Protein Kinase CK2
' Is Induced by Serum as a
Delayed Early Gene and Cooperates with Ha-ras in Fibroblast
Transformation*
Maurizio
Orlandini
,
Francesca
Semplici
,
Rebecca
Ferruzzi
,
Flavio
Meggio§,
Lorenzo A.
Pinna§, and
Salvatore
Oliviero
¶
From the
Dipartimento di Biologia Molecolare,
Università di Siena IRIS, via Fiorentina 1, 53100 Siena and the
§ Dipartimento di Chimica Biologica e Centro di Studio sulle
Biomembrane del CNR, Università di Padova, viale Colombo
3, 35121 Padova, Italy
 |
ABSTRACT |
Protein kinase CK2 is an ubiquitous and
pleiotropic Ser/Thr protein kinase composed of two catalytic (
and/or
') and two noncatalytic (
) subunits forming a
heterotetrameric holoenzyme involved in cell growth and
differentiation. Here we report the identification, cloning, and
oncogenic activity of the murine CK2
' subunit. Serum
treatment of quiescent mouse fibroblasts induces CK2
'
mRNA expression, which peaks at 4 h. The kinetics of
CK2
' expression correlate with increased kinase activity
toward a specific CK2 holoenzyme peptide substrate. The ectopic
expression of CK2
' (or CK2
) cooperates
with Ha-ras in foci formation of rat primary embryo
fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts
transformed with Ha-ras and CK2
' show a
faster growth rate than cells transformed with Ha-ras
alone. In these cells the higher growth rate correlates with an
increase in calmodulin phosphorylation, a protein substrate
specifically affected by isolated CK2 catalytic subunits but not by CK2
holoenzyme, suggesting that unbalanced expression of a CK2 catalytic
subunit synergizes with Ha-ras in cell transformation.
 |
INTRODUCTION |
Protein kinase CK2 (previously known as casein kinase II) is an
ubiquitous Ser/Thr kinase present in the cytoplasm and the nucleus of
eukaryotic cells (for review, see Refs. 1-5). CK2 holoenzyme consists
of two catalytic (
and/or
') and two regulatory (
) subunits
assembled as stable heterotetramers, which in vitro do not
dissociate unless under denaturing conditions. CK2 is unique among
Ser/Thr protein kinases for its ability to use GTP, besides ATP, as
phosphate donor and for its unusual site specificity, which is
determined by multiple acidic and/or previously phosphorylated residues
downstream (n+3) from the phosphoacceptor amino acid, determining the minimum consensus
(S/T-X-X-E/D/Yp/Sp) (6).
More than 160 cellular proteins have been reported to be phosphorylated
by CK2, and several are implicated in signal transduction, transcriptional activation, cell cycle progression, and cell
differentiation. The nuclear proteins that are CK2 substrates
includes: c-Myc (7), Max (7), c-Myb (8), serum response factor (SRF)
(9), DNA ligase I (10), DNA topoisomerase 2 (11), p53 (12), and c-Fos
(13). In mammalian cells phosphorylation of nuclear factors dependent
on CK2 could be relevant for cell growth regulation and the progression
into the cell cycle. A direct role of CK2 activity in cell cycle
progression has been demonstrated by antibody-mediated CK2 depletion
and by gene inactivation in Saccharomyces cerevisiae (14,
15). Although hundreds of papers have been published on the subject, it
is still unknown how the enzyme is regulated in vivo (4, 5,
16). CK2
undergoes stoichiometric autophosphorylation and both
CK2
and CK2
(but not CK2
') are phosphorylated in
vitro and in vivo by p34Cdc2
kinase (17). However, these phosphorylations do not correlate with any
regulation of activity. Moreover, it is not clear whether the
holoenzyme represents an up- or a down-regulated form of the kinase,
because some substrates are preferentially phosphorylated by the
tetramer, but others, like calmodulin, are phosphorylated only by the
free catalytic subunits (6).
In transgenic mice it was possible to demonstrate that in T cells the
overexpression of the catalytic CK2
subunit enhanced the
onset of lymphomas induced by either c-myc or
tal-1 (18, 19). These results shed new light on the previous
observations that cattle infected by the parasite Theileria
parva developed T cell lymphomas, because parasite-infected cells
show increased CK2 activity (20-22). Opposite results were obtained by
the overexpression of CK2
in NIH 3T3 mouse fibroblasts.
In these cells CK2
overexpression resulted in
deactivation of the mitogen-activated protein kinase kinase and
suppression of ras-dependent cell transformation
(23).
