Inducible Expression of Protein Kinase CK2 in Mammalian Cells
EVIDENCE FOR FUNCTIONAL SPECIALIZATION OF CK2 ISOFORMS*

Greg VilkDagger , Ronald B. SaulnierDagger §, Rebecca St. Pierre, and David W. Litchfield

From the Department of Biochemistry, Health Sciences Center, University of Western Ontario, London, Ontario N6A 5C1, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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. CK2alpha or CK2alpha ') together with the regulatory CK2beta 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 CK2alpha or CK2alpha ' (i.e. GK2alpha (K68M) or CK2alpha '(K69M)) together with CK2beta . 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 CK2alpha or CK2alpha ' 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 CK2alpha differed significantly from the effects of induced expression of kinase-inactive CK2alpha '. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha '(K69M), but not following induction of CK2alpha (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

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 CK2alpha 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 CK2alpha 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. CK2alpha or CK2alpha ') 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.

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 (alpha ) and two regulatory (beta ) 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 CK2alpha and CK2alpha ', that are the products of distinct genes localized to different chromosomes (29-31). Between species, CK2alpha and CK2alpha ' exhibit remarkable conservation; between humans and chickens, CK2alpha exhibits 98% identity and CK2alpha ' 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, CK2alpha and CK2alpha ' exhibit nearly 90% identity. By comparison, CK2alpha 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 CK2alpha '. 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 CK2alpha and CK2alpha ' 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 CK2alpha and CK2alpha ' 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 CK2alpha is phosphorylated by p34cdc2 during mitosis, whereas CK2alpha ' is not phosphorylated (37, 38). Moreover, we have recently identified cellular proteins that bind to CK2alpha but not CK2alpha '.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.

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 CK2alpha or CK2alpha ') together with myc epitope-tagged regulatory CK2beta subunit (39-41). To specifically interfere with signaling events involving CK2alpha and CK2alpha ', we also established cell lines expressing kinase-inactive mutants of CK2alpha or CK2alpha ' (i.e. HA epitope-tagged CK2alpha (K68M) or CK2alpha '(K69M)) along with myc-CK2beta (36). Contrary to the expectation that increased CK2 expression would enhance proliferation (9, 18), increased expression of either CK2alpha or CK2alpha ' resulted in modest decreases in proliferation. Induced expression of kinase-inactive CK2alpha differed significantly from the effects observed following induction of similar levels of kinase-inactive CK2alpha '. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha '(K69M), but not following induction of CK2alpha (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

Plasmid Constructs-- A construct encoding CK2alpha 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 CK2alpha from the hT4.1 plasmid (29) was subcloned into this TA cloning vector that contained the modified HA sequence. The CK2alpha -HA cDNA was then subcloned into the HindIII/ApaI sites of pRc/CMV (Invitrogen) to generate pRc/ CMV/CK2alpha -HA, which was designated pZW6.

The kinase-dead mutant of CK2alpha -HA (i.e. CK2alpha (K68M)-HA) was generated by first subcloning CK2alpha -HA from pZW6 into the Bluescript pSK+ cloning vector (Stratagene) using HindIII and ApaI. A BstBI and NcoI fragment from pRc/CMV/HA-CK2alpha (K68M) (36) was then used to replace the wild-type CK2alpha sequence in pSK+/CK2alpha -HA to generate pSK+/CK2alpha (K68M)-HA, which was designated pGV14. A HindIII/ApaI fragment from pGV14 encoding the CK2alpha (K68M)-HA cDNA was subcloned into the HindIII and ApaI sites of pRc/CMV to generate pRc/CMV/CK2alpha (K68M)-HA, which was designated pGV15. All constructs were verified by DNA sequencing (42).

The bidirectional constructs encoding both CK2alpha -HA or CK2alpha (K68M)-HA were generated as follows. The myc-CK2beta 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-CK2beta . The CK2alpha -HA cDNA was isolated from pZW6 using HindIII and ApaI and ligated into the SalI (MCS II) site of pBI/myc-CK2beta to generate pBI/myc-CK2beta /CK2alpha -HA, which was designated pRS3. To generate the bidirectional construct containing kinase-dead CK2alpha , 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-CK2beta /CK2alpha (K68M)-HA (designated pGV13). All constructs were verified by restriction endonuclease digests or DNA sequencing (42).

