From the
Department of Biochemistry, College of
Natural Sciences, Kyungpook National University, Daegu 702-701, Korea and the
Department of Microbiology, College of Natural
Sciences, Kyungpook National University, Daegu 702-701, Korea
Received for publication, March 13, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has been suggested that CKII plays a significant role in the control of
cell proliferation and transformation. CKII is known to catalyze the
phosphorylation of a broad spectrum of substrates, which are involved in cell
growth and proliferation, including DNA-binding proteins, nuclear
oncoproteins, and transcription factors
(13).
The expression level of CKII is greatly enhanced in a variety of tumor or
leukemic cells
(1013).
Overexpression of CKII in the T cells of transgenic mice results in a
high predisposition for lymphoma formation, and coexpression with c-Myc
results in the rapid development of leukemia
(14). Overexpression of
CKII
or CKII
' exhibits cooperativity with Ras in the
transformation of rat embryo fibroblasts and Balb/c 3T3 cells
(15). Microinjection of
antibodies directed against either CKII
or CKII
inhibits cell
cycle progression in response to serum stimulation in human IMR-90 cells
(1618).
An important role of CKII in cell cycle control has also been demonstrated in
the yeast Saccharomyces cerevisiae. The analysis using
temperature-sensitive mutants for the CKII gene has shown that CKII
is required for cell cycle progression in both G1 and
G2/M phases of the cell cycle
(19). These observations
suggest that CKII plays a significant role in cell proliferation and cell
cycle control. However, its precise role in cell cycle progression remains
largely unknown.
In our previous study, we reported that a protein called CKBBP1
(CKII-binding protein 1) is a cellular interaction partner of the
subunit of CKII (20). This
protein has also been identified by others and given the name SAG (sensitive
to apoptosis gene) (21).
CKBBP1/SAG is a Ring-H2 finger motif-containing protein with a molecular
weight of 12.6 kDa. It is localized in both the nucleus and the cytoplasm of
cells (21). CKBBP1/SAG was
subsequently found to be the second member of regulator of cullin (ROC)/RING
box protein (Rbx)/Hrt. ROC1/Rbx1/Hrt1 has been originally identified as the
fourth component of SCF (Skip1-Cullin-F-box protein) E3 ubiquitin ligase
complex as well as von Hippel-Lindau (VHL) tumor suppressor complex
(2225).
Like ROC1/Rbx1/Hrt1, CKBBP1/SAG has E3 ubiquitin ligase activity when complex
with Cullin-1 (26). SCF E3
ubiquitin ligase complex promotes ubiquitination and degradation of
I
B
, cyclins, and cyclin-dependent kinase inhibitors and is
required for G1/S transition of cell cycle
(22,
23,
2729).
In the present study, we show the evidence that CKBBP1 is phosphorylated on
threonine residue at position 10 by CKII in vitro and in
vivo. Most importantly, disruption of this phosphorylation in CKBBP1
results in accumulation of IB
and p27Kip1 in
HeLa cells and inhibits cell proliferation that appears to be linked to
defects in G1/S transition. To our knowledge, this is the first
study reporting physiological significance of the post-translational
modification of CKBBP1/SAG/ROC2/Rbx2.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis and Plasmid ConstructionsThe bacterial expression vector pET14b-CKBBP1, which expresses the full-length CKBBP1 with a hexahistidine (His) tag to the N terminus, was described previously (20). Point mutations in CKBBP1 were made using either the QuickChange site-directed mutagenesis kit (Stratagene) or PCR method. Desired mutations were incorporated into oligonucleotide primers. The primers used for threonine to alanine mutagenesis at positions 10 and 49 were 5'-AGACGGAGAGGAAGCATGCGCCCTGGCCT-3' and 5'-GACGTGGAGTGCGATGCATGCGCCATCTG-3', respectively. The primers used for serine to alanine mutagenesis at positions 24 and 42 were 5'-TCCGGGAGCTCAGGCTCCAAGGCGGGAGGCG-3' and 5'-GCGGTGGCCATGTGGGCCTGGGACGTGGAGTGC-3', respectively. The mutation sites are underlined. The mutagenesis was performed as described by the manufacturer. After digestion of the mutation products with NdeI and XhoI, the fragment ligated into the NdeI/XhoI sites of pET14b (Clontech). To replace threonine 10 with glutamic acid, CKBBP1 cDNA was PCR-amplified by using N-terminal mutagenic primer (5'-ACGCCATATGGCCGACGTGGAAGACGGAGAGGAAGAGTGCGC-3') and C-terminal reverse primer (5'-CTAACTCGAGTCATTTGCCGATTCTTT-3'). The mutation site is underlined. After digestion of the PCR products with NdeI and XhoI, the fragment was ligated into the NdeI/XhoI sites of pET14b.
