Protein Kinase CK2 Activity Research Articles

Wild-type p53 inhibits protein kinase CK2 activity.

The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory beta-subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy-terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild-type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose-dependent and was seen for various CK2 substrates. Experiments with heat-denatured p53 and the conformational mutant p53(R175H) revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild-type and of mutant p53 into p53-/- cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild-type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down-regulation of protein kinase CK2.

Stress-induced Activation of Protein Kinase CK2 by Direct Interaction with p38 Mitogen-activated Protein Kinase

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.

Time-co-ordinated control of glycogen synthase, protein phosphatase 2A and protein kinase CK2 during culture growth in Yarrowia lipolytica in relation to glycogen metabolism

In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells. There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form. During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed. GS activity proved to be a good indicator of early culture growth phase. Experiments carried out with enzymes purified from Y. lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2. GS activity would be controlled by the sequential action of PP2A and CK2.

Activation of protein kinase CK2 by LPS is mediated by the MAP kinase pathway

Stimulation of macrophages by Gram-negative bacterial lipopolysaccharide (LPS) rapidly leads to the activation of several protein kinases and the subsequent phosphorylation of transcription factors that regulate LPS-inducible genes. Several investigators have shown that the MAP kinases (MAPKs) are rapidly activated following LPS stimulation. We previously reported that LPS stimulation also up-regulates the enzymatic activity of protein kinase CK2, a ubiquitous serine/threonine kinase, although the signaling cascade that leads to CK2 activation in macrophages is unknown. Because MAPKs are known to be rapidly activated by LPS, we performed additional studies in order to determine if CK2 activation was a downstream target of MAPKs. Our studies revealed that CK2 activity was rapidly and transiently up-regulated in LPS-stimulated RAW264.7 murine macrophages. We found that PD98059, an inhibitor of ERK kinase activation by the MAP kinase MEK-1, blocked LPS-induced up-regulation of CK2 activity. In contrast, the p38 kinase inhibitor SB203580 did not block CK2 activation by LPS. This finding suggests that CK2 activation was mediated by the ERK kinase signaling cascade, but not by the p38 kinase cascade. We also found that LPS stimulation resulted in the rapid serine phosphorylation of the catalytic α and α′ subunits of CK2. This contrasts with other studies using growth factor-stimulated fibroblasts in which only phosphorylation of the regulatory β subunit of CK2 was observed. Thus, LPS stimulation leads to rapid activation and cell type-specific phosphorylation of CK2 in macrophages.

Modulation of nuclear matrix protein phosphorylation by histones: Possible involvement of NM‐associated protein kinase CK2 activity

Nuclear matrix (NM), a proteinaceous network of filaments, dictates nuclear morphology and the structure/function of DNA. Phosphorylation of NM proteins is a potential signal for regulating matrix functions. Histones also are intimately involved in DNA structure and transcription. Here, we report that various histones enhanced 32P incorporation into certain NM proteins. Modulation of NM protein phosphorylation by histones is mediated through regulation of protein kinase CK2, a messenger-independent serine/threonine kinase, which is significantly associated with the NM. The stimulatory effect of histones was mitigated by prior incubation of histones with DNA in the reaction. Phosphorylation of NM proteins was extensively reduced when an excess of the CK2-specific peptide substrate was included in the phosphorylation reaction as a competitor. Also, enhancement in the NM-associated CK2 activity by histones was blocked by inhibitors of CK2. Histone H1 effect appeared to be mediated mainly by charge effect since a stretch of polylysine induced a similar effect. Various histones also differentially affected the autophosphorylation of NM-associated CK2 subunits. This may contribute to the observed effects of histones on the NM, resulting in an enhancement and differential pattern of NM protein phosphorylation. Such a regional modification of NM protein phosphorylation might influence the nuclear functions that require histone displacement, namely, replication and transcription. J. Cell. Biochem. 72:242–250, 1999.

