FAS-associated factor 1 interacts with protein kinase CK2 in vivo upon apoptosis induction.

We show here that in several different cell lines protein kinase CK2 and Fas-associated factor 1 (FAF1) exist together in a complex which is stable to high monovalent salt concentration. The CK2/FAF1 complex formation is significantly increased after induction of apoptosis with various DNA damaging agents. Interestingly this effect is only seen in cell lines with an embryonic origin and not when cells have entered a differentiated state. It is further shown that the CK2 specific phosphorylation sites in the FAF1 molecule, i.e. serines 289 and 291 influence this complex formation. Mutation of the CK2 phosphorylation sites in the FAF1 molecule to alanine leads to a 1.5 to 2.0-fold higher association between CK2 and FAF1. Since the CK2 activity did not increase concomitantly with the complex formation we conclude that the FAF1 becomes to the CK2 enzyme so that a normal enzyme catalysis does not take place anymore. Subcellular localization experiments involving CK2 subunits and FAF1 show a co-localization of both CK2 subunits and FAF1 in the peri-nuclear cytoplasm. The majority of CK2 subunits is found in the nucleus. FAF1 is also found in the nucleoli. The results obtained further support the view that protein kinase CK2 plays an important role in certain steps of apoptosis.

Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites.

We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit. Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2. Here, we report the full length human FAF1 cDNA sequence, expression of FAF1 in Escherichia coli and purification and characterization of FAF1 as a substrate for CK2. FAF1 as well as an N-terminal 40 kDa degradation product serve as substrates for both the recombinant CK2 holoenzyme (km 100 microM) and the isolated catalytic alpha subunit (km 200 microM). Despite the high k(m) values, we obtained evidence that CK2 is the major cellular kinase responsible for FAF1 phosphorylation, using tissue extracts as kinase sources. By MALDI-MS we identified the two serine residues at positions 289 and 291 as the major in vitro CK2 phosphorylation sites. These data may help us elucidate the functions of FAF1 and the involvement of CK2 mediated phosphorylation in processes such as apoptotic signaling, ubiquitination, nuclear translocation and embryonic development.

The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds.

The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.

Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system.

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.

CK2: a protein kinase in need of control.

Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regulatory properties of CK2 are poorly understood; it is insensitive to any known second messenger and displays high basal activity. To gain insight into CK2 regulation and to understand its unusual properties, site-directed mutagenesis experiments on both subunits and X-ray crystallographic studies of the catalytic alpha-subunit were performed. The noncatalytic beta-subunit has at least three functions: (1) it protects the alpha-subunit against denaturing agents or conditions; (2) it alters the substrate specificity of the alpha-subunit; and (3) it modulates the activity of the enzyme, i.e., depending on the substrate, it increases or decreases the activity of the alpha-subunit. Mutagenesis experiments revealed that an acidic stretch between amino acids 55 and 64 has a down-regulatory and autoinhibitory function. Mutational analysis of the alpha-subunit has revealed a network of unique basic residues that are responsible for the recognition of phosphoacceptor substrates and for down-regulation by the beta-subunit and by polyanionic inhibitors. The resolution of the crystal structure of Zea mays CK2 alpha-subunit has disclosed the structural features that are responsible for high basal activity and for unusual response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential.

Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin.

Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after cisplatin and carboplatin treatment, the RNA level of p53 dropped by 59% +/- 9% and 86% +/- 8% respectively, whereas the observed level of p53 protein rose to 7 and 10 times over the untreated control, respectively. Treatment with 33 microM cisplatin prompted apoptosis as early as 4 h after drug treatment. More than 50% apoptotic cells were seen after 6 h. We conclude that cisplatin and its second generation drug carboplatin act similarly i.e. both drugs cause a concomitant decrease in p53 mRNA and an increase in p53 protein level. After 4 h treatment with either of the two drugs, p53 levels reach a threshold which leads to the initiation of apoptosis.

Interactions of protein kinase CK2beta subunit within the holoenzyme and with other proteins.