To identify genes potentially involved in cell growth, we performed a
differential screening for the isolation of transcripts induced by
mitogenic stimuli within the G1 phase. Here we report the
identification and cloning of the murine lower molecular weight catalytic subunit, CK2
'. We observed that in mouse
fibroblasts CK2
' is induced by serum treatment as a
slow-early gene. Together with CK2
' also
CK2
and CK2
are induced. Cotransfection of
an expression vector containing CK2
' together with a
vector expressing Ha-ras induced foci formation in rat
primary embryo fibroblasts. Moreover, ras-transformed BALB/c
3T3 fibroblasts overexpressing CK2
' showed a faster
growth rate than cells transformed with Ha-ras alone.
ras-Transformed fibroblasts overexpressing
CK2
' also exibited increased phosphorylating activity
toward calmodulin, which is a specific substrate of CK2 catalytic
subunits. These findings suggest that unbalanced expression of either
CK2
' or CK2
plays a role in fibroblast cell
transformation.
 |
EXPERIMENTAL PROCEDURES |
Cells and Cell Culture--
NIH 3T3 and BALB/c 3T3 fibroblasts
were grown at 37 °C in
DMEM1 supplemented with 10%
heat-inactivated FCS, penicillin-streptomycin, and glutamine. The cells
were expanded by trypsin-EDTA treatment and subcultured at a ratio of
1:3 every 2-3 days. Rat embryo fibroblasts were isolated as described
previously (24). Briefly, 14-day CDF(F344) rat embryos were sacrificed,
rinsed, and trypsinized for 30 min at 37 °C. DMEM containing 10%
FCS was added, and the cells were centrifuged, dispersed, counted, and
plated on 100-mm tissue culture dishes at a density of 2 × 106/dish. After 48 h the cells were trypsinized, and
aliquots were frozen in liquid nitrogen.
Differential Display and Cloning of the Murine CK2
'
cDNA--
To induce a relatively quiescent cell population,
subconfluent NIH 3T3 fibroblasts were incubated for 48 h in DMEM
plus 0.5% FCS. Cells were then treated for 2 and 4 h with DMEM
supplemented with 10% FCS. Total cellular RNA was extracted using the
guanidinium thiocyanate method (25) from quiescent and serum-treated
fibroblasts and subjected to the differential display technique as
described previously (26, 27). The amplified cDNA fragments were
compared in nondenaturing polyacrylamide gels. A serum-induced cDNA
fragment, named L-0401, was excised, recovered by boiling, reamplified, and cloned into pGEM-T vector (Promega). The L-0401 cDNA fragment (230 bp), whose corresponding mRNA was homologous to human
CK2
', was labeled with [32P]dCTP by random
primer labeling and used to screen a mouse fibroblast cDNA library
(27). The positive clones, inserted into the pBluescript SK vector,
were sequenced on both strands either automatically using a
Perkin-Elmer model 373 DNA sequencer or manually using a Sequenase 2.0 kit (U. S. Biochemical Corp). A positive clone, named pBS38
'
(FS304), contained the full-length mouse CK2
' cDNA.
Northern Blot Analysis--
Total RNA (10 µg) was run on
denaturing formaldehyde-agarose gels and stained with ethidium bromide
to verify that each lane contained similar amounts of undegraded rRNA.
RNA was transferred onto nylon membranes and cross-linked by UV
irradiation. Filters were hybridized with 32P-labeled
probes and washed as described (27). The mouse CK2
' probe
was obtained from the full-length cDNA (pBS38
'). The cDNA fragments of murine CK2
(base pairs 421-841) and
CK2
(base pairs 912-1321) were obtained by cDNA
amplification of a mouse fibroblast cDNA library (27). The
sequences of the primers used for amplification (5'-GCTTCGATATGACCGTCACG-3' and 5'-GACTCAACTACTAAATCCG-3' for CK2
and 5'-GTACCAGCAGGGAGACTTTGGCTAC-3' and
5'-CATAGACTTCCTGAAAGGGTGGCAG-3' for CK2
), were
obtained from EBI Nucleotide Sequence Data Base under accession number
U17112 for CK2
and X56502 for CK2
. The
amplified cDNA fragments were sequenced, labeled with
[32P]dCTP, and used in Northern blot.
Construction of Expression Vectors--
CK2
' open
reading frame (from P-2 to R-350) was amplified by PCR from
pBS38
' with a 5' primer containing a BamHI restriction site (5'-CGCGGATCCCGGCCCGGCCGCG-3') and a 3' primer containing a
KpnI restriction site (5'-CGGGGTACCTCATCGTGCTGCGGT-3'). The PCR product was digested with BamHI and KpnI and
cloned into the BamHI/KpnI site of a bacterial
expression vector (FS310) made by subcloning the
XbaI/XhoI fragment of pQE-31 (Qiagen) into the XbaI/XhoI site of pBluescript SK to give
pQEBS
' (FS311). The construct expressing murine CK2
'
under the control of the cytomegalovirus promoter, pcDNA
', was
made by subcloning a StuI/partial
XhoI-digested fragment from pBS38
' into the
EcoRV/XhoI site of pcDNA3 (Invitrogen). To
obtain murine CK2
under the control of the
cytomegalovirus promoter, the 5' end of CK2
was amplified
by PCR from pT7-7CKII
(28) with a 5' primer containing a
HindIII restriction site and an optimal Kozak sequence (29)
(5'-GAGAAAGCTTCCACCGCCATGTCGGGACCCGTGCC-3') and a 3' primer downstream
from an XhoI restriction site (5'-CTTGATTTCCCCATTCCACC-3'). The PCR product was digested with HindIII and
XhoI and cloned into the HindIII/XhoI
site of pcDNA3 to give pcDNA
5' (MO339). A
XhoI/blunt-ended HindIII fragment, corresponding
to the 3' end of CK2
, was released from pT7-7CKII
and
subcloned into the XhoI/blunt-ended ApaI site of
pcDNA
5' to give pcDNA
(MO346). All constructs were sequenced on both strands.