Modulation of nuclear matrix protein phosphorylation by histones: Possible involvement of NM-associated protein kinase CK2 activity

Nuclear matrix (NM), a proteinaceous network of filaments, dictates nuclear morphology and the structure/function of DNA. Phosphorylation of NM proteins is a potential signal for regulating matrix functions. Histones also are intimately involved in DNA structure and transcription. Here, we report that various histones enhanced 32P incorporation into certain NM proteins. Modulation of NM protein phosphorylation by histones is mediated through regulation of protein kinase CK2, a messenger-independent serine/threonine kinase, which is significantly associated with the NM. The stimulatory effect of histones was mitigated by prior incubation of histones with DNA in the reaction. Phosphorylation of NM proteins was extensively reduced when an excess of the CK2-specific peptide substrate was included in the phosphorylation reaction as a competitor. Also, enhancement in the NM-associated CK2 activity by histones was blocked by inhibitors of CK2. Histone H1 effect appeared to be mediated mainly by charge effect since a stretch of polylysine induced a similar effect. Various histones also differentially affected the autophosphorylation of NM-associated CK2 subunits. This may contribute to the observed effects of histones on the NM, resulting in an enhancement and differential pattern of NM protein phosphorylation. Such a regional modification of NM protein phosphorylation might influence the nuclear functions that require histone displacement, namely, replication and transcription.

Biochemical evidence that the N-terminal segments of the α subunit and the β subunit play interchangeable roles in the activation of protein kinase CK2

The concept that the amino-terminal segment plays a role in conferring high basal activity to protein kinase CK2 α subunit has been validated by generating two mutants (Y26F and Δ2–6) which are defective both in catalytic activity and in thermal stability. The additional finding that the activity of the two mutants is fully restored upon association with the regulatory β subunit, in conjunction with the observation that synthetic peptides reproducing the N-terminal segment (1–30) and the activation loop (175–201) of CK2α counteract the functional effects of the C-terminal domain of the β subunit, is consistent with a mechanism of activation of CK2 where the N-terminal domain of α and the C-terminal domain of β play interchangeable roles.

Protein kinase CK2 is altered in insulin-resistant genetically obese ( fa/fa) rats

Hepatic insulin receptor levels in 6-week-old obese ( fa/fa) rats were about 2-fold lower than those from lean ( Fa/−) rats, which agrees with their insulin-resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese ( fa/fa) rats were similar to those of lean ( Fa/−) animals but the cytosolic levels were reduced to half, due to a decrease in the 39-kDa catalytic subunit. Marked increases in activity, due to rises in the 44-kDa and 39-kDa catalytic subunits, were seen in the 16 000× g sediments (M1) from insulin-resistant rats, with moderate changes in the 100 000× g sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin-resistant rats.

Effect of protein kinase ck2 on topoisomerase I from plasmodia of the slime mold Physarum polycephalum.

Relaxing activity of Physarum topoisomerase I was increased by calf thymus protein kinase ck2, similarly as was the activity of mammalian topoisomerase I, despite a pronounced difference between amino-acid sequences of non-conserved domains of Physarum and mammalian enzymes. This feature of Physarum topoisomerase I was cancelled in nuclear extracts isolated from dibutyryl-cAMP treated plasmodia in which the activity of protein kinase ck2 was elevated.

A surface-plasmon-resonance analysis of polylysine interactions with a peptide substrate of protein kinase CK2 and with the enzyme.

The mechanism of protein kinase CK2 (CK2) activity stimulation by polylysine has been studied by surface plasmon resonance (SPR). The kinetics of the polylysine interaction with a peptide substrate of the enzyme, and with the enzyme itself, have been investigated. A peptide containing a threonine (T) residue surrounded by a cluster of negatively charged acidic [arginine (R) and glutamic acid (E)] residues, RRREEETEEE, and specifically phosphorylated by CK2, was selected. Polylysine interacts with both the enzyme and the peptide substrate. The rate constant, the stoichiometry of the polylysine-peptide substrate interaction and the kinetic parameters of the stimulated enzyme were used to calculate the polylysine-dependent stimulation of CK2. The results are in agreement with experimentally determined polylysine-dependent stimulation. The polylysine-enzyme interaction is too slow to account for enzyme stimulation. The behaviour of polylysine is not reproduced by the polyamine spermine. The results are consistent with a substrate-mediated mechanism of CK2 stimulation by polylysine, and they suggest that the CK2 stimulation by polyamines occurs by a different mechanism.