Protein kinase CK2 is a ubiquitous, highly conserved protein kinase with a tetrameric alpha2beta2 structure. For the formation of this tetrameric complex a beta-alpha dimer seems to be a prerequisite. Using the two-hybrid system and a series of CK2beta deletion mutants, we mapped domains involved in alpha-beta and beta-beta interactions. We also detected an intramolecular beta interaction within the amino acid stretch 132-165. Using CK2beta as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor suppressor protein Doc-1, the Fas-associated protein FAF1, the mitochondrial translational initiation factor 2 and propionyl CoA carboxylase beta subunit.

Extraribosomal function of the acidic ribosomal P1-protein YP1alpha from Saccharomyces cerevisiae.

The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function. The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast. Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator. This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1. Additionally, truncated proteins of YP1alpha and YP1beta were analyzed. The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not. Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.

Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles.

The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits alpha (catalytic) and beta (regulatory). The alpha' subunit was only detected in brain and testicles. By contrast, Northern blot analyses of the CK2alpha mRNA revealed a somewhat different picture. Here, the strongest signals were obtained for brain, liver, heart and lung. In kidney, spleen and testicles mRNAs were only weakly detectable. For CK2alpha' mRNA distribution strong signals were observed for lung, liver and testicles. In the case of CK2beta mRNA the highest signals were found for testicles, kidney, brain and liver. The amount of CK2beta mRNA in testicles was estimated to be about 6-fold higher than in brain. The strongest CK2beta signals in the Western blot were found for testicles and brain. The amount of CK2beta protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2-3-fold higher whereas the ratio of CK2beta between testicles and brain was estimated to be 3-4-fold. Results from the immunoprecipitation experiments support the notion for the existence of free CK2beta population and/or CK2beta in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2beta was observed. This is the first physiological evidence for the existence of a 'free CK2beta' (or in complex with proteins other than CK2a) in normal animal tissue apart from the hitherto dogmatic association with CK2alpha in a tetrameric holoenzyme complex.

A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit.

In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited in the EMBL database under the accession numbers R08806 and Z17360, for the ribosomal protein L5 and for A-Raf kinase. All isolated clones except the one for CK2 beta showed no interaction with the catalytic alpha subunit of CK2. A-Raf kinase is a new interesting partner of CK2 beta. The isolated A-Raf clone represented amino acids 268-606, but also a full length A-Raf clone interacted with CK2 beta. At the site of CK2 beta, residue 175 and amino acids between residues 194 and 200 are likely to be involved in direct interaction.

Structure of protein kinase CK2: dimerization of the human beta-subunit.

Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. Using the two-hybrid system we could show that the alpha- or alpha'-subunits of CK2 can interact with the beta-subunits of CK2, but not with other alpha- or alpha'-subunits. By comparison, the beta-subunit of CK2 can interact with another beta-subunit. Important amino acids for successful dimerization of the beta-subunit were localized between amino acid residues 156 and 165. Furthermore, we identified residues between amino acid 170 and 180 which antagonize the dimerization.

Mapping of the interaction sites of the growth suppressor protein p53 with the regulatory beta-subunit of protein kinase CK2.

p53 plays an essential role in cellular growth control. Some of its distinct biological functions are regulated by interaction with cellular proteins. We have previously (Wagner et al., 1994) shown that p53 binds to the regulatory subunit of protein kinase CK2. Using C-terminal protein fragments of p53 we now demonstrate that the region between amino acids 287 and 340 on the polypeptide chain of p53 is critical for binding of p53 to the beta-subunit of CK2. Neither phosphorylation at the p34cdc2 site (aa315) nor at the CK2 site (aa392) is necessary for binding of p53 to the beta-subunit of CK2. Using deletion mutants of the beta-subunit of CK2 we also show that an internal region between amino acids 72 and 149 of the beta-subunit of CK2 is necessary for binding to p53. Thus, this study defines new functional regions on the polypeptide chains of p53 and of protein kinase CK2.

Structure of the gene encoding the murine protein kinase CK2 beta subunit.

