Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

M-phase-specific cdc2 protein kinase phosphorylates the beta subunit of casein kinase II and increases casein kinase II activity

The M-phase-specific cdc2 (cell division control) protein kinase (a component of the M-phase-promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5-5-fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non-hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The beta subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the beta subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M-phase and therefore be involved in the pleiotropic effects of M-phase-promoting factor.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

Molecular cloning of the human casein kinase II alpha subunit

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.     

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.     

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

The casein kinase II beta subunit binds to Mos and inhibits Mos activity.

Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation. Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK). We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos. The Mos-interacting region of CKII beta was mapped to the C terminus. Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta as well as in unfertilized Xenopus eggs. CKII beta inhibited Mos-mediated MAPK activation in rabbit reticulocyte lysates and repressed MKK activation by v-Mos in a coupled kinase assay. In addition, microinjection of CKII beta mRNA into Xenopus oocytes inhibited progesterone-induced meiotic maturation and MAPK activation, presumably by binding of CKII beta to Mos and thereby inhibiting MAPK activation. Moreover, this inhibitory phenotype could be rescued by another protein that binds to CKII beta, CKII alpha. The ability of ectopic CKII beta to inhibit meiotic maturation and the detection of a complex between endogenous Mos and CKII beta suggest that CKII beta may act as an inhibitor of Mos during oocyte maturation, perhaps setting a threshold beyond which Mos protein must accumulate before it can activate the MAPK pathway.     

Functional and structural conservation of Schizosaccharomyces pombe dTMP kinase gene.

We describe the isolation and identification of the Schizosaccharomyces pombe dTMP kinase gene by the complementation of a Saccharomyces cerevisiae cell cycle mutant cell, cdc8. The isolated cDNA contains an open reading frame which can encode a protein with the molecular weight of 24,151. The deduced protein sequence is highly conserved among known dTMP kinase sequences from different organisms. The isolated gene should facilitate our study of its enzymatic activity, as well as nucleotide metabolism and cell cycle regulation in this organism.     

Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy.

Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition, comparison of the far-UV CD spectrum of reconstituted CK-2 with the spectra of the subunits indicates that conformational changes occur in the backbone region upon association. Such changes may explain the increased enzyme activity of the holoenzyme relative to that of the alpha subunit itself. In contrast, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin, presumably by its binding to the polylysine stretch at amino acid positions 74-77. Heat denaturation experiments (25-90 degrees C) support the notion that heparin may provide a local protective function. A similar but much larger effect was also observed in the presence of the beta subunit only, which supports previous suggestions of a protective function for this subunit. These results indicate that the protection provided by the beta subunit and the increased enzyme activity of the holoenzyme may arise, in part, from a stabilization of the conformation of the enzyme complex and an increase in alpha-helical content.     

Molecular cloning and mapping of casein kinase 2 alpha and beta subunit genes in barley.

Casein kinase 2 (CK2) is a ubiquitous, highly pleiotropic, constitutively active, and messenger-independent Ser/Thr protein kinase. It is found in two different forms: the heterotetrameric CK2, composed of two alpha catalytic subunits and two beta regulatory subunits, and the monomeric CK2 alpha, consisting of the alpha catalytic subunit. In the present study, we isolated barley cDNA clones of the CK2 alpha and beta subunit genes, designated HvCK2A and HvCK2B, respectively. Chromosome assignment, using a set of wheat-barley disomic chromosome addition lines, and RFLP mapping, using two doubled haploid populations, showed that HvCK2A was duplicated on the short arm of chromosome 2H and the long arm of chromosome 5H (designated HvCK2a-2H and HvCK2a-5H, respectively), and a single copy of HvCK2B was located on the long arm of chromosome 1H (designated HvCK2b). A PCR-Southern hybridization experiment demonstrated that the HvCK2A sequence originated from the HvCK2a-5H locus, showing that at least HvCK2a-5H was expressed. The present cDNA sequences and genomic organization of the two subunits will facilitate further functional analysis of CK2 in barley.     

The resistance to alkali treatment of the phosphorylation of beta casein versus alpha casein is specific for casein kinase II

The phosphorylation of mixed casein by casein kinase II shows resistance on beta casein after partial alkali hydrolysis of the proteins separated by gel electrophoresis. This property is specific for casein kinase II among the protein kinases tested and can be used for casein kinase II detection in biological extracts and for characterization of purified casein kinase II.     

In-vitro phosphorylation activity by recombinant alpha and beta subunits of Bombyx mori casein kinase 2

To clarify the control mechanism of the catalytic activity of casein kinase 2 (CK2) during early embryonic development in the silkworm, Bombyx mori, we attempted an in-vitro functional analysis by using the recombinant alpha and beta subunits of B. mori CK2 (rBmCK2alpha and rBmCK2beta) produced in a bacterial system. The renatured rBmCK2alpha possessed protein kinase activity. When rBmCK2alpha and rBmCK2beta were reconstituted in an approximate 1:1 molar ratio, the catalytic activity was almost the same as that of rBmCK2alpha alone. The catalytic activity of rBmCK2alpha was inhibited by polylysine, which is one of the activators of CK2 activity. However, when using the reconstituted rBmCK2alpha and rBmCK2beta (rBmCK2), activation by polylysine was observed. We examined the influence of sorbitol and 3-hydroxykynurenine (3-OHK), which are contained mainly in diapause eggs, on the phosphorylation activity of rBmCK2. Three-OHK inhibited rBmCK2 activity, but sorbitol had no effect on it. Furthermore, a functional analysis using rBmCK2alpha and beta subunits of Drosophila melanogaster CK2 revealed that a difference in the C-terminal amino acid of the CK2beta subunit influenced the phosphorylation activity of rBmCK2alpha. These results may provide new insights for clarifying the control mechanism of B. mori casein kinase 2 in eggs.     

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the alpha subunit.

The human serine/threonine protein casein kinase II (CK II) contains two distinct catalytic subunits, alpha and alpha 1, which are encoded by different genes. A combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization have localized the human CK II-alpha DNA sequence to two loci: 11p15.5-p15.4 and 20p13. In contrast, the CK II-alpha' gene has been mapped to chromosome 16 by somatic cell hybrid analysis. Taken together with our previous assignment of the CK II regulatory beta-subunit gene to 6p12-p21, these results indicate that although the products of these genes form a single biological complex, they are encoded on different human chromosomes. Further analysis should determine whether both loci of CK II-alpha are functional, or perhaps one of the two constitutes a pseudogene.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.