From the Department of Biochemistry, Health Sciences Center, University of Western Ontario, London, Ontario N6A 5C1, Canada
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ABSTRACT |
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Protein kinase CK2 (formerly casein kinase II)
exhibits elevated expression in a variety of cancers, induces
lymphocyte transformation in transgenic mice, and collaborates with
Ha-Ras in fibroblast transformation. To systematically examine the
cellular functions of CK2, human osteosarcoma U2-OS cells
constitutively expressing a tetracycline-regulated transactivator were
stably transfected with a bidirectional plasmid encoding either
catalytic isoform of CK2 (i.e. CK2 Protein kinase CK21
(formerly casein kinase II) is a ubiquitously distributed and highly
conserved protein serine/threonine kinase that is essential for
viability in eukaryotes (1, 2). Although its precise functions remain
poorly understood, there is mounting evidence to suggest that CK2 plays
an important role in the control of cell proliferation and
transformation (3-6). Alterations in the expression of CK2 have been
observed in a variety of tumor or leukemic cells (7-13), and in the
lymphocytes of cattle that develop T cell lymphomas following infection
with the parasite Theileria parva (14). Furthermore, the
targeted overexpression of CK2 CK2 has been detected in the nucleus and in the cytoplasm of cells and
is believed to phosphorylate regulatory proteins in each of these
compartments (5, 6). The substrate specificity of CK2 is somewhat
unique and has aided in the identification of candidate substrates for
CK2. Dozens of proteins have in fact been classified as likely
physiological substrates of CK2 because CK2 can phosphorylate a site or
sites that are phosphorylated in cells (6). Of particular interest to a
role in proliferation and/or transformation is the demonstration that
the products of proto-oncogenes (i.e. c-Myc (20), c-Jun
(21), c-Myb (22)) and the tumor suppressor p53 (23) are phosphorylated
in vitro by CK2 at sites that are phosphorylated in cells.
However, as it has not been possible to reproducibly manipulate the
expression of CK2 in cells (24), it has not been possible to confirm
that alterations in the cellular activity of CK2 result in alterations in the phosphorylation of its potential substrate proteins. Only in
yeast that harbor temperature-sensitive alleles of CK2 is there rigorous genetic evidence confirming the identity of some of its cellular targets (25-27).
CK2 is generally a tetrameric enzyme composed of two catalytic ( To systematically examine the role of CK2 in cellular regulation, we
have developed cell lines where we can reproducibly manipulate the
expression of either CK2 isoform. To increase the expression of CK2,
human osteosarcoma U2-OS cells that stably express the tetracycline-regulated transcriptional activator were transfected with
a bidirectional plasmid encoding a catalytic subunit of CK2 (i.e. HA epitope-tagged CK2 Plasmid Constructs--
A construct encoding CK2
The kinase-dead mutant of CK2
The bidirectional constructs encoding both CK2
Bidirectional constructs encoding HA-CK2 Antibodies--
Polyclonal anti-CK2 Cell Culture and Transfections--
UTA6 cells were derived from
the human osteosarcoma cell line U2-OS and express the
tetracycline-regulated transcriptional activator fusion protein
(generous gift from Dr. Christoph Englert, Forschungszentrum Karlsruhe,
Germany) (41). The UTA6 cells were maintained in Dulbecco's modified
Eagle's medium with 10% fetal calf serum, antibiotic supplements (0.1 mg/ml streptomycin and 100 units/ml penicillin), and 460 µg/ml G418
(all obtained from Life Technologies Inc.). UTA6 cells were
cotransfected with 5 µg of pTK-hyg plasmid
(CLONTECH) and 30 µg of pRS3, pGV13, pGV7, or
pRS2 using the standard calcium phosphate method (47), with the
exception of pGV13 which was cotransfected using the Fugene reagent as per manufacturer's instructions (Roche Molecular
Biochemicals). Fifteen hours after addition of DNA, the cells were
washed and fresh medium was added. Forty-eight hours after the removal
of the DNA precipitate, the cells were re-fed with supplemented medium containing 500 µg/ml hygromycin (Roche Molecular Biochemicals). Colonies that developed after 2 weeks of drug selection were isolated and expanded in preparation for analysis of inducible expression of the
epitope-tagged CK2 constructs using Western blotting. Clonal cell lines
RS3.22 (expressing CK2 Cell Extracts and Immunoprecipitations--
Cells were washed
twice in PBS, washed once in extraction buffer (50 mM
Tris-Cl (pH. 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol), and then extracted with 500 µl of
extraction buffer supplemented with protease and phosphatase inhibitors
(30 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM
Na3VO4, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). The cells were sonicated
twice for 10 s and then centrifuged at 55,000 rpm in a Beckman
TL100.2 rotor for 20 min at 4 °C. The supernatants were transferred
to fresh tubes and kept on ice for immediate use or frozen at
Kinase Assay--
CK2 activity was measured in RS3.22, GV13.35,
RS2.31, and GV7.21 cell extracts and immunoprecipitates using the
synthetic peptide substrate (RRRDDDSDDD) that has been described
previously (48). Assays were performed for 5 min at 30 °C in a final
reaction volume of 30 µl containing 50 mM Tris-Cl (pH
7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 0.1 mM ATP (specific
activity 500-700 cpm/pmol) and RRRDDDSDDD (0.1 mM).
Reactions were initiated by addition of 9 µl of cell extract or
immunocomplex and were terminated by spotting 10 µl on P81
phosphocellulose paper as described previously (48). The papers were
washed four times in 1% phosphoric acid and once in 95% ethanol. Once
dry, papers were immersed in scintillant and counted in a Beckman LS
5801 scintillation counter.
Western Blotting--
Cells were washed twice with PBS and lysed
in T-buffer (20 mM Tris-Cl (pH 7.4), 50 mM
NaCl, 2% Nonidet P-40, 0.5% deoxycholate, 0.2% SDS, 30 µg/ml
aprotinin, 20 µg/ml leupeptin, 1 mM
Na3VO4, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). The protein
concentration was determined using the BCA assay (Pierce), and equal
amounts of lysates were separated by 12% SDS-PAGE. The proteins were
transferred to a polyvinylidene difluoride membrane for 1 h at 100 V in blotting buffer (25 mM Tris-Cl, 190 mM
glycine, 20% methanol) as described by Towbin et al. (49).