To generate a Myc-His-tagged CKBBP1 expression construct, the wild-type and mutant CKBBP1 cDNAs were PCR-amplified using primers 5'-CCGGAATTCCGCCATGGCCGACGTGGAAGA-3' and 5'-GCGGGATCCATTTGCCGATTCTTTGGACCA-3'. The PCR fragments were digested with EcoRI and BamHI, and then subcloned into the EcoRI/BamHI site of pcDNA3.1/Myc-His vector (Invitrogen). To generate a GFP-tagged CKBBP1 expression construct, the wild-type and mutant CKBBP1 cDNAs were PCR-amplified using primers 5'-CGCGGCCTCGAGATGGCCGACGTGGAAG-3' and 5'-CTATCGGATCCCCTTTGCCGATTCTTTG-3'. The PCR fragments were digested with XhoI and BamHI, and then subcloned into XhoI/BamHI sites of pEGFP-N1 vector (Clontech). The sequences of all constructs were confirmed by nucleotide sequencing.
Purification of CKII and CKBBP1Human CKBBP1, CKII
holoenzyme, and CKII were expressed and purified in Escherichia
coli as described previously
(20,
30).
CKII Activity AssayThe standard assay for
phosphotransferase activity of CKII was conducted in a reaction mixture
containing 20 mM Tris-HCl, pH 7.5, 120 mM KCl, 10
mM MgCl2, and 100 µM
[-32P]ATP in the presence of 1 mM synthetic
peptide substrate (RRREEETEEE) in a total volume of 30 µl at 30 °C. The
reactions were started by the addition of purified CKII, HeLa cell lysates, or
CKBBP1 co-precipitates and incubated for 15 min. The reaction was stopped by
the addition of trichloroacetic acid to a final concentration of 10% and
centrifuged, and 10 µl of supernatant was then applied to P-81 paper. The
paper was washed in 100 mM phosphoric acid, and the radioactivity
was measured by scintillation counting.
Phosphorylation of CKBBP1 by CKIIPhosphorylation of
His-tagged CKBBP1 by CKII was performed in a reaction mixture containing 20
mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 100
µM [-32P]ATP, and 6 µg of His-tagged CKBBP1
in a total volume of 30 µl. The reactions were started by the addition of
purified CKII or HeLa cell lysates and incubated for 15 min at 30 °C. The
samples were then separated on 15% SDS-polyacrylamide gel. The gel was stained
with Coomassie blue, dried, and subjected to autoradiography.