Ornithine Decarboxylase Expression Leads to Translocation and Activation of Protein Kinase CK2 in Vivo

Ornithine decarboxylase (ODC) is the key initial enzyme in the biosynthesis of polyamines. Since polyamines have been shown to enhance protein kinase CK2 activity in vitro, ODC was overexpressed to examine the role of polyamines in CK2 regulation in vivo. Infection of Balb/MK cells with an ODC retrovirus to elevate ODC and polyamine levels increased overall protein phosphorylation as well as CK2 protein levels and enzyme activity in mimosine- or nocodazole- arrested cells. Immunofluorescence microscopy and enzyme analyses of subcellular fractions from ODC-overexpressing cells demonstrated translocation of CK2 from the cytoplasm to the nucleus with no apparent loss of cytoplasmic CK2 activity, suggesting polyamine activation of the remaining cytoplasmic enzyme. Similarly, K6/ODC transgenic mice exhibited higher ODC and CK2 enzyme activities than their normal littermates. ODC-immunostained cells in the transgenic skin also stained intensely for CK2 protein. Primary cultures of K6/ODC keratinocytes also exhibited increased ODC and CK2 enzyme activities compared with those from normal littermates. However, the addition of difluoromethylornithine, a specific ODC inhibitor, to the transgenic keratinocytes reduced both intracellular polyamine levels and CK2 enzyme activity. These results suggest that polyamines regulate the CK2 enzyme by affecting its cellular distribution as well as its enzyme activity and levels.

Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation.

We hypothesized that malignant transformation of normal mucosa of the upper aerodigestive tract to squamous cell carcinoma of the head and neck (SCCHN) might be associated with altered CK2 activity in the chromatin compartment of these tumors. We measured CK2 activity in the cytosol and chromatin of 7 surgical specimens of SCCHN, and 5 specimens of normal oropharyngeal mucosa from non-smokers/non-drinkers. CK2 activity in SCCHN tumors was significantly elevated in both the nuclear chromatin (P < 0.0005) and cytosolic (P <0.04) compartments relative to normal mucosa. These data suggest that activation of dysregulation of the chromatin-associated CK2 signal may play a role in the pathobiology od SCCHN.

Changes in the Activity of Nuclear Protein Kinase CK2 During Rat Liver Regeneration

Protein kinase CK2 has been found in two nuclear fractions obtained after treatment of purified rat liver nuclei with nucleases (S1 fraction) and subsequently with 1.6 M NaCl (S2 fraction). In both fractions three isoforms of the α subunit were identified. Two of them corresponded to the classical α and α′ subunits, whereas the identity of the third one (α3) remains unknown. In the S1 fraction two peaks of CK2 activity were detected at 6 h (5.5 fold) and 24 h (1.9 fold) after partial hepatectomy, whereas no significant changes were found in the S2 fraction. At 6 h after laparatomy a much lower increase of CK2 in S1 fraction was also detected (2.5 fold). The increases in CK2 activity found at 6 h after hepatectomy or laparatomy were accompanied with rises in the amount of the α3subunit.

Association of Elevated Protein Kinase CK2 Activity with Aggressive Behavior of Squamous Cell Carcinoma of the Head and Neck

