Search

Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms. Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae. We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits. Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits. Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function., (Copyright 2001 Academic Press.)

The regulatory subunit of S. cerevisiae casein kinase II (CKII) is encoded of two genes, CKB1 and CKB2. Strains harboring deletions of either or both genes exhibit specific sensitivity to high concentrations of Na+ or Li+. Na+ tolerance in S. cerevisiae is mediated primarily by transcriptional induction of ENA1, which encodes the plasma membrane sodium pump, and by conversion of the potassium uptake system to a higher affinity form that discriminates more efficiently against Na+. To determine whether reduced ENA1 expression plays a role in the salt sensitivity of ckb mutants, we integrated an ENA1-lacZ reporter gene into isogenic wild-type, ckb1, ckb2, and ckb1 ckb2 strains and monitored beta-galactosidase activity at different salt concentrations. In all three mutants transcription from the ENA1 promoter remained salt-inducible, but both basal and salt-induced expression was depressed approximately 3- to 4-fold. The degree of reduction in ENA1 expression was comparable to that observed in an isogenic strain carrying a null mutation in protein phosphatase 2B (calcineurin), which is also required for salt tolerance. These results suggest that reduced expression ofENA1 contributes to the salt sensitivity of ckb strains. Consistent with this conclusion, overexpression of ENA1 from a heterologous promoter (GAL1) completely suppressed the salt sensitivity of ckb mutants. Induction of ENA1 expression by alkaline pH is also depressed in ckb mutants, but unlike calcineurin mutants, ckb strains are not growth inhibited by alkaline pH.

Casein kinase II (CKII) of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', that are encoded by the CKA1 and -2 genes, respectively. We have constructed conditional alleles of the CKA1 gene. In contrast to cka1 cka2(ts) strains, which exhibit a defect in both G1 and G2/M cell cycle progression, cka1(ts) cka2 strains continue to divide for three cell cycles after a shift to restrictive temperature and then arrest as a mixture of budded and unbudded cells with a spherical morphology. Arrested cells exhibit continued growth, a nonpolarized actin cytoskeleton, delocalized chitin deposition, and a significant fraction of multinucleate cell bodies, confirming the presence of a cell polarity defect in cka1(ts) strains. The presence of budded as well as unbudded cells in the arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both processes is also possible. The terminal phenotype of cka1(ts) strains bears a strong resemblance to that of orb5 strains of Schizosaccharomyces pombe, which carry a temperature-sensitive CKII catalytic subunit mutation, but the underlying mechanism appears to be different in the two cases. These results establish a requirement for CKII in cell polarity in S. cerevisiae and provide the first evidence for functional specialization of CKA1 and -2.

Casein kinase II (CKII) is a highly conserved serine/threonine protein kinase that is ubiquitous in eukaryotic organisms. This review summarizes available data on CKII of the budding yeast Saccharomyces cerevisiae, with a view toward defining the possible physiological role of the enzyme. Saccharomyces cerevisiae CKII is composed of two catalytic and two regulatory subunits encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively. Analysis of null and conditional alleles of these genes identifies a requirement for CKII in at least four biological processes: flocculation (which may reflect an effect on gene expression), cell cycle progression, cell polarity, and ion homeostasis. Consistent with this, isolation of multicopy suppressors of conditional cka mutations has identified three genes that have a known or potential role in either the cell cycle or cell polarity: CDC37, which is required for cell cycle progression in both G1 and G2/M; ZDS1 and 2, which appear to have a function in cell polarity; and SUN2, which encodes a protein of the regulatory component of the 26S protease. The identity and properties of known CKII substrates in S. cerevisiae are also reviewed, and advantage is taken of the complete genomic sequence to predict globally the substrates of CKII in this organism. Although the combined data do not yield a definitive picture of the physiological role of CKII, it is proposed that CKII serves a signal transduction function in sensing and/or communicating information about the ionic status of the cell to the cell cycle machinery.

The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% large-budded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G1. Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G1, at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.

The published 'charge shuttle' mechanism of enolase (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822) assigns Glu-211 the task of orienting a water molecule that serves as the catalytic base which removes the proton from carbon-2 of the substrate. We prepared the E211Q mutant of yeast enolase 1 by site-directed mutagenesis. It appears to be folded correctly and to respond similarly to many of the normal ligands of enolase: it is stabilized against thermal denaturation by conformational Mg2+ and by Mg2+ and substrate and binds the chromophoric substrate analogue D-tartronate semialdehyde-2-phosphate (TSP) with affinity comparable to that of the native enzyme. However, it has only 0.01% (10(-4)) of the activity of native enolase under standard assay conditions and does not exhibit significantly more activity at various pH values or higher concentrations of substrate and Mg2+. Its ability to produce the form of enzyme-bound and reacted TSP that absorbs at shorter wavelengths is greatly slowed, while the longer wavelength absorbing form is produced rapidly. Overall, these observations are consistent with the hypothetical mechanism.