Phosphoamino Acid AnalysisPhosphoamino acid analysis was
performed essentially as described by others
(31). Briefly, the His-tagged
CKBBP1 protein was phosphorylated CKII and separated by SDS-polyacrylamide gel
electrophoresis, as described above. After staining of the gel with Coomassie
blue, the His-tagged CKBBP1 protein band was excised and the protein was
extracted in 50 mM NH4HCO3, pH 7.4,
containing 0.5% -mercaptoethanol and 0.1% SDS. The proteins were
precipitated in 15% trichloroacetic acid with 20 µg of bovine serum albumin
as carrier, washed in 100% ethanol, dried, and hydrolyzed in 6 N
HCl for 1 h at 110 °C. The hydrolysate was lyophilized and resuspended in
7 µl of pH 1.9 buffer (7.8% acetic acid and 2.2% formic acid) containing
phosphoamino acid standards. The samples were spotted on cellulose thin-layer
plates, and electrophoresis was performed using pH 1.9 buffer. Standards were
visualized with ninhydrin and 32P-labeled phosphoamino acids were
detected by autoradiography.
Determination of Kinetic ConstantsKm and Vmax values were calculated from Lineweaver-Burk transformations of initial rates using the computer program Sigma Plot.
Cell Culture and Establishment of Stable Cell LineHeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. For serum starvation, HeLa cell monolayers were washed with ice-cold phosphate-buffered saline and then grown in DMEM supplemented with 0.2% fetal bovine serum for 36 h prior to harvest.
To establish CKBBP1-expressing stable cell lines, HeLa cells were transfected with the Myc-His-tagged CKBBP1 constructs or the vector control by LipofectAMINE (Invitrogen) as described by the manufacturer. One day later, the cells were cultured in the presence of 1 mg/ml G418. After 2 weeks, the clones were picked and grown in the same medium in the presence of 100 µg/ml. Stable clones were examined for protein expression by Western blotting.
Preparation of HeLa Cell ExtractFor Western blotting and Ni
pull-down assay, 1 x 106 HeLa cells in 100-mm dishes
were washed with ice-cold phosphate-buffered saline, collected by scraping
with a rubber policeman, and lysed in 100 µl of ice-cold RIPA buffer (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, 1 µg/ml aprotinin, 1
µg/ml leupeptin, 1 µg/ml pepstatin). For CKII activity assay, cells were
lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 20 mM
NaCl, 1 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40,
0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 mM sodium orthovanadate, 1 mM sodium
pyrophosphate, and 4 mM p-nitrophenyl phosphate) by
sonication. The particulate debris was removed by centrifugation at 12,000
x g. The volumes of the supernatants were adjusted for equal
protein concentration.
Western BlottingProteins were separated on polyacrylamide
gels in the presence of SDS, transferred electrophoretically to nitrocellulose
membrane. The membrane was blocked with 5% skim milk in TBST (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 h and then
incubated with specific antibodies. The membrane was washed three times in
TBST, and then treated with enhanced chemiluminescence (ECL) system (Amersham
Biosciences). Some membranes were stripped in stripping buffer (2% SDS, 100
mM -mercaptoethanol, and 50 mM Tris-HCl, pH 7.0)
at 50 °C for 1 h with gentle shaking and reprobed with anti-
-actin
antibody as a control for protein loading.
Pull-down AssaysNi-NTA agarose beads and lysates from HeLa
cells that were transfected with the pcDNA3.1/Myc-His-CKBBP1 were incubated in
200 µl of binding buffer (20 mM Tris-HCl, pH 7.4, 150
mM NaCl, 1 mM PMSF). The reaction was allowed to proceed
for 1 h while rocking at 4 °C. After the beads were washed three times
with washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM PMSF, 20 mM imidazole), the bound proteins
were eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 150
mM NaCl, 1 mM PMSF, 200 mM imidazole),
denatured in 4x SDS reducing protein gel loading buffer, and then
resolved using SDS-polyacrylamide gel. The eluted proteins were visualized by
Western blotting with anti-CKII, anti-CKII
, or anti-CKBBP1
antibodies.
Metabolic 32P LabelingHeLa cells transfected with Myc-His-CKBBP1 were labeled with [32P]orthophosphate at 0.4 mCi/ml in phosphate-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 24 h at 37 °Cin5%CO2. Cells were lysed in RIPA buffer and precipitated with Ni-NTA agarose. After the beads were washed three times with washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 20 mM imidazole), the bound proteins were eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 200 mM imidazole), denatured in 4x SDS reducing protein gel loading buffer, and then resolved using 15% SDS-polyacrylamide gel. The gel was stained with Coomassie blue, dried, and subjected to autoradiography.
