Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight.

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 µM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 µM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.

Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A.

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 µM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as "denitro NBC", dNBC), the inhibitory power toward CK2 is almost entirely lost (IC(50) > 30 µM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC(50) values with NBC and dNBC are 15 and 0.60 µM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.

Urolithin as a converging scaffold linking ellagic acid and coumarin analogues: design of potent protein kinase CK2 inhibitors.

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase; its abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other relevant diseases. Previously, using different in silico screening approaches, two potent and selective CK2 inhibitors were identified by our group: ellagic acid, a naturally occurring tannic acid derivative (K(i)=20 nM) and 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC, K(i)=60 nM). Comparing the crystallographic binding modes of both ellagic acid and DBC, an X-ray structure-driven merging approach was taken to design novel CK2 inhibitors with improved target affinity. A urolithin moiety is proposed as a possible bridging scaffold between the two known CK2 inhibitors, ellagic acid and DBC. Optimization of urolithin A as the bridging moiety led to the identification of 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one as a novel, potent and selective CK2 inhibitor, which shows a K(i) value of 7 nM against the protein kinase, representing a significant improvement in affinity for the target compared with the two parent fragments.

Unprecedented selectivity and structural determinants of a new class of protein kinase CK2 inhibitors in clinical trials for the treatment of cancer.

5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer, is representative of a new class of CK2 inhibitors with K(i) values in the low nanomolar range and unprecedented selectivity versus other kinases. Here we present the crystal structure of the complexes of CX-4945 and two analogues (CX-5011 and CX-5279) with the catalytic subunit of human CK2. Consistent with their ATP-competitive mode of inhibition, all three compounds bind in the active site of CK2 (type I inhibitors). The tricyclic scaffold of the inhibitors superposes on the adenine of ATP, establishing multiple hydrophobic interactions with the binding cavity. The more extended scaffold, as compared to that of ATP, allows the carboxylic function, shared by all three ligands, to penetrate into the deepest part of the active site where it makes interactions with conserved water W1 and Lys-68, thus accounting for the crucial role of this negatively charged group in conferring high potency to this class of inhibitors. The presence of a pyrimidine in CX-5011 and in CX-5279 instead of a pyridine (as in CX-4945) ring is likely to account for the higher specificity of these compounds whose Gini coefficients, calculated by profiling them against panels of 102 and/or 235 kinases, are significantly higher than that of CX-4945 (0.735 and 0.755, respectively, vs 0.615), marking the highest selectivity ever reported for CK2 inhibitors.

Understanding protein kinase CK2 mis-regulation upon F508del CFTR expression.

We review areas of overlap between nucleoside diphosphate kinase (NDPK; nm23) and two proteins manifesting an equivalent diversity of action, each with many thousands of publications. The first is a constitutively active protein kinase, CK2 (formerly casein kinase 2), that includes NDPK amongst its hundreds of targets. The second is an enigmatic member of the ATP-binding cassette (ABC) family of membrane pumps that normally hydrolyse ATP to transport substrates. Yet our unusual family member (ABCC7) is not a pump but, uniquely, acts as a regulated anion channel. ABCC7 is the cystic fibrosis transmembrane conductance regulator (CFTR), and we discuss the highly prevalent CFTR mutation (F508del CFTR) in terms of the uncertainties surrounding the molecular basis of cystic fibrosis that cloud approaches to corrective therapy. Using lysates from cells stably expressing either wild-type or F508del CFTR, incubated with the CK2 substrate GTP, we show that the phosphoproteome of F508del CFTR-expressing cells both differs from wild-type CFTR-expressing cells and is significantly enhanced in intensity by ∼1.5-fold (p < 0.05, paired t test with Bonferroni correction, n = 4). Phosphorylation is about 50% attenuated with a specific CK2 inhibitor. We propose that a new function may exist for the CFTR region that is commonly mutated, noting that its sequence (PGTIKENIIF(508)GVSYDEYRYR) is not only highly conserved within the C sub-family of ABC proteins but also a related sequence is found in NDPK. We conclude that a latent path may exist between mutation of this conserved sequence, CK2 hyperactivity and disease pathogenesis that might also explain the heterozygote advantage for the common F508del CFTR mutant.

The role of the N-terminal domain in the regulation of the "constitutively active" conformation of protein kinase CK2α: insight from a molecular dynamics investigation.

