Yeast Protein Asf1 Possesses Modulating Activity towards Protein Kinase CK2.

Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2ß, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.

CK2 and protein kinases of the CK1 superfamily as targets for neurodegenerative disorders.

Casein kinases are involved in a variety of signaling pathways, and also in inflammation, cancer, and neurological diseases. Therefore, they are regarded as potential therapeutic targets for drug design. Recent studies have highlighted the importance of the casein kinase 1 superfamily as well as protein kinase CK2 in the development of several neurodegenerative pathologies, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. CK1 kinases and their closely related tau tubulin kinases as well as CK2 are found to be overexpressed in the mammalian brain. Numerous substrates have been detected which play crucial roles in neuronal and synaptic network functions and activities. The development of new substances for the treatment of these pathologies is in high demand. The impact of these kinases in the progress of neurodegenerative disorders, their bona fide substrates, and numerous natural and synthetic compounds which are able to inhibit CK1, TTBK, and CK2 are discussed in this review.

Organofluorine Hydrazone Derivatives as Multifunctional Anti-Alzheimer's Agents with CK2 Inhibitory and Antioxidant Features.

A set of novel hydrazone derivatives were synthesized and analyzed for their biological activities. The compounds were tested for their inhibitory effect on the phosphorylating activity of the protein kinase CK2, and their antioxidant activity was also determined in three commonly used assays. The hydrazones were evaluated for their radical scavenging against the DPPH, ABTS and peroxyl radicals. Several compounds have been identified as good antioxidants as well as potent protein kinase CK2 inhibitors. Most hydrazones containing a 4-N(CH3 )2 residue or perfluorinated phenyl rings showed high activity in the radical-scavenging assays and possess nanomolar IC50 values in the kinase assays.

Compounds from Natural Sources as Protein Kinase Inhibitors.

The advantage of natural compounds is their lower number of side-effects when compared to most synthetic substances. Therefore, over the past several decades, the interest in naturally occurring compounds is increasing in the search for new potent drugs. Natural compounds are playing an important role as a starting point when developing new selective compounds against different diseases. Protein kinases play a huge role in several diseases, like cancers, neurodegenerative diseases, microbial infections, or inflammations. In this review, we give a comprehensive view of natural compounds, which are/were the parent compounds in the development of more potent substances using computational analysis and SAR studies.

Inhibitory influence of natural flavonoids on human protein kinase CK2 isoforms: effect of the regulatory subunit.

CK2 is a pleiotropic, constitutively active protein kinase responsible for the phosphorylation of more than 300 physiological substrates. Typically, this enzyme is found in tetrameric form consisting of two regulatory subunits CK2ß and two catalytic subunits CK2α or CK2α'. Several natural occurring flavonoids were tested for their ability to inhibit both CK2 holoenzymes, CK2α2ß2 and CK2α'2ß2. We identified few substances selectively inhibiting only the α' subunit. Other compounds showed similar effect towards all four isoforms. In some cases, like chrysoeriol, pedalitin, apigenin, and luteolin, the α2ß2 holoenzyme was at least six times better inhibited than the free α subunit. Otherwise, we have found a luteolin derivative decreased the kinase activity of CK2α' with an IC50 value of 0.8 µM, but the holoenzyme only with 9.5 µM.

Selected flavonoid compounds as promising inhibitors of protein kinase CK2α and CK2α', the catalytic subunits of CK2.

CK2 is a ubiquitous protein kinase involved in many cell functions. During the last years it became an interesting target in cancer research. A series of flavonoid compounds was tested as inhibitors of protein kinase CK2. Several substances were found to be highly active against both catalytic subunits with IC50 values below 1 µM in case of CK2α'. The most promising inhibitor we identified is chrysoeriol with IC50 values of 250 and 34 nM for CK2α and CK2α', respectively.

In Search of the Antimicrobial Potential of Benzimidazole Derivatives.

A broad series of 4,5,6,7-tetrahalogenated benzimidazoles and 4-(1H-benzimidazol-2-yl)-benzene-1,3-diol derivatives was tested against selected bacteria and fungi. For this study three plant pathogens Colletotrichum sp., Fusarium sp., and Sclerotinia sp., as well as Staphylococcus sp., Enterococcus sp., Escherichia sp., Enterobacter sp., Klebsiella spp. , and Candida spp. as human pathogens were used. MIC values and/or area of growth reduction method were applied in order to compare the activity of the synthesized compounds. From the presented set of 22 compounds, only 8, 16, 18 and 19 showed moderate to good inhibition against bacterial strains. Against Candida strains only compound 19 with three hydroxyl substituted benzene moiety presented high inhibition at nystatin level or lower.