Blocking was performed with 3% gelatin in TBS (20 mM
Tris-Cl (pH 7.5), 500 mM NaCl) at room temperature for
1 h. The membrane was washed twice in TBST (0.05% Tween-20 in
TBS) and reacted with either polyclonal anti- Growth Curves--
UTA6, RS3.22, GV13.35, RS2.31, and GV7.21
cells were seeded in six-well dishes at a starting density of 20,000 cells/well in the presence or absence of 1.5 µg/ml tetracycline.
Every 24 h, cells were harvested with PBS and 5 mM
EDTA and counted with a hemocytometer. Cells were harvested from three
individual wells for each cell line. Trypan blue was used to
distinguish viable from non-viable cells. The medium was changed every
3 days.
Cell Cycle Analysis--
RS2.31 and GV7.21 cells were seeded at
1 × 105 cells in 10-cm plates in the presence or
absence of 1.5 µg/ml tetracycline. The medium was changed every 3 days. Adherent cells were collected on days 2 and 4 using PBS, 5 mM EDTA and pooled with the detached cells from the medium.
Together, these cells were fixed in 70% ethanol overnight at 4 °C.
After washing away the ethanol, cells were resuspended in a staining
solution containing 0.1% sodium citrate, 0.1% Triton X-100, 50 µg/ml propidium iodide (Sigma), and 0.1 mg/ml DNase-free RNase A and
incubated for 20 min at room temperature. Immediately before analysis,
the stained cells were passed through a 30-µm nylon mesh to remove
cell clumps. The stained cells were analyzed within 24 h on a
fluorescence-activated cell sorter (FACScan) using CellQuest version
3.0 software (Becton Dickinson). A minimum of 30,000 cells was counted.
The cell cycle profiles were generated using ModFit LT version 2.0 modeling software (Verity Software House, Topsham, ME).
Establishment of Inducible CK2 Cell Lines--
To systematically
examine the role of each CK2 isoform in the control of cell
proliferation, we undertook efforts to establish cell lines exhibiting
inducible expression of CK2. As our previous studies (36) had
demonstrated that optimal expression of CK2 requires expression of both
catalytic and regulatory CK2 subunits, we utilized a bidirectional
plasmid with dual promoters under the control of tetracycline that was
first described by Baron et al. (40) (Fig.
1). To increase the expression of each
CK2 isoform, we generated stable cell lines by transfecting U2-OS/UTA6 cells that stably express the tetracycline transactivator fusion protein (41) with the pBI plasmid encoding CK2 Expression of Exogenous CK2 in UTA6 Cells--
Inasmuch as the
production of stable cell lines for the inducible expression of CK2 had
not been previously achieved and because the bidirectional plasmid had
only been used for the expression of reporter constructs (40), we
initially performed detailed characterization of the induced expression
of CK2 to ensure catalytic and regulatory subunits of CK2 were
coordinately expressed. Four cell lines that were selected for detailed
examination (i.e. RS3.22, GV13.35, RS2.31, and GV7.21) were
cultured in the presence or absence of tetracycline for 48 h. The
cells were lysed in sample buffer and the lysates resolved by SDS-PAGE
and Western blotting. We detected three bands when probing the blots
with 12CA5 (anti-HA) and 9E10 (anti-myc) antibodies (Fig.
2A). Moreover,
tetracycline-regulated expression of HA-tagged CK2
Cell lysates were also probed with anti-
To further characterize the inducible expression of CK2, we examined
the modulation of CK2 expression by different concentrations of
tetracycline. Fig. 3 (A-C)
demonstrates that HA-CK2
We were also interested in examining the time course of induction
following tetracycline withdrawal. Expression of both HA-CK2 CK2 Kinase Activity--
To examine the kinase activity of the
exogenously expressed CK2
To examine the extent to which the induced expression of the various
CK2 constructs alters total cellular CK2 levels, we performed kinase
assays using a specific substrate of CK2 (48). Kinase assays performed
on whole cell extracts from RS3.22 and RS2.31 cells that express
CK2 Growth Curves and Cell Cycle Analysis--
Based on a number of
observations (3-6, 51, 52) that CK2 is involved in cell cycle
progression and that transient expression of CK2 accelerates cell
proliferation (18), we were interested in examining the effect of
induced alterations in the expression of CK2 on cell proliferation.
Growth curves were performed on the parent UTA6 cells and the four
stably transfected cell lines in the presence or absence of
tetracycline for 10 days. The parent untransfected UTA6 cell line did
not show any change in cell growth in the presence or absence of
tetracycline (Fig. 6A).
Contrary to expectations from recent transfection studies in
fibroblasts (18), increased expression of CK2
Having demonstrated that cell proliferation was attenuated by altered
expression of HA-CK2 There is mounting evidence to suggest that CK2 is involved in the
control of proliferation in mammalian cells (3-6). In general, increased levels of CK2 have been correlated with increased
proliferation. However, there are also results that contradict this
suggestion (19). Therefore, to systematically examine the role of CK2
in cell proliferation and to examine the mechanisms by which CK2 influences cell proliferation, we developed cell systems exhibiting the
regulated expression of CK2. Moreover, based on the possibility that
the two isozymic forms of CK2 exhibit functional specialization in
mammals as has been observed with the two isoforms of CK2 in S. cerevisiae (51, 53), we were interested in establishing systems
where the manipulation of only one isoform of CK2 is possible. Cells
exhibiting coordinate induced expression of catalytic (i.e. CK2 In overexpressing kinase-dead forms of CK2 The observation that induced expression of HA-CK2 In light of recent evidence suggesting that CK2 It is also important to recognize that the studies described in this
paper were performed utilizing epitope-tagged forms of CK2 Based on the prediction that the increased expression of CK2 In the case of HA-CK2 Overall, by manipulating the expression of each CK2 isoform or by
interfering with their individual functions, these studies suggest that
CK2 may have a complex series of functions associated with various
aspects of the control of cell proliferation. Moreover, the results
obtained by expression of similar levels of kinase-inactive CK2 or CK2
') together
with the regulatory CK2
subunit in order to increase the cellular
levels of either CK2 isoform. To interfere with either CK2 isoform,
cells were also transfected with kinase-inactive CK2
or CK2
'
(i.e. GK2
(K68M) or CK2
'(K69M)) together with
CK2
. In these cells, removal of tetracycline from the growth medium
stimulated coordinate expression of catalytic and regulatory CK2
subunits. Increased expression of active forms of CK2
or CK2
'
resulted in modest decreases in cell proliferation, suggesting that
optimal levels of CK2 are required for optimal proliferation. By
comparison, the effects of induced expression of kinase-inactive CK2
differed significantly from the effects of induced expression of
kinase-inactive CK2
'. Of particular interest is the dramatic
attenuation of proliferation that is observed following induction of
CK2
'(K69M), but not following induction of CK2
(K68M). These
results provide evidence for functional specialization of CK2 isoforms
in mammalian cells. Moreover, cell lines exhibiting regulatable
expression of CK2 will facilitate efforts to systematically elucidate
its cellular functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in the T cells of transgenic mice
results in the development of lymphomas (15). By crossing different
lines of transgenic mice, there is evidence for collaboration between
the dysregulated expression of CK2
and the c-Myc and Tal-1 oncogenes
in lymphoma development (16). Accelerated lymphomagenesis is also
observed when the mice that overexpress CK2 in T cells are crossed with mice deficient in the functional expression of p53 (17). There is also
evidence that CK2 contributes to the transformation of primary and
established fibroblasts. Overexpression of either catalytic isoform of
CK2 (i.e. CK2
or CK2
') exhibited cooperativity with
Ha-Ras in the transformation of rat embryo fibroblasts and Balb/c 3T3
cells (18). All of these observations suggest that the increased
expression of CK2 contributes to transformation. By comparison, Heriche
et al. (19) observed diminished Ras-dependent transformation when the expression of CK2 was increased in NIH 3T3 cells. Collectively, although there are unresolved
discrepancies in the precise role of CK2 in transformation, these
studies all demonstrate the potent capacity of CK2 to influence the
behavior of cells.