Transfection of Cells and Foci Formation--
Subconfluent cells
were fed with culture medium 1-2 h before transfection. Cells were
cotransfected with 5 µg of activated Ha-ras and 10 µg of
each construct for 8 h by standard CaPO4 precipitation procedures (24). Where necessary vector plasmid (pcDNA3) was added
to reach the total amount of 15 µg of DNA per transfected plate. At
least two different cesium chloride DNA preparations of each construct
were independently transfected. The cells were rinsed with PBS and
re-fed with culture medium 15 h posttransfection. The cells were
trypsinized 24 h after transfection and split 1:3. When the cells
reached confluence they were re-fed with DMEM containing 2% FCS, and
the medium was changed every 2 days. Stable cell transformants were
visible after 7-10 days. Individual foci from BALB/c 3T3 cells
transformed by Ha-ras or by Ha-ras plus
CK2
' were picked and examined for their growth
properties. For growth rate analysis cells were plated in duplicate at
1 × 105 cells on 60-mm Petri dishes in DMEM plus 2%
FCS. Cells were counted every day using a hemacytometer and the medium
was changed every 3 days. For tumor growth assays, 106
cells in midlog-phase growth were harvested, washed with PBS, resuspended in 200 µl of PBS, and injected subcutaneously into the
scapular region of BALB/c nude mice. After 2 weeks of growth, the mice
were sacrificed, and the tumors were surgically removed and
weighed.
Cell Extracts and Phosphorylation Assays--
Whole-cell
extracts were prepared by rinsing cultures grown on Petri dishes with
PBS followed by harvesting with a rubber policeman. Cells were pelleted
by brief centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.05% Triton X-100) containing a protease
inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A). The
cell lysates were centrifuged at 40,000 × g for 20 min
at 4 °C, and the supernatant was used in phosphorylation assays.
Phosphorylation experiments were performed by incubating the substrate
(200 µM peptide RRRADDSDDDDD or 10 µM
calmodulin) in a buffer containing 50 mM Tris-HCl, pH 7.5, 12 mM MgCl2, 100 mM NaCl, and 20 µM [
-32P]dATP for 10 min at 37 °C.
NaCl was omitted when calmodulin was used as substrate. The reaction
was stopped by cooling in ice followed, in the case of calmodulin, by
SDS-polyacrylamide gel electrophoresis, staining with Coomassie Blue
and either autoradiographed or directly scanned on Instant Imager
Apparatus (Canberra-Packard). 32P incorporated into the
peptide substrate was evaluated by the phosphocellulose paper procedure
(30).
 |
RESULTS |
Isolation and Characterization of the Murine CK2
'
cDNA--
In cultured mouse fibroblasts growth factor depletion
leads the cell to exit from the cell cycle and become quiescent. Serum treatment induces re-entry into the cell cycle, which is likely because
of the induction of early genes. To identify new serum-induced genes,
we used the mRNA differential display technique (26, 27). NIH 3T3
fibroblasts were serum-starved for 48 h and the RNAs were
collected either from starved cells or from cells treated with serum at
different time points. Several cDNAs, obtained only from
serum-induced cells, were amplified and sequenced. Comparison of the
cDNA fragments with the EBI Nucleotide Sequence Data Base revealed
that a cDNA induced at 4 h after serum treatment was highly
similar to the human protein kinase CK2
'. To clone the full-length cDNA coding for CK2
', a mouse fibroblast
cDNA library (27) was screened. The few positive clones were
sequenced using internal sequencing primers, and the nucleotide
sequence of one clone of 1877 base pairs in length revealed a single
open reading frame coding for a putative protein of 350 amino acid
residues. The mouse and human predicted protein products shared 98.9%
amino acid identity. The strong similarity of the murine CK2
'
deduced protein sequence with its corresponding human homologue
suggests a highly conserved function of CK2
'. Murine CK2
' protein
shows a lower degree of identity with CK2
. The two proteins share
82.4% identity over 347 amino acids overlap (Fig.
1). The greatest difference is in the
C-terminal domains, because the deduced CK2
' protein sequence is 41 amino acids shorter and thus lacks the p34Cdc2
sites phosphorylated during the cell cycle (17). CK2
' also lacks the
HEHRKL amino acid residues (166-171 of CK2
) that have been
implicated in the interaction with protein phosphatase 2A (23).

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 1.
Comparison of the deduced amino acid sequence
of mouse CK2 ' with the murine CK2 protein sequence (accession
number U17112). Mouse CK2 ' amino acid sequence was determined
from the cDNA clone pBS38 ', obtained by screening a fibroblast
cDNA library as described under "Experimental Procedures."
Vertical lines indicate identical amino acids,
colons indicate a conservative amino acid change, and
dashes represent a gap introduced to maximize sequence
alignment. The HEHRKL amino acid sequence of CK2 (residues 166-171)
is underlined.
|
|
To confirm that the cDNA encoded a biologically active CK2
'
enzyme we expressed the cDNA in Escherichia coli.
Recombinant CK2
' showed a molecular mass of about 41 kDa consistent
with the predicted size of the protein, and the nondenatured soluble bacterial extract containing the immunoreactive CK2
' was able to
phosphorylate the CK2 peptide substrate RRRADDSDDDDD in
vitro (not shown).