Bidirectional constructs encoding HA-CK2alpha ' or HA-CK2alpha '(K69M) and myc-CK2beta were generated as follows. The HA-CK2alpha ' fragment was digested pRc/CMV/HA-CK2alpha ' (36) with HindIII and ligated into pBI/myc-CK2beta at the SalI site (MCS II) to generate pBI/myc-CK2beta /HA-CK2alpha ' (designated pRS2). The pBI/myc-CK2beta /HA-CK2alpha '(K69M) plasmid, designated pGV7, was constructed by replacing the HindIII fragment encoding HA-CK2alpha ' from pRS2 with a HindIII fragment encoding HA-CK2alpha '(K69M) from pRc/CMV/HA-CK2alpha ' (36). All constructs were verified by restriction endonuclease digests or DNA sequencing (42).

Antibodies-- Polyclonal anti-CK2alpha antiserum directed against the C-terminal synthetic peptide alpha 376-391, polyclonal anti-CK2alpha ' antiserum directed against the C-terminal synthetic peptide alpha '333-350, and polyclonal anti-CK2beta antiserum directed against the C-terminal synthetic peptide beta 198-215 have been described previously (37, 43). Polyclonal anti-CK2alpha antiserum directed against an N-terminal synthetic peptide alpha 2-19 were similarly prepared and was used for the detection of CK2alpha -HA as the HA epitope interfered with recognition by the antiserum directed against the C-terminal alpha 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.

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 CK2alpha -HA and myc-CK2beta ), GV13.35 (expressing CK2alpha (K68M)-HA and myc-CK2beta ), RS2.31(expressing HA-CK2alpha ' and myc-CK2beta ), and GV7.21 (expressing HA-CK2alpha '(K69M) and myc-CK2beta ), 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).

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 -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.

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-alpha (1/1000), polyclonal anti-alpha ' (1/400), polyclonal anti-beta (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).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 CK2alpha -HA and myc-CK2beta (RS3.22 cells) or with the pBI plasmid encoding HA-CK2alpha ' and myc-CK2beta (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 CK2alpha (K68M)-HA and myc-CK2beta (GV13.35 cells) or with the pBI plasmid encoding kinase-inactive HA-CK2alpha '(K69M) and myc-CK2beta (GV7.21 cells). Epitope tags incorporated into the sequence of CK2alpha , CK2alpha ', and CK2beta 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. CK2alpha -HA, CK2alpha (K68M)-HA, HA-CK2alpha ', or HA-CK2alpha '(K69M)) or regulatory (i.e. myc-CK2beta ) 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 CK2beta subunit are illustrated.

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 CK2alpha or CK2alpha ' as well as myc-CK2beta 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-CK2beta 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-CK2beta (36), the upper band corresponds to the phosphorylated form of CK2beta , indicating that expression of HA-CK2alpha '(K69M), and CK2alpha (K68M)-HA to a lesser degree, diminishes the phosphorylation of myc-CK2beta . We were unable to detect expression of HA-tagged CK2alpha or CK2alpha ' 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 CK2alpha -HA and myc-CK2beta ), GV13.35 cells (expressing CK2alpha (K68M)-HA and myc-CK2beta ), RS2.31 cells (expressing HA-CK2alpha ' and myc-CK2beta ), and GV7.21 cells (expressing HA-CK2alpha '(K69M) and myc-CK2beta ) 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-alpha antibodies (B), anti-alpha ' antibodies (C), and anti-beta antibodies (D). The positions of the epitope-tagged and endogenous CK2 subunits are indicated. The position of the phosphorylated form of myc-CK2beta (designated myc-CK2beta -P) is also indicated. A nonspecific band that is detected with anti-CK2alpha ' is also marked (panel C).

Cell lysates were also probed with anti-alpha and anti-alpha ' to detect endogenous and transfected CK2alpha and CK2alpha ' 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 CK2alpha (Fig. 2B). By comparison, with RS2.31 and GV7.21 cells, we observed in both cell lines that expressed HA-tagged CK2alpha ' or CK2alpha '(K69M) was significantly higher than endogenous CK2alpha ' (Fig. 2C). As levels of CK2alpha -HA and HA-CK2alpha ' are comparable when probed with anti-HA antibodies, these results reflect the higher levels of endogenous CK2alpha that are observed in these cells compared with endogenous CK2alpha '. In addition, expression of HA-CK2alpha ' in RS2.31 does appear to be higher than the HA-CK2alpha '(K69M) in GV7.21 cells (Fig. 2, A and C). When each of the cell lysates were probed with anti-beta antibodies, we noted that myc-CK2beta is expressed to lower levels than endogenous CK2beta 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 CK2alpha ' and myc-CK2beta exhibit the lowest levels of induced expression (Fig. 2A). Overall, we did not observe any large changes in the expression of endogenous CK2alpha or CK2alpha ' in the presence or absence of tetracycline in any of the cell lines (Fig. 2, B-C).