RNA Binding AssayWild-type and mutant CKBBP1 were incubated with poly(U)-agarose (Sigma) in RNA binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% Nonidet P-40, 50 µM ZnCl2, 2% glycerol, and 1 mM dithiothreitol) at 4 °C for 1 h. After an extensive washing, the immobilized proteins were recovered by an elution with 500 mM NaCl, separated on a 15% SDS-polyacrylamide gel, and then immunoblotted with anti-CKBBP1 antibody.
Subcellular Localization of CKBBP1To determine the subcellular localization of the wild-type and mutant CKBBP1, GFP-tagged CKBBP1 was transiently expressed in HeLa cells. Green fluorescence images were obtained using a confocal microscope after 48 h of transfection.
Growth CurvesHeLa cells stably expressing the wild-type and mutant CKBBP1 were seeded in 6-well dishes at a starting density of 5,000 cells/well, with duplicate wells for each cell line. Every 24 h, cells were trypsinized and counted in triplicate using a hemocytometer. Trypan blue was used to distinguish viable cells from non-viable cells.
FACS AnalysisHeLa cells (2 x 105) were seeded in 100-mm dishes containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. 36 h later, the cells were collected in phosphate-buffered saline containing 2% fetal bovine serum, fixed in 65% ethanol for 1 h at 4 °C, and then incubated in 50 µg/ml DNase-free RNase A (Sigma), 25 µg/ml propidium iodide (Sigma), and 0.6% sodium citrate for 30 min at 37 °C. Flow cytometric determination of cellular DNA content was performed on a Coulter Elite ESP Cell Sorter (Beckman). The forward and side scatter gates were set to exclude any dead cells from the analysis; 10,000 events within this gate were acquired per sample.
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RESULTS AND DISCUSSION |
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In order to further characterize the nature of CKBBP1 phosphorylation by CKII, a kinetic analysis was performed. The Km was 6.25 µM, and the Vmax was 232.6 pmol/min/mg with CKBBP1 as a substrate of CKII (Fig. 1B). These parameters were comparable with reports of CKII kinetics with other protein substrates. For example, the Km value obtained for CKBBP1 was greater than the value obtained for topoisomerase II (0.4 µM), but it was lower than the value obtained for tubulin (20 µM) (33, 34). With a Km in the submicromolar range, we conclude that CKBBP1 is an efficient substrate for CKII.
CKII Is a Predominant CKBBP1 Kinase in HeLa Cell ExtractTo determine whether CKII is a predominant CKBBP1 kinase in mammalian cells, we performed an in vitro kinase assay using purified His-CKBBP1 and whole cell extract from HeLa cells with and without the addition of CKII inhibitors heparin and DRB. Following the kinase reaction, the His-CKBBP1 protein was precipitated with Ni-NTA agarose and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. As shown in Fig. 2, His-CKBBP1 was phosphorylated by HeLa cell extract and the CKBBP1 phosphorylation was significantly inhibited by CKII inhibitors heparin and DRB. Quantification by liquid scintillation counting of the 32P-labeled CKBBP1 bands revealed that 0.3 µM heparin and 50 µM DRB inhibited 89 and 70% of the phosphorylation by HeLa cell extract, respectively. Coomassie blue staining of the gel showed that CKBBP1 was equivalent in each reaction (left panel of Fig. 2). These results demonstrate that CKBBP1 is phosphorylated predominantly by a heparin- and DRB-sensitive kinase in cell extract.