CK2α and CK2α' subunits differ in their sensitivity to 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazole derivatives.

The goal of this study was to test the inhibitory activity of a series of tetrahalogenobenzimidazoles, including a number of novel derivatives, on individual catalytic subunits of human CK2. 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazoles and their newly obtained N(1)- and 2-S-carboxyalkyl derivatives showed potent inhibitory activity against both these subunits. CK2α' was up to 6 times more sensitive to the studied compounds than CK2α. The investigated iododerivatives showed, in most cases, stronger inhibitory properties than the respective brominated congeners, but the differences showed considerable dependence on the protein substrate used.

Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (ß) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2ß subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2ß subunit, that also possess the conserved features of regulatory CK2ß subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2ß proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

Interactions between subunits of protein kinase CK2 and their protein substrates influences its sensitivity to specific inhibitors.

Five isoforms of CK2 may exist simultaneously in yeast cells: free catalytic subunits CK2α', CK2α and three holoenzymatic structures composed of αα'ßß', α(2)ßß' and α'(2)ßß'. Each isolated and purified form exhibits properties typical for CK2, but they differ in substrate specificity as well as in sensitivity to specific modulators. All five isoforms of protein kinase CK2 from Saccharomyces cerevisiae were examined for their binding capacity with ATP/GTP and two commonly used ATP-competitive inhibitors TBB and TBI. Enzymes were tested with protein substrates differently interacting with CK2 subunits: Elf1, Fip1, Svf1, P2B and synthetic peptide. Obtained results show that K(m) for ATP varies from 2.4-53 µM for Elf1/CK2α' and Svf1/CK2α, respectively. Similar differences can be seen in case when GTP was used as phosphate donor. The inhibitory effect depends on composition of CK2/substrate complexes. Highest sensitivity to TBB shows all complexes containing αα'ßß' isoform with K (i) values between 0.2 and 1.1 µM. The prospect that TBB and TBI could be utilized to discriminate between different molecular forms of CK2 in yeast cells was examined. Both inhibitors, TBB as well as TBI, decreases cell growth to extents devoting interactions with different CK2 isoforms present in the cell but the presence of ß/ß'-dimer has a high importance towards sensitivity. Conceivably, a given inhibitor concentration can inhibit only selected CK2-mediated processes in the cell.

Isolation of a CK2α subunit and the holoenzyme from the mussel Mytilus galloprovincialis and construction of the CK2α and CK2ß cDNAs.

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (ß) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2ß subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2ß subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2ß subunits have similar features to their mammalian orthologs.

Catalytic activity of mutants of yeast protein kinase CK2alpha.

Yeast CK2 is a highly conserved member of the protein kinase CGMC subfamily composed of two catalytic (alpha and alpha') and two regulatory (beta and beta') subunits. The amino-acid sequences of both catalytic subunits are only 60% homologous. Modelling of the tertiary structure of the CK2alpha displays additional alpha-helical structures not present in the CK2alpha' subunit, connecting the ATP-binding loop with the catalytic and activation loops. Deletion of this part causes drastic structural and enzymatic changes of the protein (CK2alpha(Delta91-128)) with characteristics similar to yeast CK2alpha' (low sensitivity to salt, heparin and spermine). Additionally, the deletion causes an over 5-fold decrease of the binding affinity for ATP and ATP-competitive inhibitors (TBBt and TBBz). The structural basis for TBBt and TBBz selectivity is provided by the hydrophobic pocket adjacent to the ATP/GTP binding site, which is smaller in CK2 than in the majority of other protein kinases. The importance of hydrophobic interactions in the binding of specific inhibitors was investigated here by mutational analysis of CK2alpha residues whose side chains contribute to reducing the size of the hydrophobic pocket. Site-directed mutagenesis was used to replace Val67 and Ile213 by Ala. The kinetic properties of the single mutants CK2alpha(Val67Ala) and CK2alpha(Ile213Ala), and the double mutant CK2(Val67Ala Ile213Ala) were studied with respect to ATP, and both inhibitors TBBt and TBBz. The K(m) values for ATP did not change or were very close to those of the parental kinase. In contrast, all CK2alpha mutants analysed displayed higher K(i) values towards the inhibitors (10 to 12-fold higher with TBBt and 3 to 6-fold with TBBt) comparing to recombinant wild-type CK2alpha.