)
and two regulatory (
) subunits (28). The regulatory subunit is
generally required for optimal expression of activity and appropriate
regulation of substrate specificity (6). In mammals, there are two
isozymic forms of the catalytic subunit of CK2, designated CK2
and
CK2
', that are the products of distinct genes localized to different
chromosomes (29-31). Between species, CK2
and CK2
' exhibit
remarkable conservation; between humans and chickens, CK2
exhibits
98% identity and CK2
' exhibits 97% identity (29, 32). The two
isoforms are also closely related to each other. Within the domain that
contains all of the conserved consensus motifs for members of the
protein kinase family, CK2
and CK2
' exhibit nearly 90% identity.
By comparison, CK2
has a C-terminal extension of approximately 60 amino acids that is completely unrelated to the smaller
(i.e. approximately 20 amino acids) C-terminal domain of
CK2
'. In the yeast Saccharomyces cerevisiae, there are
also two distinct forms of the catalytic CK2 subunit, designated CKA1
and CKA2 (1, 33). Genetic studies in this organism have demonstrated
that CKA1 and CKA2 can compensate for each other but also demonstrates
functional specialization for the two CK2 isoforms. Although few direct
studies of the independent functional properties of CK2
and CK2
'
have been performed in mammalian cells, there is mounting evidence to
suggest that they may also exhibit functional specialization. For
example, striking differences in the subcellular localization of CK2
and CK2
' were observed in HeLa cells (34). However, less obvious
differences in the localization of CK2 isoforms were subsequently
observed in chicken hepatoma cells or fibroblasts (35) and in
transfected COS-7 cells (36), indicating that the control of CK2
localization is complex. There are also indications that the two
isoforms of CK2 are independently regulated during cell cycle
progression as CK2
is phosphorylated by
p34cdc2 during mitosis, whereas CK2
' is not
phosphorylated (37, 38). Moreover, we have recently identified cellular
proteins that bind to CK2
but not
CK2
'.2 Collectively, these
results provide indications that the two isozymic forms of CK2 could
have independent cellular functions. However, efforts to elucidate the
precise functions of CK2 have been hindered by the lack of reproducible
strategies for manipulating its expression in cells.
or CK2
') together with
myc epitope-tagged regulatory CK2
subunit (39-41). To specifically
interfere with signaling events involving CK2
and CK2
', we also
established cell lines expressing kinase-inactive mutants of CK2
or
CK2
' (i.e. HA epitope-tagged CK2
(K68M) or
CK2
'(K69M)) along with myc-CK2
(36). Contrary to the expectation
that increased CK2 expression would enhance proliferation (9, 18),
increased expression of either CK2
or CK2
' resulted in modest
decreases in proliferation. Induced expression of kinase-inactive
CK2
differed significantly from the effects observed following
induction of similar levels of kinase-inactive CK2
'. Of particular
interest is the dramatic attenuation of proliferation that is observed following induction of CK2
'(K69M), but not following induction of
CK2
(K68M). These results provide the first direct evidence for
functional specialization of CK2 isoforms in mammalian cells. Moreover,
by establishing tetracycline-regulatable systems for the control of CK2
expression, we are now in a position for the first time to examine the
mechanisms by which CK2 influences proliferation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
with a
C-terminal HA epitope tag was generated by first introducing a
BlpI site and a stop codon at the 5' and 3' ends,
respectively, of the HA coding sequence (5'-GGCCGCATCTTTTACCCATACGATGTTCCTGACTATGCGGGCTATGACGTCCCGGACTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCTGCTAGTGCGGCCGC-3') using a polymerase chain reaction strategy. The following primers were
used: 5'-GCTCAGCAGTACCCATACGATGTT-3' (sense primer) and 5 '-TTAAGCGTAATCTGGAACGTC-3' (antisense primer). The sequence was amplified, subcloned into the TA cloning vector (Invitrogen), and
verified by DNA sequencing (42). The TA vector encoding the modified HA
sequence was digested with SacI/BlpI, and the SacI/BlpI fragment of CK2
from the hT4.1
plasmid (29) was subcloned into this TA cloning vector that contained
the modified HA sequence. The CK2
-HA cDNA was then subcloned
into the HindIII/ApaI sites of pRc/CMV
(Invitrogen) to generate pRc/ CMV/CK2
-HA, which was designated pZW6.
-HA (i.e. CK2
(K68M)-HA)
was generated by first subcloning CK2
-HA from pZW6 into the
Bluescript pSK+ cloning vector (Stratagene) using HindIII
and ApaI. A BstBI and NcoI fragment
from pRc/CMV/HA-CK2
(K68M) (36) was then used to replace the
wild-type CK2
sequence in pSK+/CK2
-HA to generate pSK+/CK2
(K68M)-HA, which was designated pGV14. A
HindIII/ApaI fragment from pGV14 encoding the
CK2
(K68M)-HA cDNA was subcloned into the HindIII and
ApaI sites of pRc/CMV to generate pRc/CMV/CK2
(K68M)-HA, which was designated pGV15. All constructs were verified by DNA sequencing (42).