The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2 beta gene. Both genes contain a first untranslated exon. Also, the promoter regions from the human and murine CK2 beta gene share some common features, e.g., they contain neither a TATA nor a CAAT box, exon 1 is flanked by a cluster of CpG dinucleotides and recognition sequences for the HpaII restriction endonuclease, and several blocks of sequence in the 5' flanking region are conserved between mouse and human. Despite all of these common features, one of the most striking differences found concerns the human CK2 alpha subunit binding domain at position -170 to -239 of the human gene. This domain has no counterpart in the murine gene. Hence, regulation of transcription of the CK2 beta gene by the catalytic CK2 alpha subunit as was described by Robitzki et al. (J. Biol. Chem. 268: 5694-5703, 1993) for the human gene cannot be considered a general regulatory mechanism.

Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors.

A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta-subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127.

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.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

Dissection of the dual function of the beta-subunit of protein kinase CK2 ('casein kinase-2'): a synthetic peptide reproducing the carboxyl-terminal domain mimicks the positive but not the negative effects of the whole protein.

The dual function of the regulatory beta-subunit of protein kinase CK2 is highlighted by its ability to abolish calmodulin phosphorylation in contrast to its stimulatory effect on the phosphorylation of peptide substrates. Here we show that a synthetic peptide reproducing the C-terminal region of the beta-subunit (beta[170-215]) stimulates to a similar extent the phosphorylation of either the peptide substrate or calmodulin and also protects the catalytic alpha-subunit against thermal inactivation as efficiently as full-length beta-subunit. These data show that the positive and negative functions of the beta-subunit reside in physically separated domains and that the elements responsible for positive regulation are located in the C-terminal region.

Characterization of the cDNA and three processed pseudogenes from the murine protein kinase CK2 alpha subunit.

Seven protein kinase CK2 alpha clones were isolated from a murine genomic DNA library. They were assigned to four different genomic loci (A,B,C,D). Locus D was previously identified as a processed pseudogene (Boldyreff et al. (1992) Biochem. Biophys. Res. Commun. 186, 723-730). Here we present sequences of genomic loci B and C and the murine CK2 alpha cDNA. Loci B and C are like locus D processed pseudogenes, however, with considerable differences among each other and to the cDNA, especially with respect to the lengths of the putative gene products. Genomic locus D would code for a protein of 82 amino acids, locus B for a protein of 132 amino acids and locus C product for the full length product of 391 amino acids as the murine cDNA.

Mapping the residues of protein kinase CK2 implicated in substrate recognition: mutagenesis of conserved basic residues in the alpha-subunit.

Six mutants of protein kinase CK2 alpha subunit in which basic residues have been mutated into alanines were assayed for their capability to phosphorylate the peptide RRRADDSDDDDD. Two mutants (R228A and R278K279R280A) behaved more or less as alpha wild type and one (H160,166A) was nearly inactive, hampering the calculation of kinetic parameters. In contrast 3 mutants (K74-77A, K79R80K83A and R191,195K198A) phosphorylated the peptide with reduced efficiency accounted for by increased Km and decreased Vmax values. By using derivatives of the RRRADDSDDDDD peptide in which individual aspartyl residues were variably replaced by alanine(s) and two peptide substrates derived from I-2 (KYRIREQESSGEEDSDL and RRKDLHDDEEDEEMSETADGE) it was shown that mutations in the 191-198, 74-77 and 79-83 regions were the least detrimental whenever the acidic determinants were lacking at positions +1, +4/+5 and +3, respectively. These data support the conclusion that the basic residues present in the p+1 loop of CK2 alpha specifically recognize the acidic determinant adjacent to the C-terminal side of serine, while the specificity determinants located more down-stream are variably recognized by different residues of the unique basic cluster spanning between Lys74 and Lys83.

PstI identifies biallelic DNA polymorphism of the human casein kinase 2 alpha gene (CSNK2A1).

cDNA probe of the casein kinase 2 alpha subunit gene detects a biallelic PstI polymorphism. This restriction fragment length polymorphism is the first known genetic marker of this gene.