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CKII Phosphorylates Threonine at Residue 10 within CKBBP1 in VitroTo determine the nature of the phosphorylated residue, we performed phosphoamino acid analysis of CKBBP1 labeled with 32P in vitro. Phosphoamino acid analysis revealed that phosphorylation of CKBBP1 occurred exclusively on threonine, but not on serine or tyrosine residues (Fig. 3A).
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There are only two threonine residues at positions 10 and 49 within the polypeptide chain of CKBBP1. In order to determine the CKII-phosphorylation site within CKBBP1, both threonine residues were replaced by alanine. For the control experiment, serine residues at positions 24 and 42 were substituted by alanine. This resulted in the following CKBBP1 mutants: CKBBP1T10A, CKBBP1T49A, CKBBP1S24A, and CKBBP1S42A. The mutant CKBBP1 proteins were expressed and purified in E. coli and subjected to phosphorylation by CKII. As shown in Fig. 3B, the CKII-mediated phosphorylation was completely abolished in CKBBP1T10A, but not in CKBBP1T49A, showing that the threonine 10 was the only CKII phosphorylation site in CKBBP1. Serine to alanine mutations had no significant effect on the CKBBP1 phosphorylation by CKII.
To determine whether threonine at residue 10 within CKBBP1 is phosphorylated by HeLa cell extract-derived kinases, His-CKBBP1WT and His-CKBBP1T10A were added to HeLa cell extract and subjected to an in vitro kinase assay. After precipitation with Ni-NTA agarose beads, the CKBBP1 proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. As shown in Fig. 3C, alanine mutation of threonine 10 completely abolished the CKBBP1 phosphorylation by HeLa cell extract-derived kinases. These results demonstrate that threonine at residue 10 is the only site for CKBBP1 phosphorylation in the whole cell extract.
CKII preferentially phosphorylates serine/threonine residues followed by a stretch of acidic residues on the immediate C-terminal side (+1 to +3). Among these acidic residues, the third position after the phosphoacceptor is the most improtant determinant. However, further analyses have indicated that acidic residues located at positions 2 to +7 can also serve as specificity determinants for CKII phosphorylation (1, 35). Our current observation also indicates that CKII phosphorylates serine/threonine residue without any acidic amino acids at positions +1 to +3 after phosphoacceptor. In case of CKII-mediated CKBBP1 phosphorylation, instead, two acidic residues locate at positions 1 and 2 before the phosphoacceptor (Fig. 3D).
Threonine 10 within CKBBP1 Is Phosphorylated by CKII in VivoTo determine whether the CKBBP1 protein is phosphorylated on threonine at residue 10 in vivo, HeLa cells were transfected with the plasmids encoding Myc-His-CKBBP1WT or Myc-His-CKBBP1T10A. The empty vector (pcDNA3.1/Myc-His) was used as a control. After selection for G418 resistance, the stable expression of CKBBP1 was examined by immunoblotting with anti-CKBBP1 antibody. As shown in Fig. 4A, the stable expression of Myc-His-CKBBP1 was detectable in HeLa cells that were transfected with pcDNA3.1/Myc-His-CKBBP1. However, Myc-His-CKBBP1 was not detectable in cells that were transfected with pcDNA3.1/Myc-His vector alone.
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Using these stable cell lines, we metabolically 32P-labeled
CKBBP1 in HeLa cells. Myc-His-CKBBP1WT and
Myc-His-CKBBP1T10A were precipitated from these cells with Ni-NTA
agarose resin and the precipitates were resolved on SDS-polyacrylamide gel and
autoradiographed. As shown in Fig.
4B, the phosphorylated CKBBP1 band was observed in HeLa
cells that were transfected with pcDNA3.1/Myc-His-CKBBP1WT.