-HA or CK2
(K68M)-HA were generated as follows. The myc-CK2
construct was digested from pRc/CMV (36) with NotI and ApaI.
After gel purification, the overhangs on the fragment were filled in,
and the fragment was ligated into the EcoRV site (MCS I) of
the bidirectional pBI vector (CLONTECH) to generate
pBI/myc-CK2
. The CK2
-HA cDNA was isolated from pZW6 using
HindIII and ApaI and ligated into the SalI (MCS II) site of pBI/myc-CK2
to generate
pBI/myc-CK2
/CK2
-HA, which was designated pRS3. To generate the
bidirectional construct containing kinase-dead CK2
, pRS3 was
digested with Bsu36I and BstBI and fragments of
3493 and 2350 base pairs isolated. The third fragment generated in that
digestion (containing the sequence that codes for catalytic residue
K68) was discarded. Next, pGV15 was digested with BstBI and
Bsu36I and the DNA fragment containing the K68M mutation was
isolated and triple ligated with the 3493- and 2350-base pair fragments
to generate pBI/myc-CK2
/CK2
(K68M)-HA (designated pGV13). All
constructs were verified by restriction endonuclease digests or DNA
sequencing (42).
' or HA-CK2
'(K69M) and
myc-CK2
were generated as follows. The HA-CK2
' fragment was
digested pRc/CMV/HA-CK2
' (36) with HindIII and ligated into pBI/myc-CK2
at the SalI site (MCS II) to generate
pBI/myc-CK2
/HA-CK2
' (designated pRS2). The
pBI/myc-CK2
/HA-CK2
'(K69M) plasmid, designated pGV7, was
constructed by replacing the HindIII fragment encoding HA-CK2
' from pRS2 with a HindIII fragment encoding
HA-CK2
'(K69M) from pRc/CMV/HA-CK2
' (36). All constructs were
verified by restriction endonuclease digests or DNA
sequencing (42).
antiserum directed
against the C-terminal synthetic peptide
376-391,
polyclonal anti-CK2
' antiserum directed against the C-terminal synthetic peptide
'333-350, and polyclonal anti-CK2
antiserum directed against the C-terminal synthetic peptide
198-215 have been described previously (37, 43).
Polyclonal anti-CK2
antiserum directed against an N-terminal
synthetic peptide
2-19 were similarly prepared and was
used for the detection of CK2
-HA as the HA epitope interfered with
recognition by the antiserum directed against the C-terminal
376-391 peptide. The monoclonal antibody 12CA5, which
reacts against the HA epitope (44, 45), was purchased from BabCO
(Berkeley, CA). The hybridoma producing the 9E10 monoclonal antibodies
directed against the myc epitope (46) was injected into mice and
ascites fluid was collected and purified by ammonium sulfate
precipitation. Goat anti-rabbit or goat anti-mouse secondary antibodies
conjugated with alkaline phosphatase (AP) were purchased from
Bio-Rad.
-HA and myc-CK2
), GV13.35 (expressing CK2
(K68M)-HA and myc-CK2
), RS2.31(expressing HA-CK2
' and
myc-CK2
), and GV7.21 (expressing HA-CK2
'(K69M) and myc-CK2
),
exhibiting high levels of tightly regulated expression, were maintained
in medium containing 460 µg/ml (active) G418, 500 µg/ml hygromycin, and 1.5 µg/ml tetracycline (Sigma).
80 °C. Cells designated for immunoprecipitation were washed as
described above and lysed in Nonidet P-40 lysis buffer (1% Nonidet
P-40, 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, and
all of the protease and phosphatase inhibitors as above). The extracts
were sonicated and spun at 12,000 × g for 15 min to
remove debris. The protein concentration was determined using the BCA
protein assay (Pierce). Equal amounts of protein were aliquoted in
tubes and volumes equilibrated. The antibodies used were 12CA5 (8 µl/reaction) and 9E10 (0.5 µl/reaction) and 20 µl of a 50%
protein A-Sepharose solution (Amersham Pharmacia Biotech) was added to
the mixture. After incubation by tumbling for 1 h at 4 °C, the
protein A-Sepharose was isolated by centrifugation and washed once in
lysis buffer and three times in kinase buffer (50 mM
Tris-Cl (pH 7.5), 150 mM NaCl, 10 mM
MgCl2, 1 mM dithiothreitol). The pellet was
resuspended in kinase buffer and used for kinase assays.
(1/1000), polyclonal
anti-
' (1/400), polyclonal anti-
(1/500), 12CA5 monoclonal
antibody (1/500), or 9E10 monoclonal antibody (1/500) in 1% gelatin in
TBST for 1 h at room temperature. The membrane was washed twice
with TBST. Secondary antibody was goat anti-rabbit or goat anti-mouse
antibody conjugated with AP and diluted 1/3000 with 1% gelatin in
TBST. After reaction for 1 h at room temperature, the membrane was
washed twice with TBST and once with TBS. Visualization was performed
by developing with 5-bromo-4-chloro-3-indoyl phosphate
p-toluidine salt and p-nitro blue tetrazolium
chloride (Bio-Rad) in AP development buffer (100 mM Tris-Cl
(pH 9.5), 0.5 mM MgCl2).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-HA and myc-CK2
(RS3.22 cells) or with the pBI plasmid encoding HA-CK2
' and
myc-CK2
(RS2.31 cells). To interfere specifically with the functions
of each CK2 isoform, the UTA6 cells were stably transfected with the
pBI plasmid encoding kinase-inactive CK2
(K68M)-HA and myc-CK2
(GV13.35 cells) or with the pBI plasmid encoding kinase-inactive HA-CK2
'(K69M) and myc-CK2
(GV7.21 cells). Epitope tags
incorporated into the sequence of CK2
, CK2
', and CK2
permitted
detection of the exogenously expressed subunits in the presence of
endogenous CK2. Importantly, deleterious effects of the epitope tags on
the functions of CK2 have not been observed in previous studies from this (36) and other (19) laboratories.
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Fig. 1.