Protein kinase CK2 (also known as casein kinase 2) is a messenger-independent protein serine/threonine kinase ubiquitously distributed in eukaryotes. CK2 has been found to phosphorylate a wide variety of cytosolic and nuclear substrates which are intimately involved in regulation of DNA, RNA, and protein synthesis, and differentiation. We therefore addressed the hypothesis that malignant transformation of upper aerodigestive tract mucosa to squamous cell carcinoma of the head and neck (SCCHN) might be associated with altered CK2 activity. To this end, we subjected surgical specimens of SCCHN tumors and of normal oropharyngeal mucosa to subcellular fractionation. We then quantitated CK2 activity in cytosol and nuclei of these specimens using a CK2-specific peptide substrate (Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu). We found that CK2 activity was significantly elevated in both nuclear (p < 0.0005) and cytosolic (p < 0.0034) compartments of SCCHN tumors, relative to normal oropharyngeal mucosa. Moreover, CK2 activity in the cellular cytosolic fraction of SCCHN tumors was associated with less differentiated histologic grade (p < 0.037), positive nodal metastatic status (p < 0.056), and a poor clinical outcome (p < 0.028). Kaplan-Meier cumulative survival analysis revealed greatly reduced survival in the high-CK2 activity patient group, with high statistical significance (p < 0.023). These preliminary data reveal that malignant transformation of the upper aerodigestive tract mucosa is associated with altered CK2 activity. The results further suggest that dysregulation of this protein kinase may play a significant role in the pathobiology of SCCHN, and that CK2 activity may be a prognostic indicator in this malignancy.

Phosphorylation and activation of protein kinase CK2 by p34cdc2 are independent events.

Recombinant isolated beta-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km = 1.7 microM, Vmax = 20 nmol.min-1.mg-1). Two synthetic peptides reproducing the 170-215 and the 206-215 C-terminal fragments of the beta-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 microM, respectively). In contrast, both the beta-subunit associated with the alpha-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 beta-subunit phosphorylation observed in intact cells occurs prior to beta-subunit incorporation into the holoenzyme. The isolated CK2 alpha-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the beta-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to beta-subunit, p34cdc2/cyclin B also protects the alpha-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated alpha-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated beta-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the beta or the alpha-subunit. These data indicate that in vitro CK2 alpha-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.

Phosphorylation and Activation of Protein Kinase Ck2 by p34cdc2 are Independent Events

Recombinant isolated β‐subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km= 1.7 μM, Vmax= 20 nmol · min−1· mg−1). Two synthetic peptides reproducing the 170–215 and the 206–215 C‐terminal fragments of the β‐subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 μM, respectively). In contrast, both the β‐subunit associated with the α‐subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 β–subunit phosphorylation observed in intact cells occurs prior to β‐subunit incorporation into the holoenzyme. The isolated CK2 α‐subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the β‐subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to β‐subunit, p34cdc2/cyclin B also protects the α‐subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated α‐subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated β‐subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the β or the α‐subunit. These data indicate that in vitro CK2 α‐subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.

Influence of Indomethacin and difluoromethylornithine on human tumour growth in nude mice

Biopsy material from six human colorectal carcinomas was transplanted to 114 nude mice. A treatment protocol was established which included no treatment (control, C), indomethacin (I), difluoromethylornithine (D) or a combination of both (ID). The influence of the various drugs on tumour weight and protein kinase CK2 activity was monitored. CK2 activity was measured because in all tumours examined so far the enzyme activity was found to be enhanced several-fold when compared to the non-neoplastic tissue of the same patient. More than half of the investigated tumours showed a conspicuous reduction in weight after drug treatment, and I and the combination of D/I were significantly effective using the mixed model analysis. Furthermore, we have tried to discover whether there is a change in the subcellular localisation of protein kinase CK2 subunits associated with drug treatment. We analysed the tumours and the non-neoplastic control tissues by immunohistochemistry using antibodies directed against the CK2 subunits and against the proliferation marker Mib. In addition, we have also investigated the behaviour of the nucleolar protein B23 which has also been shown to be enhanced several-fold in rapidly proliferating tissue and which is also a substrate for CK2. The immunohistochemical data suggest that, irrespective of the drug treatment and the observed reduction in CK2 activity, the CK2 subunits remain localised in the nucleus.

Asymmetric Expression of Protein Kinase CK2 Subunits in Human Kidney Tumors

Renal clear cell carcinomas and the corresponding ipsilateral control tissues were investigated for protein kinase CK2 activity and subunit ratio. The average protein kinase CK2 activity from 21 different kidney samples was 318 U/mg and that from the corresponding tumors 610 U/mg. The subunit ratio of protein kinase CK2α in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2β (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation. This at least partly owing to the presence of excess enzymatically active protein kinase α-subunit but also to a significantly higher presence of the non-catalytic β-subunit.