However, no phosphorylated CKBBP1 band was observed in HeLa cells that were
transfected with pcDNA3.1/Myc-His-CKBBP1T10A or pcDNA3.1/Myc-His
vector alone. Because similar amounts of Myc-His-CKBBP1WT and
Myc-His-CKBBP1T10A were co-precipitated (data not shown, see
Fig. 5B), these
results apparently indicate that CKBBP1 is phosphorylated on threonine residue
at position 10 by CKII in HeLa cells. Since both the and
subunits of CKII have been shown to be autophosphorylated in the cells, the
co-precipitated phosphoprotein bands of 44 and 28 kDa are thought to represent
the
and
subunits of CKII, respectively. Indeed, Western blots
of the co-precipitates probed anti-CKII
and anti-CKII
antibodies
showed that CKII
and CKII
were co-precipitated with CKBBP1 in
HeLa cells (see below).
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Effects of Threonine 10 Phosphorylation within CKBBP1 on CKBBP1 Binding
to RNA and CKII, Subcellular Localization of CKBBP1, and CKII
ExpressionAlthough the biological importance remains to be
elucidated, it has been reported that CKBBP1 binds to RNA in vitro
(36). To examine whether the
CKII-mediated phosphorylation of CKBBP1 modulates its binding activity to RNA,
we generated a variant of CKBBP1, CKBBP1T10E, with substitution of
the CKII phosphorylation site to glutamic acid, which may mimic the spatial
and charge contributions of phosphorylated threonine
(37,
38). Wild-type or mutant
CKBBP1 proteins, CKBBP1T10A and CKBBP1T10E were
incubated with poly(U)-agarose beads and the immobilized proteins were
visualized by Western blotting with anti-CKBBP1 antibody. As shown in
Fig. 5A,
CKBBP1T10A and CKBBP1T10E maintained the full capacity
of CKBBP1 to bind to RNA as compared with the wild-type CKBBP1 protein.
To examine the effect of CKBBP1 phosphorylation on the binding to
CKII, HeLa cells were transfected with
pcDNA3.1/Myc-His-CKBBP1T10E and a stable cell line was obtained as
above. After selection for G418 resistance, the stable expression of CKBBP1
was examined by immunoblotting with anti-CKBBP1 antibody (data not shown).
HeLa cells stably transfected with pcDNA3.1/Myc-His-CKBBP1WT,
pcDNA3.1/Myc-His-CKBBP1T10A, pcDNA3.1/Myc-His-CKBBP1T10E
or pcDNA3.1/Myc-His (vector control) were lysed and the complexes of CKBBP1
and CKII were precipitated with Ni-NTA agarose beads. Western blots of the
co-precipitates probed anti-CKII
and anti-CKII
antibodies showed
that similar amounts of the respective CKII subunit were co-precipitated in
these cells (Fig. 5B).
From this observation, we speculate that threonine 10 within CKBBP1 is not
involved in the CKII
binding. This is consistent with our previous study
in which the C-terminal region containing the Ring-H2 finger motif within
CKBBP1 is sufficient for CKII
binding
(20).
CKII phosphorylation is known to regulate the nuclear translocation of some proteins (39, 40). To examine the differences in the subcellular localization of the wild-type and mutant CKBBP1 proteins, we fused CKBBP1WT, CKBBP1T10A, or CKBBP1T10E to GFP, and expressed transiently the fusion proteins in HeLa cells. Confocal fluorescence microscopy indicated that wild-type and mutant CKBBP1 localized to both the nucleus and the cytoplasm (data not shown).
It has been shown that ROC1/Rbx1 and CKBBP1/SAG/ROC2 are components of SCF
E3 ubiquitin ligases that mediate the degradation of substrate proteins
(2226).
Because CKBBP1 is a CKII-binding protein, we hypothesized that
CKII
could be degraded by ubiquitin-mediated proteolysis. To test this
hypothesis, we examined the protein levels of CKII
and CKII
in
HeLa cells stably expressing Myc-His-CKBBP1WT,
Myc-His-CKBBP1T10A, or Myc-His-CKBBP1T10E by Western
blot analysis of whole cell extracts using anti-CKII
and
anti-CKII
antibodies. As shown in
Fig. 5C, the
expression levels of both CKII
and CKII
are not altered in these
stable cell lines. When CKII activity in these cell lysates was assessed using
the synthetic peptide substrate RRREEETEEE, similar levels of CKII activity
were detected (Fig.