Protein kinase CK2 is an extremely well-conserved pleiotropic protein kinase with a growing list of substrates, the majority of which are proteins implicated in signal transduction, gene expression, and transcription-related functions. Protein kinase CK2 is a ubiquitous heterotetrameric serine/threonine protein kinase made up of two α or α' catalytic subunits and two ß regulatory subunits. Moreover, protein kinase CK2 is defined as a "constitutively active" protein kinase in contrast to most other protein kinases characterized by the presence of distinct conformations associated with the active and inactive states. As previously demonstrated by in vitro mutation studies, CK2 activity is substantially regulated by the interaction between the N-terminal tail and the kinase domain. In fact, progressive deletions of the N-terminal tail show a decrease in the activity of the kinase. Even if the detrimental effects of Δ2-12 deletion can be partially reversed by the addition of a CK2ß subunit, deletions Δ2-12 and Δ2-30 progressively decrease the basal activity of CK2. In particular, as experimentally demonstrated, the Δ2-12 N-terminal deletion affects both the K(M) value for ATP and for the substrate peptide, and the k(cat) value of CK2α. In this work, molecular dynamics (MD) simulations were carried out on wild-type (wt), Δ2-12 and Δ2-30 deletion mutants of CK2α in order to explore the role of the N-terminal tail on the conformational behavior of CK2. Furthermore, classical MD simulations were carried out to assess the anticipated impact of conformational changes in a novel set of CK2α mutant forms, such as the triple mutant Y206F-R10A-Y261F and the single mutant Y125F.

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature beta-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in beta-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 beta-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.

The pleiotropic protein kinase CK2 phosphorylates HTLV-1 Tax protein in vitro, targeting its PDZ-binding motif.

The HTLV-1 transactivator Tax is an oncoprotein capable of deregulating the expression of many cellular genes and interfering with signalling pathways. Here we show that Tax-1 is phosphorylated in vitro by the pleiotropic human serine/threonine kinase CK2 at three residues, Ser-336, Ser-344 and Thr-351, close to and within its C-terminal PDZ-binding motif. We also show that the mutation of Thr-351 to aspartate abolishes Tax-1 binding to the scaffold protein hDlg, a tumour suppressor factor, while having no effect on transactivation. These results suggest that CK2, whose constitutive activity is often hijacked by viruses to sustain their vital cycle, could modulate Tax-1 oncogenic interactions.

Cystic fibrosis transmembrane regulator fragments with the Phe508 deletion exert a dual allosteric control over the master kinase CK2.

Cystic fibrosis mostly follows a single Phe508 deletion in CFTR (cystic fibrosis transmembrane regulator) (CFTRDeltaF508), thereby causing premature fragmentation of the nascent protein with concomitant alterations of diverse cellular functions. We show that CK2, the most pleiotropic protein kinase, undergoes allosteric control of its different cellular forms in the presence of short CFTR peptides encompassing the Phe508 deletion: these CFTRDeltaF508 peptides drastically inhibit the isolated catalytic subunit (alpha) of the kinase and yet up-regulate the holoenzyme, composed of two catalytic and two non-catalytic (beta) subunits. Remarkable agreement between in silico docking and our biochemical data point to different sites for the CFTRDeltaF508 peptide binding on isolated CK2alpha and on CK2beta assembled into the holoenzyme, suggesting that CK2 targeting may be perturbed in cells expressing CFTRDeltaF508; this could shed light on some pleiotropic aspects of cystic fibrosis disease.

Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2.

A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiodobenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodobenzimidazole (TIBI) is one of the most efficient CK2 inhibitors (K(i)=23 nM) described to date.

A modified KESTREL search reveals a basophilic substrate consensus for the Saccharomyces cerevisiae Npr1 protein kinase.

The Saccharomyces cerevisiae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the Tor (target of rapamycin) signaling pathway. Inhibition of the Tor proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. As an alternative to peptide arrays, the substrate requirement of Npr1 was probed with a peptide library that was generated by cleaving yeast cell extracts with CNBr, and after reverse-phase chromatography, the individual fractions were phosphorylated in vitro with recombinant Npr1. In this way, the ribosomal protein Rpl24a was found to be an excellent in vitro substrate for Npr1. Synthetic peptides tailored around the phosphorylation site of Rpl24a show that Npr1 is a Ser/Thr protein kinase with an absolute requirement for a basic residue at the P-3 position and a strong preference for basic P + 1 residues, whereas proline at P + 1 is strongly disfavored. The results obtained with synthetic peptides suggest a (K/R)-X-X-S-(K/R) consensus sequence for Npr1. The availability of a consensus sequence allows a targeted search for physiologically relevant Npr1 substrates involved in the regulation of yeast amino acid permeases.

Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a moderately potent and poorly selective inhibitor of protein kinase CK2, one of the most pleiotropic serine/threonine protein kinases, implicated in neoplasia and in other global diseases. By virtual screening of the MMS (Molecular Modeling Section) database, we have now identified quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) as an inhibitor of CK2 that is more potent and selective than emodin. CK2 inhibition by quinalizarin is competitive with respect to ATP, with a Ki value of approx. 50 nM. Tested at 1 microM concentration on a panel of 75 protein kinases, quinalizarin drastically inhibits only CK2, with a promiscuity score (11.1), which is the lowest ever reported so far for a CK2 inhibitor. Especially remarkable is the ability of quinalizarin to discriminate between CK2 and a number of kinases, notably DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase), PIM (provirus integration site for Moloney murine leukaemia virus) 1, 2 and 3, HIPK2 (homeodomain-interacting protein kinase-2), MNK1 [MAPK (mitogen-activated protein kinase)-interacting kinase 1], ERK8 (extracellular-signal-regulated kinase 8) and PKD1 (protein kinase D 1), which conversely tend to be inhibited as drastically as CK2 by commercially available CK2 inhibitors. The determination of the crystal structure of a complex between quinalizarin and CK2alpha subunit highlights the relevance of polar interactions in stabilizing the binding, an unusual characteristic for a CK2 inhibitor, and disclose other structural features which may account for the narrow selectivity of this compound. Tested on Jurkat cells, quinalizarin proved able to inhibit endogenous CK2 and to induce apoptosis more efficiently than the commonly used CK2 inhibitors TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole).

Identification of novel protein kinase CK1 delta (CK1delta) inhibitors through structure-based virtual screening.

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer's disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).

Modulation of protein kinase CK2 activity by fragments of CFTR encompassing F508 may reflect functional links with cystic fibrosis pathogenesis.

Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500-518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2alpha catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500-518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the beta-subunit. Such a stimulatory efficacy of the CFTR 500-518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2.

The regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide.

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.

The selectivity of inhibitors of protein kinase CK2: an update.

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.

Coumarin as attractive casein kinase 2 (CK2) inhibitor scaffold: an integrate approach to elucidate the putative binding motif and explain structure-activity relationships.

Casein kinase 2 (CK2) is an ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other diseases. Recently, using different virtual screening approaches, we have identified several novel CK2 inhibitors. In particular, we have discovered that coumarin moiety can be considered an attractive CK2 inhibitor scaffold. In the present work, we have synthetized and tested a small library of coumarins (more than 60), rationalizing the observed structure-activity relationship. Moreover, the most promising inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC), has been also crystallized in complex with CK2, and the experimental binding mode has been used to derive a linear interaction energy (LIE) model.

Linear motifs in the C-terminus of D. melanogaster cryptochrome.

The C-terminus of cryptochrome (CRY) regulates light responses in Drosophila. These include the light-dependent binding of Drosophila dCRY to the clock proteins PERIOD and TIMELESS in a yeast two-hybrid system, which we proved to be a convenient and reliable readout of the behavior of dCRY in vivo. In this study, we present a combination of in silico analysis and experimental validation in yeast, to identify novel functional motifs in the C-terminal region of dCRY. Our results suggest that linear motifs are present in this small region, which is a likely hotspot for molecular interactions.

Tetrabromocinnamic acid (TBCA) and related compounds represent a new class of specific protein kinase CK2 inhibitors.

Abnormally high constitutive activity of protein kinase CK2, levels of which are elevated in a variety of tumours, is suspected to underlie its pathogenic potential. The most widely employed CK2 inhibitor is 4,5,6,7-tetrabromobenzotriazole (TBB), which exhibits a comparable efficacy toward another kinase, DYRK1 a. Here we describe the development of a new class of CK2 inhibitors, conceptually derived from TBB, which have lost their potency toward DYRK1 a. In particular, tetrabromocinnamic acid (TBCA) inhibits CK2 five times more efficiently than TBB (IC50 values 0.11 and 0.56 microM, respectively), without having any comparable effect on DYRK1 a (IC50 24.5 microM) or on a panel of 28 protein kinases. The usefulness of TBCA for cellular studies has been validated by showing that it reduces the viability of Jurkat cells more efficiently than TBB through enhancement of apoptosis. Collectively taken, the reported data support the view that suitably derivatized tetrabromobenzene molecules may provide powerful reagents for dissecting the cellular functions of CK2 and counteracting its pathogenic potentials.

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.