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-CK2alpha '(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-beta (Fig. 3C). In Fig. 3 (B and C), we noted that expression of endogenous CK2alpha ' or CK2beta , respectively, appeared unaltered during increasing expression of the transfected constructs. Similar results were obtained with RS2.31 cells expressing HA-CK2alpha ' (data not shown). These results demonstrate modulation of the expression of HA-CK2alpha ' and HA-CK2alpha '(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-CK2alpha '(K69M) expression. GV7.21 cells (expressing HA-CK2alpha '(K69M) and myc-CK2beta ) 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-CK2alpha '(K69M) and myc-CK2beta was monitored by Western blotting. A-C, 12CA5 and 9E10 antibodies (A), anti-alpha ' antibodies (B), and anti-beta antibodies (C) were used in monitoring expression. Endogenous CK2alpha ' and CK2beta are indicated in B and C, respectively. Phosphorylated (myc-CK2beta -P) and non-phosphorylated forms of myc-CK2beta 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-CK2alpha ' as in Fig. 2.

We were also interested in examining the time course of induction following tetracycline withdrawal. Expression of both HA-CK2alpha ' (Fig. 4A) and HA-CK2alpha '(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-CK2alpha '(K69M) was decreased after 72 h. This observation was seen in three independent experiments and may in part explain the lower levels of HA-CK2alpha '(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 CK2alpha -HA and CK2alpha (K68M)-HA and noted that expression of both proteins, unlike HA-CK2alpha '(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-CK2alpha ' and myc-CK2beta ) and GV7.21 cells (B, expressing HA-CK2alpha '(K69M) and myc-CK2beta ) 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.

CK2 Kinase Activity-- To examine the kinase activity of the exogenously expressed CK2alpha -HA, CK2alpha (K68M)-HA, HA-CK2alpha ', and HA-CK2alpha '(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 CK2alpha -HA or HA-CK2alpha ' than from cells expressing CK2alpha (K68M)-HA or HA-CK2alpha '(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-CK2beta into complexes containing exogenous and endogenous catalytic CK2 subunits. Importantly, these immunocomplex kinase activities provide indications that the expression levels of constructs encoding CK2alpha and CK2alpha ' 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 CK2alpha -HA and myc-CK2beta ), from GV13.35 cells (expressing CK2alpha (K68M)-HA and myc-CK2beta ), from RS2.31 cells (expressing HA-CK2alpha ' and myc-CK2beta ), and from GV7.21 cells (expressing HA-CK2alpha '(K69M) and myc-CK2beta ) 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.

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 CK2alpha -HA and HA-CK2alpha ', 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.

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 CK2alpha -HA did not enhance cell proliferation and in fact seemed to induce a slight decrease in proliferation (Fig. 6B). Increased expression of HA-CK2alpha ' also resulted in a modest decrease in proliferation (Fig. 6D). By comparison, induced expression of kinase-inactive CK2 isoforms had dramatically different consequences. Whereas CK2alpha (K68M)-HA was without significant effect (Fig. 6C), HA-CK2alpha '(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-CK2alpha '(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-CK2alpha '(K69M) were, if anything, lower than that observed with CK2alpha (K68M)-HA (see Fig. 2), we believe that these results reflect a potent ability of HA-CK2alpha '(K69M) to influence cell proliferation in a manner that is distinct from the CK2alpha (K68M)-HA. Importantly, these results provide the first direct evidence for a role of CK2alpha ' in cell proliferation in mammalian cells and provide evidence for functional differences between CK2alpha and CK2alpha '.


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Fig. 6.   Cell proliferation profiles of the inducible CK2 cell lines. Parental UTA6 cells (A), RS3.22 cells (expressing CK2alpha -HA and myc-CK2beta ) (B), GV13.35 cells (expressing CK2alpha (K68M)-HA and myc-CK2beta ) (C), RS2.31 cells (expressing HA-CK2alpha ' and myc-CK2beta ) (D), and GV7.21 cells (expressing HA-CK2alpha '(K69M) and myc-CK2beta ) (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.

