Inhibition of protein kinase CK2 expression and activity blocks tumor cell growth

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous serine/threonine kinase. It is a multifunctional and pleiotropic protein kinase implicated in the regulation of cell proliferation, survival, and differentiation. Deregulation of CK2 is observed in a wide variety of tumors. It has been the focus of intensive research efforts to establish the cause–effect relationship between CK2 and neoplastic growth. Here, we further validate the role of CK2 in cancer cell growth using siRNA approach. We also screened a library of more than 200,000 compounds and identified several molecules, which inhibit CK2 with IC₅₀ < 1 μM. The binding mode of a representative compound with maize CK2 was determined. In addition, the cellular activity of the compounds was demonstrated by their inhibition of phosphorylation of PTEN Ser370 in HCT116 cells. Treatment of a variety of cancer cell lines with the newly identified CK2 inhibitor significantly blocked cell growth with IC₅₀s as low as 300 nM.     

Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.     

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.     

Heat shock mediated modulation of protein kinase CK2 in the nuclear matrix

Nuclear matrix, a key structure in the nuclear framework, appears to be a particularly responsive target during heat shock treatment of cells. We have previously shown that nuclear matrix is a preferential target for protein kinase CK2 signaling in the nucleus. The levels of CK2 in the nuclear matrix undergo dynamic changes in response to altered growth status in the cell. Here, we have demonstrated that CK2 targeting to the nuclear matrix is profoundly influenced by treatment of the cells to temperatures higher than 37 degrees C. Rapid increase in the nuclear matrix association of CK2 is observed when cells are placed at temperatures of 41 and 45 degrees C. This effect at 45 degrees C was higher than at 41 degrees C, and was time-dependent. Also, different cell lines behaved in a qualitatively similar manner though the quantitative responses differed. The modulations in the nuclear matrix associated CK2 in response to heat shock appear to be due to trafficking of the enzyme between cytosolic and nuclear compartments. In addition, it was noted that isolated nuclei subjected to heat shock also responded by a shuttling of the intrinsic CK2 to the nuclear matrix compartment. These results suggest that modulations in CK2 in the nuclear compartment in response to the heat stress occur not only by a translocation of the enzyme from the cytoplasmic compartment to the nuclear compartment, but also that there is a redistribution of the kinase within the nuclear compartment resulting in a preferential association with the nuclear matrix. The results support the notion that CK2 association with the nuclear matrix in response to heat shock may serve a protective role in the cell response to stress.     

Role of the carboxyl terminus on the catalytic activity of protein kinase CK2alpha subunit

Protein kinase CK2 (also known as casein kinase 2) has catalytic (alpha, alpha') and regulatory (beta) subunits. The role of carboxyl amino acids in positions from 324 to 328 was studied for Xenopus laevis CK2alpha. Deletions and mutations of these residues were produced in recombinant CK2alpha, which was assayed for kinase activity. Activity dropped 7000-fold upon deletion of amino acids 324-328. The key residues are isoleucine 327 and phenylalanine 324. A three dimensional model of CK2alpha indicates that these hydrophobic residues of helix alphaN may interact with hydrophobic residues in helix alphaE which is linked to the catalytic center.     

Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-alpha

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-alpha in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-gamma, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-alpha-overexpressing clones, IFN-gamma-induced COX-2 expression was up-regulated in DN PKC-alpha-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-gamma-induced COX-2 mRNA and protein levels in DN PKC-alpha-overexpressing clones. Taken together, these results suggest a role for PKC-alpha in the modulation of LPS- and IFN-gamma-induced COX-2 expression, as well as in IFN-gamma-induced PGE2 secretion.     