5D).
Taken together, these results suggest that CKII-mediated CKBBP1
phosphorylation has no effect on properties of CKBBP1 such as its ability to
bind RNA, associate with CKII, or localize to particular subcellular
compartment. Alternatively, the possibility that carboxylate group of glutamic
acid-10 within CKBBP1T10E is not functionally equivalent to
phosphate group on the phosphorylated threonine 10 cannot be ruled out. To
assess directly the effect of CKII phosphorylation on these properties of
CKBBP1, the phosphorylated CKBBP1 protein will be used in future study.
Overexpression of CKBBP1T10A Causes Accumulation of
IB
and p27Kip1Since SCF
E3 ubiquitin ligases have been known to catalyze ubiquitin-mediated
proteolysis of I
B
and p27Kip1
(22,
23,
29), we investigated the
effect of CKBBP1 phosphorylation on proteasome-dependent degradation of
I
B
and p27Kip1. The protein levels of
I
B
and p27Kip1 in HeLa cells stably
expressing Myc-His-CKBBP1WT, Myc-His-CKBBP1T10A, or
Myc-His-CKBBP1T10E were examined by Western blot analysis of whole
cell extracts using anti-I
B
and anti-p27Kip1
antibodies. As shown in Fig.
6A, there was no significant difference in the protein
level of either I
B
or p27Kip1 when the cells
were cultured in DMEM containing 10% serum. However, both I
B
and
p27Kip1 were accumulated in HeLa cells stably expressing
Myc-His-CKBBP1T10A when the cells were serum-starved.
Quantification by densitometer analysis revealed that overexpression of
CKBBP1T10A increased the protein levels of I
B
and
p27Kip1 3-fold and 2-fold, respectively. By contrast,
overexpression of CKBBP1T10E reduced 30% of the protein levels of
I
B
and p27Kip1 as compared with the
overexpression of CKBBP1WT. There was no change in levels of
-actin as a control.
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To determine whether the increased amounts of IB
and
p27Kip1 in HeLa cells expressing CKBBP1T10A are
due to reduction of proteasome-dependent degradation, the stable HeLa cell
lines were treated with the proteasome inhibitor MG132 during serum
starvation. Western blot analysis of cell extracts using anti-I
B
and p27Kip1 antibodies revealed that the amounts of
I
B
and p27Kip1 were unchanged in cells
expressing CKBBP1T10A irrespectively of the MG132 treatment.
However, MG132 blocked I
B
and p27Kip1
degradation in cells expressing control vector, CKBBP1WT, or
CKBBP1T10E, allowing that their amounts were increased to the
levels in cells expressing CKBBP1T10A
(Fig. 6B). These
observations indicate that the effect of stable expression of
CKBBP1T10A on I
B
and p27Kip1
accumulations is mainly due to the reduction in proteasome-dependent
degradation. In addition, these results suggest that the threonine 10
phosphorylation within CKBBP1 is required for efficient proteasome-dependent
degradation of I
B
and p27Kip1.
To further confirm that CKII-mediated CKBBP1 phosphorylation promotes the
degradation of IB
and p27Kip1, the stable
HeLa cell lines were treated with 20 µM DRB during serum
starvation and then the protein levels of I
B
and
p27Kip1 were examined by Western blot analysis. Treatment
with DRB resulted in the apparent induction of I
B
and
p27Kip1 accumulation in cells expressing control vector or
CKBBP1WT, allowing that their amounts were increased to the levels
in cells expressing CKBBP1T10A. In contrast, treatment of cells
expressing CKBBP1T10E with DRB did not increase the amounts of
I
B
and p27Kip1 to their levels in cells
expressing CKBBP1T10A (Fig.