Having demonstrated that cell proliferation was attenuated by altered expression of HA-CK2alpha ' or induced expression of HA-CK2alpha '(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-CK2alpha '(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-CK2alpha ' or HA-CK2alpha ' (K69M). RS2.31 cells (expressing HA-CK2alpha ' and myc-CK2beta ) (A) and GV7.21 cells (expressing HA-CK2alpha '(K69M) and myc-CK2beta ) (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

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. CK2alpha or CK2alpha ') and regulatory (i.e. CK2beta ) 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 CK2alpha constructs harboring an N-terminal HA epitope tag were expressed at levels much lower than was observed with constructs encoding CK2alpha ' with an N-terminal HA tag (data not shown). Consequently, it was necessary to generate CK2alpha 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 CK2alpha constructs with C-terminal epitope tags were expressed to levels comparable to those observed with the N-terminally tagged CK2alpha ' constructs. We do not understand why the expression levels of HA-CK2alpha are so much lower than that observed with HA-CK2alpha ' or CK2alpha -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.

In overexpressing kinase-dead forms of CK2alpha and CK2alpha ', we anticipated that we could interfere with their respective cellular functions (36, 54). As it is likely that CK2alpha and CK2alpha ' have a similar capacity to form complexes with CK2beta , 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 CK2beta . Accordingly, myc-CK2beta was co-expressed with either CK2alpha -HA or HA-CK2alpha ' 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 CK2alpha (K68M)-HA or HA-CK2alpha '(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 CK2alpha (K68M)-HA or HA-CK2alpha '(K69M) would result from their ability to compete with endogenous CK2alpha or CK2alpha ', 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 CK2alpha (K68M)-HA or HA-CK2alpha '(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.

The observation that induced expression of HA-CK2alpha '(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 CK2alpha ' resulted in diminished proliferation. Previous studies that have used antisense oligonucleotides to down-regulate the expression of CK2 focused exclusively on CK2beta or CK2alpha without attempts to down-regulate CK2alpha ' (56). Collectively, these results suggest that both CK2alpha and CK2alpha ' are required for proliferation suggesting that the two CK2 isoforms may have some overlapping functions in mammalian cells. Consistent with a role for both CK2alpha and CK2alpha ' 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 CK2alpha and CK2alpha ' do indeed have entirely redundant functions. Moreover, data in this study demonstrate that similar levels of induced kinase-inactive CK2alpha and kinase-inactive CK2alpha ' have strikingly different outcomes. Importantly, these data provide the first direct evidence for functional specialization of CK2 isoforms in mammalian cells.

In light of recent evidence suggesting that CK2alpha and CK2alpha ' behave as delayed early genes and that either CK2alpha or CK2alpha ' can enhance proliferation when overexpressed in fibroblasts (18), we were surprised to observe diminished cell proliferation in cells overexpressing CK2alpha -HA or HA-CK2alpha '. We do not have a precise explanation for the discrepancies between our studies and those published elsewhere. However, given that the expression of CK2alpha or CK2alpha ' 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 CK2alpha or CK2alpha ' than would be observed with the exclusive expression of these proteins without myc-CK2beta . 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-CK2beta exhibits the electrophoretic characteristics of autophosphorylated CK2beta when it is expressed in cells together with active CK2alpha ' (36). Inasmuch as we have previously shown in transient transfection experiments that the autophosphorylation of myc-CK2beta occurs efficiently when it forms complexes with CK2alpha ', this result suggests that the majority of CK2beta is in complexes. Therefore, we believe that the results seen here do not arise from the high expression of uncomplexed CK2beta .

It is also important to recognize that the studies described in this paper were performed utilizing epitope-tagged forms of CK2alpha and CK2alpha '. 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.

Based on the prediction that the increased expression of CK2alpha or CK2alpha ' 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 CK2alpha -HA or HA-CK2alpha ' may be different from that observed with HA-CK2alpha '(K69M). In the case of CK2alpha -HA or HA-CK2alpha ', 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.

In the case of HA-CK2alpha '(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-CK2alpha '(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 CK2alpha ' interferes with Ras-dependent signaling events. There are also indications that the induced expression of HA-CK2alpha '(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 CK2alpha ' 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.

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 CK2alpha and CK2alpha ' 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.

    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.

    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.

Dagger 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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