Schematic illustration of the bidirectional
constructs encoding the CK2 isoforms. The cDNAs encoding
catalytic (i.e. CK2 -HA, CK2
(K68M)-HA, HA-CK2
', or
HA-CK2
'(K69M)) or regulatory (i.e. myc-CK2
) subunits
of CK2 were subcloned into the respective multiple cloning sites (MCS
II and MCS I) of the pBI plasmid (40) as described under "Materials
and Methods." The pBI plasmid contains a bidirectional promoter
composed of a tetracycline response element consisting of seven copies
of the 42-base pair tet operator sequence. The tetracycline response
element is situated between two minimal CMV promoters that lack the
enhancer of a complete CMV promoter. Four constructs expressing
different forms of the CK2 catalytic subunits together with the
regulatory CK2
subunit are illustrated.
or CK2
' as
well as myc-CK2
is observed in each of the four cell lines. The
identities of these bands as HA-tagged proteins or as myc-tagged
proteins were confirmed on immunoblots performed with only one of the
antibodies (data not shown). Whereas one prominent band is detected in
each of the induced lanes with anti-HA antibodies, it is apparent that two bands are detected with anti-myc antibodies. It is noteworthy that
the lower myc-CK2
band was predominant in GV7.21 cells and to a
lesser degree in GV13.35 cells, whereas the upper band was predominant
in RS3.22 and in RS2.31 cells (Fig. 2A). As noted in our
previous examination of myc-CK2
(36), the upper band corresponds to
the phosphorylated form of CK2
, indicating that expression of
HA-CK2
'(K69M), and CK2
(K68M)-HA to a lesser degree, diminishes
the phosphorylation of myc-CK2
. We were unable to detect expression
of HA-tagged CK2
or CK2
' subunits in lysates of cells cultured in
the presence of tetracycline, demonstrating tight control of
expression. However, this does not exclude the possibility of low
levels of expression in the presence of tetracycline.
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Fig. 2.
Inducible expression of CK2.
RS3.22 cells (expressing CK2 -HA and myc-CK2
), GV13.35 cells
(expressing CK2
(K68M)-HA and myc-CK2
), RS2.31 cells (expressing
HA-CK2
' and myc-CK2
), and GV7.21 cells (expressing
HA-CK2
'(K69M) and myc-CK2
) were plated in 10-cm dishes at 1 × 106 cells/dish in the presence (+) or absence (
) of
1.5 µg/ml tetracycline. Forty-eight hours later, the cells were lysed
in sample buffer and 30 µg of protein subjected to SDS-polyacrylamide
gel electrophoresis. Protein expression was examined on Western blots
with a mixture of 12CA5 and 9E10 antibodies (A), anti-
antibodies (B), anti-
' antibodies (C), and
anti-
antibodies (D). The positions of the epitope-tagged
and endogenous CK2 subunits are indicated. The position of the
phosphorylated form of myc-CK2
(designated myc-CK2
-P) is also
indicated. A nonspecific band that is detected with anti-CK2
' is
also marked (panel C).
and anti-
' to detect
endogenous and transfected CK2
and CK2
' subunits. With RS3.22 and
GV13.35 cells, we observed that expression of the HA-tagged protein was
roughly equivalent to the levels of endogenous CK2
(Fig.
2B). By comparison, with RS2.31 and GV7.21 cells, we
observed in both cell lines that expressed HA-tagged CK2
' or
CK2
'(K69M) was significantly higher than endogenous CK2
' (Fig.
2C). As levels of CK2
-HA and HA-CK2
' are comparable
when probed with anti-HA antibodies, these results reflect the higher
levels of endogenous CK2
that are observed in these cells compared
with endogenous CK2
'. In addition, expression of HA-CK2
' in
RS2.31 does appear to be higher than the HA-CK2
'(K69M) in GV7.21
cells (Fig. 2, A and C). When each of the cell
lysates were probed with anti-
antibodies, we noted that myc-CK2
is expressed to lower levels than endogenous CK2
in all cell lines
(Fig. 2D). Together, these results demonstrate that
catalytic and regulatory subunits of CK2 are coordinately expressed in
each of the stably transfected cell lines and are tightly regulated by
tetracycline. Moreover, of the four cell lines, it is apparent that the
GV7.21 cells that inducibly express kinase-inactive CK2
' and
myc-CK2
exhibit the lowest levels of induced expression (Fig.
2A). Overall, we did not observe any large changes in the
expression of endogenous CK2
or CK2
' in the presence or absence
of tetracycline in any of the cell lines (Fig. 2, B-C).
'(K69M) is repressed to levels below
detection using Western blots at a tetracycline concentration of 100 ng/ml. Maximum expression was achieved at a concentration of 0.1 ng/ml
or less. Similar results were observed when probing with 12CA5 (Fig.
3A), 9E10 (Fig. 3B), or anti-
(Fig.
3C). In Fig. 3 (B and C), we noted that expression of endogenous CK2
' or CK2
, respectively, appeared unaltered during increasing expression of the transfected constructs. Similar results were obtained with RS2.31 cells expressing HA-CK2
' (data not shown). These results demonstrate modulation of the expression of HA-CK2
' and HA-CK2
'(K69M) in UTA6 cells by
modulating the concentration of tetracycline between 0.1 and 100 ng/ml
and provide further indications that two proteins that are encoded by
the pBI plasmid are coordinately expressed.
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Fig. 3.
Dose response of induced
HA-CK2 '(K69M) expression. GV7.21 cells
(expressing HA-CK2
'(K69M) and myc-CK2
) were plated in 10-cm
dishes at 1.5 × 106 cells/plate in increasing
concentrations of tetracycline. Forty-eight hours later, expression of
both HA-CK2
'(K69M) and myc-CK2
was monitored by Western blotting.
A-C, 12CA5 and 9E10 antibodies (A), anti-
'
antibodies (B), and anti-
antibodies (C) were
used in monitoring expression. Endogenous CK2
' and CK2
are
indicated in B and C, respectively.
Phosphorylated (myc-CK2
-P) and non-phosphorylated forms of
myc-CK2
are also indicated. Equal amounts of protein were loaded in
each lane. The unmarked upper band in
panel B is a nonspecific band that is detected
with anti-CK2
' as in Fig. 2.
' (Fig.
4A) and HA-CK2
'(K69M) (Fig.
4B) were detectable in Western blots after only 6 h in
the absence of tetracycline and reached a maximum level at 24 h.
We also noted that expression of HA-CK2
'(K69M) was decreased after
72 h. This observation was seen in three independent experiments
and may in part explain the lower levels of HA-CK2
'(K69M) expression
that were observed (Fig. 2). Interestingly, a similar phenomenon was
noted by Resnitzky (50), who observed that induced levels of cyclin D1
were not maintained after 24 h of induction. The mechanism by
which this occurs is unknown at this time. We also examined the time
course of induction of CK2
-HA and CK2
(K68M)-HA and noted that
expression of both proteins, unlike HA-CK2
'(K69M), was sustained
(data not shown). Collectively, the dose response and time course of
induction demonstrate that expression of both CK2 subunits is under
coordinate control in both cell lines, an important demonstration that
the bidirectional promoter is suitable for studies of this nature.