6C). These results indicate that the phosphorylation
state at threonine 10 is important for the degradation of I
B
and
p27Kip1, and that CKII may regulate the degradation of
I
B
and p27Kip1 through CKBBP1
phosphorylation on threonine 10.
Overexpression of CKBBP1T10A Reduces Cell GrowthSince we demonstrated that overexpression of CKBBP1T10A in HeLa cells caused accumulation of p27Kip1, which is the cyclin-dependent kinase inhibitor primarily responsible for the control of cell growth at the G1/S transition, we investigated whether overexpression of CKBBP1T10A reduces cell proliferation. To investigate the effect of overexpression of the CKBBP1 mutants on cell proliferation, growth curves were performed on the stably transfected HeLa cell lines. Compared with the vector-transfected control, stable expression of Myc-His-CKBBP1WT or Myc-His-CKBBP1T10E did not significantly change cell proliferation. However, stable expression of Myc-His-CKBBP1T10A induced an apparent decrease in cell proliferation over the time course (Fig. 7A). FACS analysis was employed to examine whether stable expression of Myc-His-CKBBP1T10A interferes with cell cycle progression. As shown in Fig. 7B, increased expression of Myc-His-CKBBP1WT or Myc-His-CKBBP1T10E did not cause a detectable change in the cell cycle profile. However, accumulation of G0/G1 peak was observed with the stable expression of Myc-His-CKBBP1T10A. These results indicate that the phosphorylation of CKBBP1 on threonine 10 plays an important role in the G1/S phase progression of the cell cycle in HeLa cells.
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Cell cycle progression from G1 to S is precisely regulated by
timely synthesis and degradation of specific regulatory proteins
(41,
42). SCF E3 ubiquitin ligase
complex promotes ubiquitination and degradation of IB
and
p27Kip1 and this proteolysis process is necessary for
G1/S transition
(22,
23,
2729).
The present study demonstrates that the phosphorylation of threonine 10 within
CKBBP1 is required for the efficient degradation of I
B
and
p27Kip1 as well as the G1/S phase progression.
Since CKII has been known to be required for G1/S transition of the
cell cycle (19), we suggest
that CKII may be involved in G1/S transition, at least in part,
through modulation of the p27Kip1 degradation by CKBBP1
phosphorylation.
It has been shown that CDC34, ubiquitin-conjugating enzyme E2, interacts
with SCF ubiquitin ligase and is involved in the ubiquitination of many
substrates including IB
and p27Kip1. CDC34
also associates with the
subunit of CKII and is phosphorylated by CKII
(43). Our previous and present
studies have shown that CKBBP1 associates with the
subunit of CKII and
is phosphorylated by CKII. All these data lead to the possibility that CKII
participates in the formation of various complex such as CKII-SCF, CKII-CDC34,
and/or CKII-SCF-CDC34-CKII and that CKII may modulate protein ubiquitination
in multiple steps. Whether CKII-mediated CKBBP1 phosphorylation regulates
directly the activity of SCF ubiquitin ligase is presently unclear. In future
study, it will be important to investigate the role of CKII in the modulation
of SCF ubiquitin ligase activity in the cells.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 82-53-950-6355; Fax: 82-53-943-2762; E-mail: ysbae{at}knu.ac.kr.
1 The abbreviations used are: CKII, protein kinase CKII (formerly casein
kinase II); CKBBP1, CKII-binding protein 1; DRB,
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole; FACS,
fluorescence-activated cell sorter; GFP, green fluorescent protein; PMSF,
phenylmethylsulfonyl fluoride; Rbx, RING box protein; ROC, regulator of
cullin; SAG, sensitive-to-apoptosis gene; SCF, Skip1-Cullin-F-box protein;
RIPA, radioimmune precipitation assay buffer; DMEM, Dulbecco's modified
Eagle's medium; NTA, nitrilotriacetic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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