Murine protein kinase CK2: Gene and oncogene

Protein kinase CK2 (casein kinase II) is a serine-threonine protein kinase with a wide range of substrates, many of which are involved in cell cycle regulation. CK2 activity is elevated in a variety of human tumors and we have used a transgenic mouse model to demonstrate that dysregulated expression of CK2 can induce lymphoma. Thus, CK2 fulfills the definition of an oncogene: A mutated, dysregulated, or mis-expressed gene that contributes to cancer in a dominant fashion. CK2 cooperates in transforming cells with other lymphoid oncogenes such as myc and tal-1, and here we show cooperativity with loss of the tumor suppressor gene p53. To understand more about the physiological and pathological role of CK2, we are cloning the murine CK2 cDNA and gene. CK2 will be used to generate transgenic and knockout mice and the regulatory elements for gene expression will be analyzed.     

CX-4945: the protein kinase CK2 inhibitor and anti-cancer drug shows anti-fungal activity

CX-4945 is a selective inhibitor of protein kinase CK2 exhibiting clinical significance. Its antitumor properties arise from the abrogation of CK2-mediated pro-survival cellular pathways. The presented data reveal the influence of CX-4945 on the growth of yeast cells showing variable potency against Saccharomyces cerevisiae deletion strains with different contents of CK2 subunits. The catalytic subunit CK2α appears to sensitize yeast to the CX-4945 action. Moreover, the compound suppresses hyphal growth and cell adhesion of Candida albicans, thereby abolishing some hallmarks of invasiveness of the pathogen. It is known that cancer patients are more prone to fungal infections. Our data unveil the dual-activity of CX-4945; when used in anti-cancer therapy, it may simultaneously prevent cancer-associated candidiasis.     

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop alpha-Thr(172) via inhibition of calcium/calmodulin-dependent protein kinase kinase-alpha and -beta, but not LKB1. These agents also enhanced phosphorylation of alpha-Ser(485/491) by the cAMP-dependent protein kinase. AMPK alpha-Ser(485/491) phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK alpha-Ser(485/491) can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.     

Cooperative modulation of protein kinase CK2 by separate domains of its regulatory beta-subunit

Protein kinase CK2 ("casein kinase 2") holoenzyme is composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. A truncated form of the beta-subunit lacking its C-terminal region (betaDelta171-215) has lost the ability to stably associate with the catalytic subunits and to display a number of properties which are mediated by structural elements still present in its sequence, notably down-regulation of catalytic activity, autophosphorylation, and responsiveness to polycationic effectors. All these functions are restored by simultaneous addition of a synthetic peptide reproducing the deleted fragment, beta170-215, which is able to associate with the catalytic subunits and to stimulate catalytic activity. This peptide includes a segment displaying significant sequence similarity with a region of cyclin A which interacts with the PSTAIRE motif of CDK2 eliciting its catalytic activity. A peptide reproducing this sequence (beta181-203), but not its derivative in which three nonpolar side chains have been replaced by polar ones, interacts with the alpha-subunit and stimulates its catalytic activity; it also partially restores the ability of truncated betaDelta171-215 to autophosphorylate. These data disclose the essential role of a structural module located between residues 181 and 203 in conferring regulatory properties to the beta-subunit of CK2.     

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co-transfected together, arguing against the existence of free subunits in CHO cells.     

Transient increase of a protein kinase activity identified to CK2 during sea urchin development

Using GST-EF-1 delta as an exogenous substrate, and EF-1 delta kinase activity was shown to increase transiently during early development of sea urchin embryos. The basal activity of EF-1 delta kinase in unfertilized eggs was 150 fmoles/min/mg protein. The activity began to increase 10 h after fertilization and reached its maximum level (8.4 x basal) at 24 h. The activity then declined to twice the basal value at 72 h post-fertilization. The EF-1 delta kinase activity was identified to a CK2-type enzyme on the basis of its substrate specificity for EF-1 delta, crude casein and beta casein, its inhibition by heparin, DRB, 2,3-bisphosphoglycerate, and its stimulation by spermine, spermidine, and polylysin. Furthermore, the activity was inhibited by the synthetic peptide RRREEETEEE specific for CK2. DRB (200 microM) and 2,3-bisphosphoglycerate (2.5 mM) blocked or delayed the transition from blastula to gastrula of the embryos, suggesting a role for the kinase in early development.     

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.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.