CK2 is an ubiquitously expressed protein kinase essential for cell
growth. Northern blot analysis with a CK2
' probe revealed two hybridizing transcripts of 2.2 and 4.2 kilobases, respectively. The
expression of CK2
' mRNA is relatively constant in all
tissues with the exception of testis where a much stronger
CK2
' signal was detected (Fig.
2). These results contrast with
CK2
expression, which is abundant in brain and barely
expressed in testis (31, 32). Thus, CK2
and
CK2
' are differentially regulated in these tissues.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of CK2a' mRNA in
mouse adult tissues. Multiple tissue Northern blot containing 2 µg of poly(A)+ RNA (CLONTECH) was
hybridized with CK2 ' cDNA. -Actin was
used as a control for RNA loading. Tissues are indicated as follows:
t, testis; k, kidney; sm, skeletal
muscle; li, liver; lu, lung; sp;
spleen; b, brain; h, heart.
|
|
CK2
' Is Induced by Serum Treatment in Cultured
Fibroblasts--
The identification of CK2
' in a
screening for mRNAs induced by serum treatment of quiescent
fibroblasts suggested that this gene is induced by mitogenic stimuli.
To measure the induction of the CK2
' transcript, we
performed Northern blot analysis on CK2
' mRNA in
fibroblasts before and after serum treatment. As can be observed in
Fig. 3A the CK2
'
mRNA was low in quiescent fibroblasts and increased about 50% in
serum-induced fibroblasts, with a peak induction at 4 h. The
quantitative analysis of the transcripts normalized to the
glyceraldheyde-3-phosphate dehydrogenase mRNA levels is reported in
Fig. 3B. The serum-induced increase in CK2
'
transcripts was not blocked by the presence of the protein synthesis
inhibitor, cycloheximide, as shown by the superinduction of
CK2
' mRNA (Fig. 3, A and B).
Thus, in mouse fibroblasts CK2
' is induced with slow
kinetics by serum treatment, and this induction is independent of
protein synthesis. Because protein kinase CK2 is a tetramer containing
two catalytic and two regulatory subunits, we also tested whether the
mRNA corresponding to CK2
and CK2
were
induced by serum treatment of quiescent cells. Northern blot analysis
of CK2
revealed three hybridizing transcripts of 1.6, 3.1, and 4.6 kilobases, respectively. The CK2
mRNA
showed a less pronounced but detectable increase at 2 h after
serum treatment. Similar to CK2
' the CK2
mRNA increase was not blocked by the inhibition of protein
synthesis. Northern blot analysis with the probe for CK2
revealed a single transcript of 1.2 kilobases that increased with the
same kinetic of CK2
', and cycloheximide treatment did not
influence its induction.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of CK2 mRNAs and
CK2 kinase activity from cell extracts of quiescent and serum-induced
fibroblasts. A, Northern blots using the probes
CK2 ', CK2 , and CK2 as
indicated. Confluent NIH 3T3 fibroblasts were serum-starved for 48 h prior to the addition of medium plus 10% FCS alone or containing 10 µg/ml of cycloheximide as indicated (CHX). At various time
points, total cellular RNA was extracted and Northern blot analysis was
performed. Numbers indicate the hours of serum induction of
quiescent fibroblasts. The relative positions of 18 and 28 S rRNA are
shown. Glyceraldheyde-3-phosphate dehydrogenase (gapdh) was
used as a control for RNA loading. B, quantitative analysis
of CK2 mRNA levels in cultured fibroblasts. The blots
were analyzed using a PhosphorImager (Molecular Dynamics), and the
values obtained were normalized to the glyceraldheyde-3-phosphate
dehydrogenase mRNA levels. C, catalytic activity of the
whole-cell extracts were determined using a specific CK2 substrate
peptide. Confluent NIH 3T3 fibroblasts were serum-starved for 48 h
and then induced with medium containing 10% FCS at the indicated time
points. Cells were lysed, and whole-cell extracts were used in the
phosphorylation assay. The CK2 activity is reported as picomoles of
32P incorporated for µg of protein. The values shown are
the mean of at least three experiments with a S.E. not exceeding
16%.
|
|
The mRNA analysis showed a moderate but measurable induction of the
CK2 subunits. To confirm the functional significance of this induction,
we determined whether the increased mRNA levels corresponded to an
increase of CK2 by testing the kinase activity of the whole-cell
extracts from quiescent and serum-induced cells, using the
phosphorylation assay with the CK2 peptide substrate. The time course
of the serum-induced CK2 activity showed a maximal increase of about
1.8-fold at 4 h compared with quiescent cells (Fig.
3C). To confirm that the activity monitored with the peptide substrate is because of CK2, the effect of heparin, a specific CK2
inhibitor, was examined. The phosphorylation of the peptide was
entirely suppressed by 1 µg/ml heparin as expected for CK2-catalyzed phosphorylation (not shown).
CK2
' Cooperates with Ha-ras in Rat Embryo Fibroblast
Transformation--
The above results showed that mouse fibroblasts
respond to mitogenic stimuli with an increase of CK2 transcripts and
kinase activity at the G0/G1 phase transition.