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Fig. 4.
Time course of induced CK2 expression.
A and B, RS2.31 cells (A, expressing
HA-CK2 ' and myc-CK2
) and GV7.21 cells (B, expressing
HA-CK2
'(K69M) and myc-CK2
) were seeded in 10-cm plates at 1 × 106 cells/plate in the absence of tetracycline. The
cells were harvested at the indicated time points and lysed in sample
buffer. Equal amounts of protein (40 µg) were loaded in each lane and
resolved by SDS-polyacrylamide gel electrophoresis and analyzed by
Western blotting. The HA- and myc epitope-tagged CK2 constructs were
detected using a mixture of 12CA5 and 9E10 antibodies as described
under "Materials and Methods." The positions of the epitope-tagged
CK2 subunits are indicated.
-HA, CK2
(K68M)-HA, HA-CK2
', and
HA-CK2
'(K69M), immunoprecipitations were performed using 12CA5 (Fig.
5A) or 9E10 (Fig.
5B) antibodies from lysates of cells cultured in the
presence or absence of tetracycline. When kinase assays were performed
on immunoprecipitates from cells cultured in the presence of
tetracycline, negligible activity was observed. By comparison, in the
absence of tetracycline, kinase activity was readily detected in both
12CA5 and 9E10 immunoprecipitates. As expected, much greater activity
was observed in 12CA5 immunoprecipitates from cells expressing
CK2
-HA or HA-CK2
' than from cells expressing CK2
(K68M)-HA or
HA-CK2
'(K69M). The observation that immunoprecipitates from cells
expressing either of the kinase-inactive proteins displayed some kinase
activity likely reflects the existence of endogenous CK2 subunits in a
minor fraction of tetrameric CK2 complexes that contain the kinase-dead
proteins. As expected, immunoprecipitates performed with 9E10
antibodies also exhibit higher activity from the cells expressing
kinase active CK2 isoforms. However, the difference was less striking
than that observed with 12CA5 antibodies. Again, this result likely
reflects the incorporation of myc-CK2
into complexes containing
exogenous and endogenous catalytic CK2 subunits. Importantly, these
immunocomplex kinase activities provide indications that the expression
levels of constructs encoding CK2
and CK2
' are similar, a result
that is in correspondence with the results of immunoblot analysis.
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Fig. 5.
Kinase activity of induced CK2. Lysates
from RS3.22 cells (expressing CK2 -HA and myc-CK2
), from GV13.35
cells (expressing CK2
(K68M)-HA and myc-CK2
), from RS2.31 cells
(expressing HA-CK2
' and myc-CK2
), and from GV7.21 cells
(expressing HA-CK2
'(K69M) and myc-CK2
) were prepared from cells
cultured in the presence (+, open bars) or
absence (
, solid bars) of tetracycline for
48 h. Kinase assays were performed on immunoprecipitates prepared
with 12CA5 (A) or 9E10 antibodies (B) as
described under "Materials and Methods." C, kinase
assays in whole cell lysates. The kinase activity results are expressed
in picomoles/min and are the average of duplicate determinations (the
ranges are indicated by the error bars). The
experiment is representative of three independent experiments.
-HA and HA-CK2
', respectively, demonstrated 2-3-fold
increases in kinase activity following induction of the respective CK2
isoform. These results indicate that similar levels of each protein are
expressed, a result that corresponds well with the results of
immunoblot analysis (Fig. 2) and the immunocomplex kinase assays (Fig.
5, B and C). Induced expression of
kinase-inactive CK2 isoforms also resulted in increases in CK2 activity
of approximately 1.5-fold (Fig. 5C). The latter result was
somewhat surprising, as these constructs encode proteins that are
devoid of kinase activity. We do not have a precise explanation for the
observed increases in CK2 activity in the GV13.35 and GV7.21 cells. It is possible that induced expression of CK2 subunits could result in
increased formation of tetrameric CK2 complexes that include endogenous
subunits. In fact, we have previously shown that CK2 subunits are
stabilized through the formation of multi-subunit complexes (36).
Alternatively, cells may have feedback control mechanisms that result
in increased expression of CK2 under circumstances where CK2 activity
is compromised. This issue has not been further examined.
-HA did not enhance
cell proliferation and in fact seemed to induce a slight decrease in
proliferation (Fig. 6B). Increased expression of HA-CK2
'
also resulted in a modest decrease in proliferation (Fig.
6D). By comparison, induced expression of kinase-inactive
CK2 isoforms had dramatically different consequences. Whereas
CK2
(K68M)-HA was without significant effect (Fig. 6C),
HA-CK2
'(K69M) induced a dramatic 4-5-fold reduction in
proliferation over the time course (Fig. 6E). We also
observed that a number of cells expressing HA-CK2
'(K69M) in the
absence of tetracycline would float off the culture dishes, suggesting that expression may affect cell viability and may possibly induce apoptosis. As levels of HA-CK2
'(K69M) were, if anything, lower than
that observed with CK2
(K68M)-HA (see Fig. 2), we believe that these
results reflect a potent ability of HA-CK2
'(K69M) to influence cell
proliferation in a manner that is distinct from the CK2
(K68M)-HA.
Importantly, these results provide the first direct evidence for a role
of CK2
' in cell proliferation in mammalian cells and provide
evidence for functional differences between CK2
and CK2
'.
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Fig. 6.
Cell proliferation profiles of the inducible
CK2 cell lines. Parental UTA6 cells (A), RS3.22 cells
(expressing CK2 -HA and myc-CK2
) (B), GV13.35 cells
(expressing CK2
(K68M)-HA and myc-CK2
) (C), RS2.31
cells (expressing HA-CK2
' and myc-CK2
) (D), and GV7.21
cells (expressing HA-CK2
'(K69M) and myc-CK2
) (E) were
seeded in six-well dishes at 2 × 104 cells/well in
medium with (open circles) or without
(closed circles) 1.5 µg/ml tetracycline. The
medium was changed every 3 days. Cell counts were obtained in
triplicate for a period up to 10 days. The results represent the
average (± S.D.) of triplicate determinations for one experiment that
is representative of three independent experiments.
