Inducible expression of the regulatory protein kinase CK2β subunit: Incorporation into complexes with catalytic CK2 subunits and re‐examination of the effects of CK2β on cell proliferation

The regulatory subunit of protein kinase CK2, designated CK2β, exists both free in cells and in complexes with the CK2 catalytic subunits. Growing evidence suggests that CK2β has functions dependent and independent of the CK2 catalytic subunits. There have been indications that CK2β has functions associated with DNA damage responses and in the control of cell proliferation. For example, transient and stable constitutive overexpression of CK2β in mammalian cells was previously shown to perturb cell cycle progression and to attenuate proliferation. To systematically investigate the molecular mechanisms responsible for these effects of CK2β on cell proliferation, we generated human osteosarcoma U2OS cell lines with tetracycline‐regulated expression of CK2β. Increased expression of CK2β results in increases in total cellular CK2 activity, but no changes in cell cycle profiles or proliferation. Furthermore, following exposure to ultraviolet radiation, p53 induction was identical regardless of the levels of CK2β in cells. Mouse 3T3‐L1 cells stably transfected with CK2β also showed no alterations in cell proliferation. The differences between these results and those previously reported emphasize the complex nature of CK2β and its cellular functions. Furthermore, these results indicate that increased expression of CK2β is not by itself sufficient to effect alterations in cell proliferation. J. Cell. Biochem. 84: 84–99, 2002. © 2001 Wiley‐Liss, Inc.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms.

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.     

Development of a stabilized form of the regulatory CK2beta subunit that inhibits cell proliferation

A number of cancers are characterized by elevated expression of CK2 (formerly casein kinase II), which has been implicated as a key component in cell proliferation and transformation. Two lines of evidence, (a) deregulated expression of CK2 and (b) CK2beta ubiquitination and degradation of these in a proteasome-dependent manner prompted further investigation of the regulation of CK2beta protein stability. We demonstrate that mutating six surface-exposed lysine residues to arginine (6KR) to interfere with ubiquitin attachment can stabilize CK2beta. Examination of 6KR expression in cells revealed increased stability over time and increased its steady-state expression level compared with CK2beta. In cells, 6KR was no longer sensitive to proteasome inhibition but maintained an elevated expression level. In our studies, 6KR functioned as a normal CK2 regulatory subunit, because it participated in CK2beta dimerization, associated with catalytic subunits, was autophosphorylated, and formed active, stable CK2 tetramers. The physiological role of CK2beta stabilization was investigated in cell proliferation assays, which showed a significant decrease in proliferation in cells expressing 6KR compared with CK2beta. Overall, our results indicate that a stabilized form of CK2beta can be used to inhibit cell proliferation.     

The regulatory beta subunit of protein kinase CK2 mediates formation of tetrameric CK2 complexes

Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (alpha and/or alpha') subunits and two regulatory (beta) subunits. Because CK2beta is synthesized in excess of CK2alpha, we hypothesized that formation of CK2beta homodimers precedes the incorporation of the catalytic subunits of CK2 into complexes. To test this hypothesis, we cotransfected cells with two epitope-tagged variants of CK2beta. The results of these cotransfection studies demonstrate that interactions between two CK2beta subunits take place in the absence of CK2alpha. Together with results from previous biosynthetic labeling studies, these results suggest that formation of CK2beta homodimers occurs before incorporation of catalytic subunits of CK2 into CK2 complexes. We also cotransfected Cos-7 cells with a deletion fragment of CK2beta (i.e. Myc-beta1-166) together with full-length hemagglutinin (HA)-tagged CK2beta and/or CK2alpha'. Although complexes between Myc-beta1-166 and HA-beta were readily detected, we obtained no evidence of direct interactions between Myc-beta1-166 and HA-CK2alpha'. These results suggest that residues within the N-terminal 166 amino acids of CK2beta are sufficient for interactions between CK2beta subunits, whereas the C-terminal domain of CK2beta is required for complex formation with the catalytic subunits of CK2. Finally, we observed that expression of full-length HA-beta promotes phosphorylation of Myc-beta1-166 by HA-CK2alpha'.     

The emerging CK2 interactome: insights into the regulation and functions of CK2

Protein kinase CK2 represents a small family of protein serine/threonine kinases implicated in a variety of biological processes including events relating to cell proliferation and survival. Notably, CK2 displays oncogenic activity in mice and exhibits altered expression in several types of cancer. Accordingly, a detailed understanding of the cellular functions of CK2 and elucidation of the mechanisms by which CK2 is regulated in cells is expected to contribute to understanding its role in tumorigenesis with the prospect of novel approaches to therapy. While CK2 has traditionally been viewed as a tetrameric complex composed of two catalytic and two regulatory subunits, mounting evidence suggests that its subunits may have functions independent of tetrameric CK2 complexes. In mammals, as is the case in the budding yeast Saccharomyces cerevisiae, there are two isozymic forms of CK2, adding additional heterogeneity to the CK2 family. Studies in yeast and in human cells demonstrate that the different forms of CK2 interact with a large number of cellular proteins. To reveal new insights regarding the regulation and functions of different forms of CK2, we have examined the emerging interactomes for each of the CK2 subunits. Analysis of these interactomes for both yeast and human CK2 reinforces the view that this family of enzymes participates in a broad spectrum of cellular events. Furthermore, while there is considerable overlap between the interactomes of the individual CK2 subunits, notable differences in each of the individual interactomes provides additional evidence for functional specialization for the individual forms of CK2.     

Maize protein kinase CK2: regulation and functionality of three beta regulatory subunits

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.     