Although the induction observed is not dramatic, we measured a
reproducible increase of CK2 activity of about 80%. Previous
experiments showed that as little as a 10% increase in
CK2
expression in lymphoid organs of transgenic mice
accelerated the onset of lymphomas induced by either c-myc
or tal-1 oncogenes (18, 19). To test whether CK2
' or CK2
play a direct role in tumor
induction, we performed standard focus formation assay transfecting
primary rat embryo fibroblasts with Ha-ras,
CK2
', or CK2
alone; the combination of
Ha-ras with each catalytic subunit. Neither
Ha-ras alone nor the CK2 catalytic subunits
transfected independently induced foci formation in primary cells
(Table I). However, transformed foci were
visible within 10 days in the plates cotransfected with
Ha-ras and either CK2
' or CK2
.
We therefore conclude that either CK2
' or
CK2
cooperate with oncogenic ras in primary
cell transformation.
View this table:
[in this window]
[in a new window]
|
Table I
Transformation of primary rat embryo fibroblasts
A standard focus assay was used to assess the ability of various
constructs to transform primary rat embryo fibroblasts. The number of
foci were counted after crystal violet staining. The experiments were
repeated four times, and the average foci number with their S.E. are
listed.
|
|
The Expression Level of CK2
' Correlates with Increased Growth
Rate of Transformed Clones--
To study further the effect of
CK2
' expression on the growth rate of
ras-transformed cells, we compared the growth behavior of
mouse fibroblasts transformed either with Ha-ras alone or
with Ha-ras and CK2
'. For this experiment we
chose immortalized mouse fibroblasts, because these cells can be
transformed with Ha-ras alone, and therefore it is possible
to obtain transformed clones either expressing or not ectopic
CK2
'. BALB/c 3T3 were used for this set of experiments,
because the efficiency of NIH 3T3 transformation with Ha-ras
alone was too high, making it difficult to quantitate the effects of
CK2
'. As shown in Fig.
4A in BALB/c 3T3 cells the
cotransfection of CK2
' and Ha-ras resulted in
approximately a 3-fold higher number of foci compared with the number
of foci induced by Ha-ras alone. Moreover, we observed that
foci generated with Ha-ras alone were smaller than those
from clones transformed with Ha-ras and CK2
',
suggesting that overexpression of CK2
' contributed to the
cell growth of transformed cells (Fig. 4B). In parallel sets
of experiments we observed a similar effect following cotransfection of
Ha-ras with the CK2
catalytic subunit. The transfection of CK2
', or CK2
, alone did not
induce foci formation (not shown).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
Analysis of foci formation in BALB/c 3T3
fibroblasts transformed either with Ha-ras or with
Ha-ras and CK2 '. BALB/c 3T3 fibroblasts
were cotransfected with Ha-ras and either the vector plasmid
(pcDNA3) or pcDNA ' (a plasmid expressing CK2 '
under the control of the cytomegalovirus promoter). Cells were
transfected, and foci formation was monitored at day 10. A,
number of foci measured in four different transfection experiments,
with error bars representing the S.D. of the measurements.
B, a representative experiment showing foci formation of
BALB/c 3T3 fibroblasts transfected with Ha-ras or with
Ha-ras and CK2 ' as indicated. Cells were
stained with crystal violet.
|
|
To analyze in more detail the growth rate of the transformed clones,
four ras-transformed clones (R-1 to R-4) and four
ras-CK2
'-transformed clones (R
'-1 to
R
'-4) were chosen for further analysis. Fig. 5A shows the growth curves
obtained by counting cells over a period of 5 days. The clones obtained
by cotransfection of Ha-ras and CK2
' showed a
marked increase in their growth rates compared with
ras-transformed clones. Eight other clones obtained from different transfections were analyzed, and those cotransfected with
CK2
' and Ha-ras also exhibited increased
growth rates (not shown).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
CK2 ' overexpression accelerates
proliferation of transformed cells. Individual foci from BALB/c
3T3 cells transformed with Ha-ras (R-1, R-2, R-3, R-4) or
with Ha-ras plus CK2 ' (R '-1, R '-2,
R '-3, R '-4) were picked and analyzed for their characteristics.
A, growth curves of isolated foci. Cells were plated at a
density of 105 cells/6-cm culture dish in medium containing
2% FCS and counted at daily intervals. The mean values of duplicate
cultures are shown plotted against time. B,
calmodulin phosphorylation by isolated foci. Confluent cells
were lysed and total cellular extracts were used in phosphorylation
assay using calmodulin as specific CK2 catalytic subunit substrate. The
reaction products were run on SDS-polyacrylamide gel electrophoresis,
and the blots were autoradiographed or directly scanned. All data are
the mean of at least three separate determinations with a S.E. of less
than 12%. C, correlation between calmodulin phosphorylation
and growth curve rates. The cell number corresponds to the number of
cells described in A at day 5 of growth. The values of
calmodulin phosphorylation are the same of the experiment described in
B. Clones transformed with Ha-ras are represented
with open circles, whereas clones transformed with
Ha-ras plus CK2 ' are represented with
black squares. D, tumor formation in nude mice.
Mice were injected with 106 log-phase cells from the
ras-transformed cells (R-1 to R-4) and from the
Ha-ras with CK2 '-transformed cells (R '-1 to
R '-4). Tumors were harvested after 2 weeks of growth and weighed.