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2.

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2alpha (Km 0.35 microM) and CK2alpha' (Km 0.18 microM) as well as CK2 holoenzyme (Km 1.1 microM). Different Km values argue that CK2beta(beta') subunit has an inhibitory effect on the activity of both CK2alpha and CK2alpha' towards surviving factor Svf1. Reconstitution of alpha'2betabeta' isoform of CK2 holoenzyme shows that beta/beta' subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of alpha2betabeta' isoform, which may be due to interaction between Svf1 and regulatory CK2beta subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.

Yeast holoenzyme of protein kinase CK2 requires both beta and beta' regulatory subunits for its activity.

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (alpha and/or alpha') subunits and two regulatory beta and beta' subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of alphaalpha'betabeta', alpha2betabeta', alpha'2betabeta' and a free alpha'-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory beta subunit cannot substitute other beta' subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the beta or beta' regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic alpha subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2beta/beta' subunits in the regulation of holoenzyme assembly and phosphotransferase activity.

Yeast elf1 factor is phosphorylated and interacts with protein kinase CK2.

One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2alpha' (K(m) 0.38 microM) and holoenzyme (K(m) 0.13 microM). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Coimmunoprecypitation experiments show that Elf1 interacts with catalytic (alpha and alpha') as well as regulatory (beta and beta') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.

Fip1--an essential component of the Saccharomyces cerevisiae polyadenylation machinery is phosophorylated by protein kinase CK2.

Since Fip1 is phosphoprotein we investigated whether it is a substrate for protein kinase CK2. According to the amino acid sequence Fip1 harbours twenty putative CK2 phosphorylation sites. Here we have report characterization of Fip1 as a substrate for both forms of CK2. Fip1 serves as a substrate for both the recombinant CK2alpha ' (Km 1.28 microM) and holoenzyme (Km 1.4 microM) but not for CK1. By MALDI-MS we identified the two serine residues at positions 73 and 77 as the possible in vitro phosphorylation sites. These data may help to elucidate the role of Fip1 in the mRNA 3'-OH polyadenylation process and the involvement of CK2 mediated phosphorylation in regulation of interactions and activity members of cleavage/polyadenylation factor (CPF) complex.

Different properties of four molecular forms of protein kinase CK2 from Saccharomyces cerevisiae.

CK2 is a pleiotropic constitutively active serine/threonine protein kinase composed of two catalytic alpha- and two regulatory beta-subunits, whose regulation is still not well understood. It seems to play an essential role in regulation of many cellular processes. Four active forms of CK2, composed of alphaalpha'betabeta', alpha(2)betabeta', alpha'(2)betabeta', and a free alpha'-subunit were isolated from wild-type yeast and strains containing a single deletion of the catalytic subunit. Each species exhibits properties typical for CK2, but they differ in substrate specificity and sensitivity to inhibitors. This suggests that each CK2 isomer may regulate different process or may differ in the way of its regulation.

TBBz but not TBBt discriminates between two molecular forms of CK2 in vivo and its implications.

Two ATP-competitive inhibitors-4,5,6,7-tetrabromo-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-benzimidazole (TBBz) have been shown to decrease activity of CK2 holoenzyme. Surprisingly it occurs that TBBz contrary to TBBt does not inhibit free catalytic subunit CK2 [Formula: see text]. Both inhibitors are virtually inactive against RAP protein kinase. The above-mentioned protein kinases phosphorylate in vitro a set of acidic ribosomal P-proteins of the 60S ribosomal subunit. Such a modification is one of the mechanisms regulating translational activity of ribosomes in vivo. Application of these two very selective inhibitors allows us to define the role of free catalytic [Formula: see text] subunit of CK2 in phosphorylation of ribosomal proteins. It occurs that CK2 [Formula: see text] but not CK2 holoenzyme is responsible for phosphorylation of P-proteins in vivo. Moreover, elimination of both forms of protein kinase CK2 (hCK2 and CK2 [Formula: see text] ) activity in living cells led to dramatic loss of the translational activity of the ribosome.

The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD1.

The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.

Selectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources.

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.