' or induced expression of HA-CK2
'(K69M), we
sought to determine whether this result arose from a specific cell
cycle perturbation. Flow cytometric analysis of both cell lines, RS2.31
(Fig. 7A) and GV7.21 (Fig.
7B), was performed at 2 and 4 days after release from
tetracycline. In neither cell line was there any indication of a
specific arrest or delay in one particular stage of the cell cycle. The
data in Fig. 7C reveal only modest changes in the cell cycle
profiles of both cell lines. There was also a modest increase in the
percentage of sub-G1 (apoptotic) population in GV7.21 cells
as compared with RS2.31 cells (data not shown). However, the proportion
of apoptotic cells observed in cells expressing HA-CK2
'(K69M) did
not exceed 10% of the total population and is not likely sufficient to
account for the dramatic decrease in proliferation. Collectively, these
results are consistent with a pleiotropic role for CK2 in regulating
the growth and survival of mammalian cells.
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Fig. 7.
Cell cycle distribution following induced
expression of HA-CK2 ' or
HA-CK2
' (K69M). RS2.31 cells (expressing
HA-CK2
' and myc-CK2
) (A) and GV7.21 cells (expressing
HA-CK2
'(K69M) and myc-CK2
) (B) were seeded in 10-cm
dishes at a concentration of 1 × 105 cells/dish with
(+) or without (
) 1.5 µg/ml tetracycline (Tet.). On the
indicated days, the cells were harvested, fixed in ethanol, and stained
with propidium iodide to examine the cell cycle distribution as
described under "Materials and Methods." Cell cycle analysis was
performed using a FACScan. C, the percentage of cells in
each phase of the cell cycle is shown for the corresponding days in the
absence or presence of tetracycline. The percentage of cells in each
cell cycle phase was determined using the ModFit modeling software
program.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or CK2
') and regulatory (i.e. CK2
) subunits of
CK2 were obtained, demonstrating the utility of a bidirectional vector for the simultaneous expression of two proteins that are destined to
form a multi-subunit complex (40). As previously noted in our transient
transfection studies (36), we observed that CK2
constructs harboring
an N-terminal HA epitope tag were expressed at levels much lower than
was observed with constructs encoding CK2
' with an N-terminal HA tag
(data not shown). Consequently, it was necessary to generate CK2
constructs encoding a C-terminal HA epitope similar to those described
by Heriche et al. (19). As noted by Western blotting and in
kinase assays, the CK2
constructs with C-terminal epitope tags were
expressed to levels comparable to those observed with the N-terminally
tagged CK2
' constructs. We do not understand why the expression
levels of HA-CK2
are so much lower than that observed with
HA-CK2
' or CK2
-HA (i.e. typically 20-fold lower).
Importantly, studies from this (36) and other laboratories (19) have
not observed any deleterious effects of the epitope tags on the
functional properties of CK2.
and CK2
', we
anticipated that we could interfere with their respective cellular functions (36, 54). As it is likely that CK2
and CK2
' have a
similar capacity to form complexes with CK2
, we wanted to ensure that the overexpression of one kinase-inactive isoform did not result
in decreased formation of complexes between the other isoform and
CK2
. Accordingly, myc-CK2
was co-expressed with either CK2
-HA or HA-CK2
' to ensure that the former would not be limiting for the
formation of tetrameric CK2 complexes. It is also important to note
that the total CK2 activity that was measured in lysates of cells with
the induced expression of CK2
(K68M)-HA or HA-CK2
'(K69M) was, if
anything, greater than that measured in cells without induced
expression. These results indicate that the expression of
kinase-inactive mutants of CK2 did not interfere directly with the
activity of endogenous CK2. Instead, we would expect that the
interference by CK2
(K68M)-HA or HA-CK2
'(K69M) would result from
their ability to compete with endogenous CK2
or CK2
',
respectively, for cellular substrates with a resultant decrease in
substrate phosphorylation. Our observation that the total cellular CK2
activity appeared to increase following induction of CK2
(K68M)-HA or
HA-CK2
'(K69M) may also reflect the existence of an autoregulatory
mechanism whereby CK2 controls its own expression. For example, there
may be cellular targets for CK2 that inhibit the expression of CK2 when
they are phosphorylated such that interfering with the phosphorylation of these targets would result in increased expression of endogenous CK2. Cells exhibiting the inducible expression of CK2 may provide a
novel opportunity for an examination of such a regulatory mechanism.
'(K69M)
attenuated cell proliferation is not unexpected as CK2 appears to be
involved in various aspects of cell cycle control (51, 52, 55).
However, it is important to note that these data are the first
demonstration where a strategy designed to specifically interfere with
CK2
' resulted in diminished proliferation. Previous studies that
have used antisense oligonucleotides to down-regulate the expression of
CK2 focused exclusively on CK2
or CK2
without attempts to
down-regulate CK2
' (56). Collectively, these results suggest that
both CK2
and CK2
' are required for proliferation suggesting that
the two CK2 isoforms may have some overlapping functions in mammalian
cells. Consistent with a role for both CK2
and CK2
' in cell
proliferation is the demonstration that the expression of both isoforms
of CK2 is induced following serum stimulation of quiescent cells (18,
57). However, as proliferation could be attenuated through a variety of
mechanisms, including increases in cell death or decreases in cell
cycle progression, it is premature to conclude that CK2
and CK2
'
do indeed have entirely redundant functions. Moreover, data in this
study demonstrate that similar levels of induced kinase-inactive CK2
and kinase-inactive CK2
' have strikingly different outcomes.
Importantly, these data provide the first direct evidence for
functional specialization of CK2 isoforms in mammalian cells.
and CK2
' behave
as delayed early genes and that either CK2
or CK2
' can enhance
proliferation when overexpressed in fibroblasts (18), we were surprised
to observe diminished cell proliferation in cells overexpressing
CK2
-HA or HA-CK2
'. We do not have a precise explanation for the
discrepancies between our studies and those published elsewhere.
However, given that the expression of CK2
or CK2
' can be
precisely controlled in the studies reported here, we are confident
that the effects do not arise from issues related to clonal variation
or differences in transfection efficiency that can occur without
inducible expression. It is possible that the difference lies in the
fact that our studies were performed by co-expression of catalytic and
regulatory CK2 subunits whereas Orlandini et al. (18)
performed overexpression of only the catalytic subunits of CK2. In this
regard, our cells would likely have lower levels of uncomplexed CK2
or CK2
' than would be observed with the exclusive expression of
these proteins without myc-CK2
. As it is apparent from biosynthetic
labeling studies that the bulk of cellular CK2 exists in tetrameric
complexes (58), we believe that our system represents a natural
situation. This conclusion is based on the observation that the
majority of the myc-CK2
exhibits the electrophoretic characteristics
of autophosphorylated CK2
when it is expressed in cells together
with active CK2
' (36). Inasmuch as we have previously shown in
transient transfection experiments that the autophosphorylation of
myc-CK2
occurs efficiently when it forms complexes with CK2
',
this result suggests that the majority of CK2
is in complexes.