The error bars represent the S.D. for three mice used for
each clone.
|
|
To examine whether the growth differences correlated with the increased
expression of CK2
' in the transformed clones, we tested
the kinase activity mediated by CK2
' by measuring the phosphorylation of calmodulin. Calmodulin is an ideal substrate for
examining the contributions of the CK2 catalytic subunits, because in
reconstitution experiments with recombinant subunits its
phosphorylation is suppressed completely by adding CK2
(5, 33-35).
Clones, transformed with both Ha-ras and CK2
',
showed higher levels of calmodulin phosphorylation when compared with ras-transformed clones (Fig. 5B). This
growth-related phosphorylation of calmodulin was inhibited (>90%) by
addition of either a molar excess (0.5 mM) of the specific
peptide substrate, or 1 µg/ml heparin (not shown). These findings, in
conjunction with the alkalilability of the phosphate incorporated into
calmodulin, which rules out the possibility of tyrosine
phosphorylation, show that calmodulin phosphorylation is entirely
because of CK2 rather than to any other protein kinase(s). Thus,
although we observed clonal variability, the enhanced growth correlated
with increased CK2
'-dependent calmodulin
phosphorylation (Fig. 5C). Finally, in mice we tested the
growth of Ha-ras versus Ha-ras plus
CK2
'-transformed clones. Exponentially growing
transformed clones were collected and injected subcutaneously into the
scapular region of nude mice, and the resultant tumors were removed
surgically after 2 weeks of growth and weighed. Consistent with the
results from the cell growth in vitro, CK2
'
was found to produce a significant enhancement of tumor growth (Fig.
5D).
 |
DISCUSSION |
Here we report the cloning of the murine CK2
'
subunit and show that its mRNA and kinase activity are induced in
response to serum stimulation of quiescent fibroblasts. Furthermore, we show that expression of CK2
' under the control of a
constitutive promoter cooperates with Ha-ras in
transformation of rat primary fibroblasts and increases cell growth of
transformed cells both in vitro and in vivo.
Our study originated from a screening for serum-induced messages in
quiescent mouse fibroblasts, which allowed us to identify the
CK2
' as an induced gene. We therefore cloned the murine
full-length CK2
' cDNA, analyzed its expression
pattern in vivo, in vitro, and activity in
cultured cells. Northern blot analysis showed a CK2
' peak
of induction at 4 h after serum treatment and that this induction
does not require new protein synthesis. Therefore CK2
',
like c-myc and MCP-1 (Refs. 36-38 and
references therein), belongs to a subset of early genes induced with a
slow kinetic. The analysis of induction revealed a lower but still
measurable increase of both CK2
and CK2
at
the same time points, suggesting that newly assembled CK2 tetramers can
be formed following serum induction. In accordance with this prediction
we observed an increased CK2-dependent phosphorylation
activity in protein extracts from serum-induced cells. An active role
of CK2 in cell cycle progression has already been suggested both in
mammalian cells and yeast (14, 15, 39). Our data show, for the first
time, that CK2 activity is indeed increased at the boundary between
G0 and G1, suggesting that new CK2 synthesis is
required at this stage of the cell cycle. Its specific induction could
be necessary for several reasons. It is possible that the kinase
already present in the cells is in a form which is not able to
phosphorylate some critical substrates. Alternatively, because we
observed a higher CK2
' induction compared with the other
catalytic subunit, a higher proportion of
'2
2 or
'
2 tetramers
could be formed. The formation of these two types of tetramer may lead
to different substrate specificity. Despite the fact that the catalytic
properties of isolated recombinant CK2
and CK2
' are very similar
(28, 34), which is consistent with their high sequence homology in
their catalytic domains, significant structural differences suggest
divergent functional commitments. Thus, the C-terminal segment of
vertebrate CK2
, which lies outside the catalytic core and includes
several phosphorylation sites affected both in vitro and
in vivo by cyclin-dependent kinases, is absent
in CK2
' (17). Likewise a motif (HEHRKL) responsible for association
of CK2
with protein phosphatase 2A (23) is substantially altered in
CK2
' (HQQKKL). This difference is especially remarkable as it occurs
inside a region that is otherwise highly conserved between CK2
and
CK2
'. Suggestive of specific function(s) of CK2
' in higher
organisms is the observation that CK2
' could not be detected in
Drosophila, Xenopus, and
Schizosaccharomyces pombe. In S. cerevisiae,
however, a somewhat atypical CK2
' subunit is found that exhibits
functional differences from CK2
(39, 40).
CK2
' cooperates with oncogenic ras in the
transformation of primary fibroblasts. Our data show that neither CK2
catalytic subunits nor Ha-ras alone induce foci formation
when transfected in primary cells, whereas transformed foci become
evident upon cotransfection with Ha-ras and either
CK2
' or CK2
. Therefore, we can conclude
that although the structural differences between CK2
and CK2
' may
reflect distinct functional roles, at least with respect to the
cooperation with ras in cell transformation, these
differences are not critical.
Recently, it was observed that exogenous expression of
CK2
suppressed cell growth and inhibited foci formation
induced by activated ras (23). Our results diverge from this
observation. The reasons for such a discrepancy are presently unclear.
It is possible that the inhibitory effect of CK2
previously observed (23) was dependent on the genetic background of the
NIH 3T3 cells used in those experiments. Alternatively, the reduction of foci observed by Hériché and collaborators (23) may have resulted from a CK2
poisoning effect because of a too
high expression of CK2
. In a standard focus formation
assay we observed cooperation between CK2 catalytic subunits
and oncogenic ras both in primary and immortalized
fibroblasts, suggesting we are observing a general phenomenon.