Serum-stimulated cell cycle entry of fibroblasts requires undisturbed phosphorylation and non-phosphorylation interactions of the catalytic subunits of protein kinase CK2

Protein kinase CK2 is a pleiotropic Ser/Thr kinase occurring as alpha2beta2, alpha'2beta2, or alphaalpha'beta2 tetramers. A requirement in serum-stimulated cell cycle entry in both the cytoplasm and the nucleus of human fibroblasts for phosphorylation(s) by CK2 has been concluded from stimulation inhibition by microinjected antibodies against the regulatory subunit (beta). We have now examined this idea more directly by microinjection-mediated perturbation of phosphorylation and non-phosphorylation interactions of the catalytic subunits (alpha and alpha'), and by verifying the supposed matching of the cellular partition of CK2 subunits in the fibroblasts employed. While immunostaining and cell fractionation indicate that the partitions of subunits indeed match each other (with their predominant location in the nucleus in both quiescent and serum-stimulated cells), microinjection of substrate or pseudosubstrate peptides competing for the CK2-mediated phosphorylation in vitro resulted in significant inhibition of serum stimulation when placed into the nucleus but not when placed into the cytoplasm. Also inhibitory were nuclear but not cytoplasmic injections of antibodies against alpha and alpha' that affect neither their kinase activity in vitro nor their complexing to beta. The data indicate that the role played by CK2 in serum-stimulated cell cycle entry is predominantly nuclear and more complex than previously assumed, involving not only phosphorylation but also experimentally separable non-phosphorylation interactions by the catalytic subunits.     

Functional conservation between the human, nematode, and yeast CK2 cell cycle genes.

Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms. Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae. We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits. Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits. Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function.     

Heat-induced relocalization of protein kinase CK2. Implication of CK2 in the context of cellular stress

Among various other roles described so far, protein kinase CK2 has been involved in cell cycle, proliferation, and development. Here, we show that in response to specific stresses (heat shock or UV irradiation), a pool of the cellular CK2 content relocalizes in a particular nuclear fraction, increasing the activity of the kinase there. Electron microscopic analysis shows that upon heat shock, CK2alpha and CK2beta subunits are both detected in similar speckle structures occurring in the interchromatin space but are differentially targeted inside the nucleolus. This CK2 relocalization process takes place in a time- and dose-dependent manner and is reversible upon recovery at 37 degrees C. Altogether, this work suggests CK2 be involved in the response to physiological stress in higher eukaryotic cells.     

Validation of protein kinase CK2 as oncological target

Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits α and/or α′ and two regulatory subunits β whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.     

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.     

An extensive survey of CK2 alpha and beta subunits in Arabidopsis: multiple isoforms exhibit differential subcellular localization

Casein kinase 2 (CK2) is a ubiquitous enzyme essential for the viability of eukaryotic cells. In the present work we analyzed the Arabidopsis thaliana genome in a search for the genes coding for all CK2 alpha and beta subunits. We found four alpha subunit and four beta subunit genes. Expression analysis showed that all CK2 subunit genes are expressed in inflorescences, stems, leaves and roots. The level of expression of these genes is very similar, except for the one that codes for an alpha subunit harboring a putative chloroplastic destination peptide (alphacp), which shows a slightly higher expression level in all tissues. Using transgenic plants and agroinfiltration, we have also characterized the subcellular localization of all proteins encoded by CK2 genes. Our results show that all alpha subunits are localized in the nucleus, with the exception of alphacp, which is only found in the chloroplasts. On the other hand, beta subunits have a more diverse distribution, with some of them localizing both to the nucleus and to the cytosol, while others are exclusively located in one of these compartments. Remarkably, no CK2beta subunit was found in the chloroplasts. Finally, by directly measuring its activity, we have demonstrated that purified Arabidopsis chloroplasts have active CK2 that can be regulated by external addition of CK2beta. This study represents a complete survey of the CK2 gene family in Arabidopsis and the first step for future studies on CK2 cellular function in this species.     

Interactions between protein kinase CK2 and Pin1. Evidence for phosphorylation-dependent interactions

The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha' subunits together with two regulatory CK2 beta subunits. Our results indicate that Pin1 can interact with CK2 complexes that contain CK2 alpha. Furthermore, Pin1 can interact directly with the C-terminal domain of CK2 alpha that contains residues that are phosphorylated in vitro by p34(Cdc2) and in mitotic cells. Substitution of the phosphorylation sites of CK2 alpha with alanines resulted in decreased interactions between Pin1 and CK2. The other catalytic isoform of CK2, designated CK2 alpha', is not phosphorylated in mitotic cells and does not interact with Pin1, but a chimeric protein consisting of CK2 alpha' with the C terminus of CK2 alpha was phosphorylated in mitotic cells and interacts with Pin1, further implicating the phosphorylation sites in the interaction. In vitro, Pin1 inhibits the phosphorylation of Thr-1342 on human topoisomerase II alpha by CK2. Topoisomerase II alpha also interacts with Pin1 suggesting that the effect of Pin1 on the phosphorylation of Thr-1342 could result from its interactions with CK2 and/or topoisomerase II alpha. As compared with wild-type Pin1, isomerase-deficient and WW domain-deficient mutants of Pin1 are impaired in their ability to interact with CK2 and to inhibit the CK2-catalyzed phosphorylation of topoisomerase II alpha. Collectively, these results indicate that Pin1 and CK2 alpha interact and suggest a possible role for Pin1 in the regulation of topoisomerase II alpha. Furthermore, these results provide new insights into the functional role of the mitotic phosphorylation of CK2 and provide a new mechanism for selectively regulating the ability of CK2 to phosphorylate one of its mitotic targets.