Therefore, we believe that the results seen here do not arise from the
high expression of uncomplexed CK2
.
and
CK2
'. To characterize the induction of CK2, it was necessary to
employ a strategy to distinguish between the expressed CK2 and the
endogenous CK2. Consequently, we utilized the HA and Myc epitopes that
have been widely utilized for the expression of proteins in mammalian
cells (44-46). For a number of reasons, we believe that the epitope
tags used in this study do not significantly affect the functional
properties of CK2. In particular, similar constructs have been utilized
in studies from this (36) and other laboratories (19). Importantly, the
epitope tags do not interfere with the ability of CK2 to form complexes
consisting of catalytic and regulatory CK2 subunits. Furthermore,
epitope-tagged CK2 exhibits the same localization as that of endogenous
CK2 (36). However, we cannot directly exclude the possibility that the
tags interfere with specific cellular functions of CK2 (i.e.
recognition and/or phosphorylation of a specific protein target).
Consequently, it is conceivable that the epitope tags contribute to the
modest decreases in proliferation that are seen upon induced
overexpression of CK2. Despite this limitation, we believe that our
data clearly suggest that increases in the expression of CK2 are not
sufficient to accelerate cell proliferation.
or
CK2
' would increase the phosphorylation of target proteins whereas
the expression of kinase-inactive CK2 isoforms could interfere with the
phosphorylation of target proteins, we would expect that the mechanisms
responsible for the attenuated proliferation observed with CK2
-HA or
HA-CK2
' may be different from that observed with HA-CK2
'(K69M).
In the case of CK2
-HA or HA-CK2
', we do not have any precise
insights into how increased expression brings about diminished
proliferation as no significant alterations in cell cycle distribution
were observed nor were there any indications of decreased viability or
increased apoptosis. However, given that CK2 is believed to play a role
at various points during the cell cycle in yeast and in mammalian
cells, we speculate that its increased expression causes delays during
multiple stages of the cell cycle with no obvious accumulations during
any specific stage. Despite all of the correlations between increased
expression of CK2 and increased proliferation, it is not inconceivable
that the increased phosphorylation of substrate proteins could result in attenuated proliferation. For example, the phosphorylation of
several potential CK2 substrates including c-Jun (21), c-Myb (22, 59),
and p53 (60) could actually result in diminished proliferative
capacity. With c-Myb and c-Jun, phosphorylation by CK2 has been shown
to inhibit their DNA binding activity, a result that would be expected
to inhibit proliferation. By comparison, there are indications that CK2
plays a role in enhancing the DNA binding activity of p53 and the
ability of p53 to induce cell cycle arrest.
'(K69M), despite the dramatic attenuation of
proliferation, it does not appear to induce a specific arrest at one
particular stage in the cell cycle. In a similar vein, it is noteworthy
that inactivating mutations of one of the isoforms of CK2 in S. cerevisiae also affected multiple stages in the cell cycle (51).
Moreover, studies in mammalian cells where microinjected antibodies
were used to interfere with CK2 also indicated that CK2 is required at
multiple points during the cell cycle (52). At present, the precise
mechanisms responsible for the effects of HA-CK2
'(K69M) remain to be
elucidated. However, there are interesting possibilities to consider.
For example, as CK2 was recently shown to exhibit cooperativity with
Ha-Ras in the transformation of fibroblasts (18), it is conceivable that the kinase-inactive form of CK2
' interferes with
Ras-dependent signaling events. There are also indications
that the induced expression of HA-CK2
'(K69M) compromises the
viability of cells as evidenced by a high number of cells that became
detached from the substrate and increases the proportion of cells that
were apoptotic (data not shown). The apoptotic population of cells that
was detected as the sub-G0/G1 peak by
fluorescence-activated cell sorting was, however, quite low (less than
10% of the total cells), suggesting that apoptosis was not by itself
responsible for the dramatic alterations in proliferation that were
observed (data not shown). Collectively, these data suggest that
CK2
' has substrates that need to be optimally phosphorylated in
order to achieve optimal cell cycle progression or to maintain cell survival. With respect to cell survival, it is interesting to note that
the substrate specificity of CK2 is strikingly similar to members of
the caspase family (61). In particular, CK2 typically phosphorylates
serine/threonine residues that are followed by a series of acidic
residues (i.e. glutamic or aspartic acids) (6), whereas the
caspases typically cleave aspartic acid residues that are often
preceded by other acidic residues (aspartic acids in particular) (61).
It is therefore likely that there will be a number of proteins where
CK2 sites are adjacent to the recognition sequences for caspases. Thus,
it is intriguing to speculate that CK2 could have a role in controlling
cell survival by regulating the ability of caspases to degrade their targets.
and
CK2
' provide the first direct evidence for functional specialization
of CK2 isoforms in mammalian cells. Finally, by establishing model
systems where the expression of CK2 can be reliably manipulated, we are
now for the first time in a position to systematically examine the
mechanisms by which CK2 influences the life and death of mammalian cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Christoph Englert for cell lines, Dr. Bernhard Lüscher for cell lines and helpful discussions, Kevin C. Graham for technical assistance and advice, and Zilong Wang for plasmid construction.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Terry Fox Foundation (to D. W. L.).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.
These authors contributed equally to this work.
§ Supported by a fellowship from the Faculty of Medicine at the University of Western Ontario with funds from the Cancer Research Society Inc.
¶ Research Scientist of the National Cancer Institute of Canada. To whom correspondence should be addressed. Tel.: 519-661-4186; Fax: 519-661-3175; E-mail: litchfi{at}julian.uwo.ca.
2 D.G. Bosc, K. C. Graham, D. Prober, R. D. Gietz, and D. W. Litchfield, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: CK2, protein kinase CK2 or casein kinase II; HA, the YPYDVPDY epitope of influenza virus hemagglutinin; MCS, Multiple Cloning Site; myc, the MASMEQKLISEEDLNN epitope of the c-Myc protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TA, pCRII vector (Invitrogen); TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween 20; AP, alkaline phosphatase.
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REFERENCES |
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