Moreover, our results are in agreement with experiments in transgenic
mice where the constitutive expression of CK2
accelerated the formation of lymphomas induced by c-myc or
tal-1 (18, 19). Comparison of the growth curves of
fibroblasts transformed with Ha-ras alone versus
fibroblasts transformed with Ha-ras and CK2
' demonstrated that transformed clones, expressing constitutively CK2
', grew faster. The enhanced growth of
Ha-ras and CK2
'-transformed clones correlates
with increased catalytic activity when monitored using calmodulin as a
phosphoacceptor substrate, symptomatic of the presence of free
catalytic subunit (33, 34). Thus, these data suggest that unbalanced
expression of CK2
' or CK2
leads to
phosphorylation of some critical target(s) necessary to accelerate the
cell cycle progression of ras-transformed cells. Therefore, it seems likely that the transforming potential of CK2 in each experimental model is because of a fraction of catalytic subunits not
combined with CK2
to form the canonical holoenzyme. This hypothesis
is supported by the phosphorylation of calmodulin, because this protein
is unaffected by the CK2 holoenzyme.
In contrast the hypothesis that following serum treatment CK2
and
CK2
' not combined with CK2
could be transiently present in
untransformed dividing cells is not consistent with available experimental data. Indeed, we observed that after serum treatment of
fibroblast, CK2
was induced with the same kinetics of the catalytic subunits. In addition, by using the specific substrate calmodulin or by titrating in with recombinant CK2
, we did not detect free catalytic subunits in cell extracts (not shown). Therefore, it is likely that during the G0/G1 progression
of the cell cycle the newly synthesized CK2
' and CK2
are rapidly
assembled into tetrameric CK2. Thus, the assembly of newly synthesized
CK2 subunits into a tetrameric enzyme could represent a mechanism for
the modulation of the too reactive free catalytic subunits necessary to
reprogram the CK2 kinase activity during the progression of the cell
cycle. Future studies aimed at the identification of specific target(s) of the CK2 catalytic subunits may unveil some target(s) critical for
cell transformation.
 |
ACKNOWLEDGEMENTS |
We thank Rino Rappuoli for hospitality in
IRIS laboratories, Beatrice Grandi for her excellent technical help,
and Nicholas Valiante for the critical reading of the
manuscript.
 |
FOOTNOTES |
*
The work was supported by grants to L. A. P. from
MURST, Consiglio Nazionale delle Ricerche (Target project ACRO),
Italian Ministero della Sanità (Project AIDS), European
Commission (BioMed-2), and AIRC, and to S. O. from Biocine spa and
AIRC.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ001420.
¶
To whom correspondence should be addressed: Dipartimento di
Biologia Molecolare Università di Siena, via Fiorentina 1, 53100 Siena, Italy. E-mail: oliviero{at}iris02.biocine.it.
The abbreviations used are:
DMEM, Dulbecco's
modified Eagle's medium; FCS, fetal calf serum; PCR, polymerase chain
reaction; PBS, phosphate-buffered saline.
 |
REFERENCES |
-
Pinna, L. A.
(1990)
Biochim. Biophys. Acta
1054,
267-284[Medline]
[Order article via Infotrieve]
-
Tuazon, P. T.,
and Traugh, J. A.
(1991)
Adv. Second Messenger Phosphoprotein Res.
23,
123-164[Medline]
[Order article via Infotrieve]
-
Issinger, O.-G.
(1993)
Pharmacol. Ther.
59,
1-30[CrossRef][Medline]
[Order article via Infotrieve]
-
Allende, J. E.,
and Allende, C. C.
(1995)
FASEB J.
9,
313-323[Abstract/Free Full Text]
-
Pinna, L. A.,
and Meggio, F.
(1997)
Prog. Cell Cycle Res.
3,
77-97[Medline]
[Order article via Infotrieve]
-
Meggio, F.,
Marin, O.,
and Pinna, L. A.
(1994)
Cell. Mol. Biol. Res.
40,
401-409[Medline]
[Order article via Infotrieve]
-
Luscher, B.,
Kuenzel, E. A.,
Krebs, E. G.,
and Eisenman, R. N.
(1989)
EMBO J.
8,
1111-1119[Abstract]
-
Luscher, B.,
Christenson, E.,
Litchfield, D. W.,
Krebs, E. G.,
and Eisenman, R. N.
(1990)
Nature
344,
517-522[CrossRef][Medline]
[Order article via Infotrieve]
-
Marais, R. M.,
Hsuan, J. J.,
McGuigan, C.,
Wynne, J.,
and Treisman, R.
(1992)
EMBO J.
11,
97-105[Abstract]
-
Prigent, C.,
Lasko, D. D.,
Kodama, K.,
Woodgett, J. R.,
and Lindahl, T.
(1992)
EMBO J.
11,
2925-2933[Abstract]
-
Cardenas, M. E.,
Dang, Q.,
Glover, C. V.,
and Gasser, S. M.
(1992)
EMBO J.
11,
1785-1796[Abstract]
-
Meek, D. W.,
Simon, S.,
Kikkawa, U.,
and Eckhart, W.
