme
forms, we deliberately used a lower amount of the holoenzyme CK2 so as
to give it the same catalytic activity as CK2
and CK2
toward the
DSD peptide substrate. Under this condition, the holoenzymes still
phosphorylated Nopp140 at four times the rate of either free
or
. If normalized to the molarity level, an approximately 20-fold
difference in phosphorylation could be estimated for the free catalytic
subunit (
or
) compared with holoenzyme CK2. This large
difference could be partly due to the association between CK2
and
Nopp140, thus favoring substrate recognition. However, because CK2 can
also associate with the phosphorylated form of Nopp140, this specific
CK2-Nopp140 interaction may also be correlated with other cellular
functions of CK2 and Nopp140.
CK2 has been shown to be a major nuclear protein (31). Its growth-related accumulation in the nucleolus has also been observed (32, 33). The finding of the association of CK2 with Nopp140 suggests that CK2 may play an important role in the nucleolus. It has been shown that many nuclear localization sequence-interacting nucleolar proteins, including nucleolin, B23, Nopp140, and its associated protein NAP57 (25), are good substrates of CK2 and can migrate back and forth between the nucleus and cytoplasm (for review, see Ref. 34). These nucleolar proteins all have NH2-terminal domains containing stretches of acidic and serine residues with numerous CK2 phosphorylation sites, and the nuclear localization sequence binding ability of the protein seems to be dependent on their phosphorylation (for review, see Ref. 35). Recently, a major nucleolar protein, nucleolin, was also shown to be able to associate with CK2 in vitro and probably in vivo (16-18). It is possible that in addition to the phosphorylation, the association between CK2 and nucleolar proteins may represent another way of regulating the latter. The association of Nopp140 with CK2, together with phosphorylation, could affect its function in ribosomal protein transport.
Interestingly, it was found recently that Nopp140 appears to be a growth-inhibiting protein, e.g. when rat Nopp140 was overexpressed in yeast, growth impairment was observed (19). Also, SRP40, a yeast homolog of Nopp140, was identified by a genetic screen for genes that cause growth arrest when overexpressed (36). Indeed, when deletion and overexpression of SRP40 were conducted in yeast, deletion caused only minor growth impairment, but its overexpression resulted in a severe growth defect (19). It would be of interest to know if the growth inhibitory function of Nopp140 is mediated both by phosphorylation and its association with CK2.
Using deletion mutants of CK2, the region at which CK2
binds to
Nopp140 was mapped to the first 20 NH2-terminal amino
acids, a domain containing the autophosphorylation sites of CK2 Ser-2 or Ser-3 (21). Since bacterially expressed GST-CK2
was used in the
binding assay of these studies, the subunit should have been in its
dephosphorylated form. On the other hand, in the overlay experiments,
CK2 was radiolabeled by the autophosphorylation reaction in which Ser-2
and/or Ser-3 of the CK2
subunit would be phosphorylated. The CK2
bound to the membrane which was detected would have been phosphorylated. This indicates that both the dephosphorylated and the
phosphorylated forms of CK2
can interact with Nopp140. From CK2
activity data (Fig. 5B), deletion of the 20 NH2-terminal amino acids gives a mutant that will still
activate CK2
as well as the wild type
does. It is reasonable to
assume that CK2 is still in its active form even when it is complexed
with Nopp140, and this was indeed what we observed when CK2 activity
toward the peptide substrate was measured in the presence of
Nopp140.
One interesting observation is that when higher amounts of GST fusion
proteins of CK2 and CK2
were used in the binding assay, CK2
did exhibit affinity for Nopp140. By contrast, CK2
bound very
weakly to Nopp140. This CK2
-Nopp140 interaction may have occurred
indirectly through a separate protein, since the association was not
detected with a lower amount of GST
. If CK2
and CK2
have a
different affinity for the hypothetical protein, this could cause the
observed difference in their binding to Nopp140. One question that
could be raised is whether
and
subunits exhibit redundancy in
vertebrates as they do in yeast (5). It is known that CK2
and
CK2
are encoded by different genes (22) and that they are
structurally very homologous (85% homology) with major differences
only in the COOH terminus (4). It has been shown that the
subunit
can act as a transcription factor to control
gene expression,
whereas
cannot (37). Also, the
subunit phosphorylates the
subunit more efficiently than
.3 It would be
interesting to know whether the two catalytic subunits are associated
with different proteins in the cell and have different functions.
So far, a number of proteins have been found which interact with CK2,
some with the catalytic or
subunit, and some with the
subunit. For example, proteins that are reported to be able to interact
with CK2
are the transcription factor ATF1 (38), nucleolin (16-18),
and the heat shock protein HSP 90 (39). Proteins that are shown to
interact with CK2
are p53 (9), DNA topoisomerase II (11),
c-Mos,2 and Nopp140. Currently, it is not clear whether the
binding properties of the different subunits are related to the
regulation of CK2.
We thank Lynda Munar for assistance with the cell culture and Drs. Yim Foon Lee and Patrick S. H. Chou for DNA oligomer synthesis.