(1990)
EMBO J.
9,
3253-3260[Abstract]
-
Meisner, H.,
and Czech, M. P.
(1991)
Curr. Opin. Cell Biol.
3,
474-483[Medline]
[Order article via Infotrieve]
-
Pepperkok, R.,
Lorenz, P.,
Ansorge, W.,
and Pyerin, W.
(1994)
J. Biol. Chem.
269,
6986-6991[Abstract/Free Full Text]
-
Hanna, D. E.,
Rethinaswamy, A.,
and Glover, C. V. C.
(1995)
J. Biol. Chem.
270,
25905-25914[Abstract/Free Full Text]
-
Litchfield, D. W.,
Dobrowolska, G.,
and Krebs, E. G.
(1994)
Cell. Mol. Biol. Res.
40,
373-381[Medline]
[Order article via Infotrieve]
-
Litchfield, D. W.,
Luscher, B.,
Lozeman, F. J.,
Eisenman, R. N.,
and Krebs, E. G.
(1992)
J. Biol. Chem.
267,
13943-13951[Abstract/Free Full Text]
-
Seldin, D. C.,
and Leder, P.
(1995)
Science
267,
894-897[Medline]
[Order article via Infotrieve]
-
Kelliher, M. A.,
Seldin, D. C.,
and Leder, P.
(1996)
EMBO J.
15,
5160-5166[Abstract]
-
ole-MoiYoi, O. K.,
Sugimoto, C.,
Conrad, P. A.,
and Macklin, M. D.
(1992)
Biochemistry
31,
6193-6202[Medline]
[Order article via Infotrieve]
-
ole-MoiYoi, O. K.,
Brown, W. C.,
Iams, K. P.,
Nayar, A.,
Tsukamoto, T.,
and Macklin, M. D.
(1993)
EMBO J.
12,
1621-1631[Abstract]
-
ole-MoiYoi, O. K.
(1995)
Science
267,
834-835[Medline]
[Order article via Infotrieve]
-
Hériché, J. K.,
Lebrin, F.,
Rabilloud, T.,
Leroy, D.,
Chambaz, E. M.,
and Goldberg, Y.
(1997)
Science
276,
952-955[Abstract/Free Full Text]
-
Oliviero, S.,
Robinson, G.,
Struhl, K.,
and Spiegelman, B. M.
(1992)
Genes Dev.
6,
1799-1809[Abstract]
-
Chomzyski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Liang, P.,
and Pardee, A. B.
(1992)
Science
257,
967-971[Medline]
[Order article via Infotrieve]
-
Orlandini, M.,
Marconcini, L.,
Ferruzzi, R.,
and Oliviero, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11675-11680[Abstract/Free Full Text]
-
Bodenbach, L.,
Fauss, J.,
Robitzki, A.,
Krehan, A.,
Lorenz, P.,
Lozeman, F. J.,
and Pyerin, W.
(1994)
Eur. J. Biochem.
220,
263-273[Abstract]
-
Kozak, M.
(1987)
Nucleic Acids Res.
15,
8125-8148[Abstract]
-
Glass, D. B.,
Masaracchia, R. A.,
Feramisco, J. R.,
and Kemp, B. E.
(1978)
Anal. Biochem.
87,
566-575[Medline]
[Order article via Infotrieve]
-
Meisner, H.,
Heller-Harrison, R.,
Buxton, J.,
and Czech, M. P.
(1989)
Biochemistry
28,
4072-4076[Medline]
[Order article via Infotrieve]
-
Lozeman, F. J.,
Litchfield, D. W.,
Piening, C.,
Takio, K.,
Walsh, K. A.,
and Krebs, E. G.
(1990)
Biochemistry
29,
8436-8447[Medline]
[Order article via Infotrieve]
-
Meggio, F.,
Boldyreff, B.,
Issinger, O.-G.,
and Pinna, L. A.
(1994)
Biochemistry
33,
4336-4342[Medline]
[Order article via Infotrieve]
-
Antonelli, M.,
Daniotti, J. L.,
Rojo, D.,
Allende, C. C.,
and Allende, J. E.
(1996)
Eur. J. Biochem.
241,
272-279[Abstract]
-
Sarno, S.,
Vaglio, P.,
Marin, O.,
Issinger, O.-G.,
Ruffato, K.,
and Pinna, L. A.
(1997)
Biochemistry
36,
11717-11724[CrossRef][Medline]
[Order article via Infotrieve]
-
Kelly, K.,
Cochran, B. H.,
Stiles, C. D.,
and Leder, P.
(1983)
Cell
35,
603-610[Medline]
[Order article via Infotrieve]
-
Muller, R.,
Bravo, R.,
Burckhardt, J.,
and Curran, T.
(1984)
Nature
312,
716-720[Medline]
[Order article via Infotrieve]
-
Freter, R. R.,
Alberta, J. A.,
Lam, K. K.,
and Stiles, C. D.
(1995)
Mol. Cell. Biol.
15,
315-325[Abstract]
-
Rethinaswamy, A.,
Birnbaum, M. J.,
and Glover, C. V. C.
(1998)
J. Biol. Chem.
273,
5869-5877[Abstract/Free Full Text]
-
Glover, C. V. C.
(1998)
Prog. Nucleic Acid Res. Mol. Biol.
59,
95-133[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.