Saccharomyces cerevisiae casein kinase II (CKII) contains two distinct catalytic (alpha and alpha') and regulatory (beta and beta') subunits. We report here the isolation and disruption of the gene, CKB1, encoding the 38-kDa beta subunit. The predicted Ckb1 sequence includes the N-terminal autophosphorylation site, internal acidic domain, and potential metal binding motif (CPX3C-X22-CPXC) present in other beta subunits but is unique in that it contains two additional autophosphorylation sites as well as a 30-amino-acid acidic insert. CKB1 is located on the left arm of chromosome VII, approximately 33 kilobases from the centromere and does not correspond to any previously characterized genetic locus. Haploid and diploid strains lacking either or both beta subunit genes are viable, demonstrating that the regulatory subunit of CKII is dispensable in S. cerevisiae. Such strains exhibit wild type behavior with regard to growth on both fermentable and nonfermentable carbon sources, mating, sporulation, spore germination, and resistance to heatshock and nitrogen starvation, but are salt-sensitive. Salt sensitivity is specific for NaCl and LiCl and is not observed with KCl or agents which increase osmotic pressure alone. These data suggest a role for CKII in ion homeostasis in S. cerevisiae.

Casein kinase II of Saccharomyces cerevisiae is composed of two distinct catalytic subunits, alpha and alpha', and two distinct regulatory subunits, beta and beta' (Padmanabha, R. and Glover, C. V. C. (1987) J. Biol. Chem. 262, 1829-1835; Bidwai, A. P., Reed, J. C., and Glover, C. V. C. (1994) Arch. Biochem. Biophys. 309, 348-355). We report here the cloning, sequencing, and disruption of the CKB2 gene encoding the beta'-subunit. The deduced amino acid sequence of Ckb2 displays only 40-45% identity to other beta-subunit sequences reported to date, allowing a better definition of conserved features of this protein. Most notable is the conservation of a cysteine-containing sequence, CPX3C-X22-CPXC, which may constitute a novel metal-binding motif. The degree of sequence divergence of Ckb2 is comparable to that of the Drosophila Stellate protein, a testis-specific protein of unknown function, suggesting that the latter may function as a second beta-subunit in Drosophila. CKB2 is located on the right arm of chromosome XV between the HIR2 and WHI2 loci and has not been previously identified genetically. Haploid and homozygous diploid cells harboring a ckb2 null allele are viable, demonstrating that the beta'-subunit does not have an essential function distinct from that of beta. Strains lacking a functional CKB2 gene appear to grow normally on both fermentable and non-fermentable carbon sources, mate and sporulate normally, and display normal resistance to nitrogen starvation and heat shock. However, haploid strains harboring disruptions of both the beta' gene and either of the catalytic subunit genes exhibit a synthetic phenotype consisting of slow growth and flocculation in rich glucose medium. The occurrence of this synthetic phenotype implies that the beta'-subunit interacts physically and/or functionally with both the alpha- and alpha'-subunits in vivo.

The subunit composition of casein kinase II (CKII) from S. cerevisiae has been difficult to define, particularly with respect to the existence and number of regulatory (beta) subunits. A single, integral beta subunit, a loosely associated beta subunit, two distinct beta subunits, and a complete absence of beta subunits have all been proposed. Our laboratory reported yeast CKII to be composed of four polypeptides of 42, 41, 35, and 32 kDa (R. Padmanabha and C. V. C. Glover, 1987, J. Biol. Chem. 262, 1829-1835). The 42- and 35-kDa polypeptides were identified as distinct catalytic subunits, alpha and alpha', on the basis of N-terminal sequencing and subsequent molecular cloning. The 41- and 32-kDa polypeptides were found to undergo autophosphorylation, a characteristic of the beta subunit in other species, but antibodies raised against the beta subunit of Drosophila CKII crossreacted only with the 41-kDa polypeptide. In order to clarify the subunit composition of yeast CKII, particularly with regard to the 32-kDa polypeptide, we have purified the enzyme to homogeneity using a modified procedure. Based on the results of autophosphorylation studies, Western blotting, peptide mapping of the 41- and 32-kDa peptides, and sequencing of subunit-specific peptides, we demonstrate that the 32-kDa polypeptide is an additional beta subunit (beta') distinct from the 41-kDa beta subunit. This represents the first demonstration of beta subunit heterogeneity in purified CKII from any species.

Analysis of casein kinase II in organisms amenable to genetic manipulation is essential to elucidating the physiological function(s) of this ubiquitous protein kinase. This paper summarizes work from our laboratory on the enzyme from Saccharomyces cerevisiae. The biochemistry, molecular biology, and genetics of S. cerevisiae casein kinase II are reviewed and discussed.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which must be encoded by separate genes (R. Padmanabha and C. V. C. Glover, J. Biol. Chem. 262:1829-1835, 1987). The gene encoding the 42-kilodalton alpha subunit has been isolated by screening a yeast genomic library with oligonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the polypeptide. This gene (designated CKA1) contains an intron-free open reading frame of 372 amino acid residues. The deduced amino acid sequence is 67% identical to the alpha subunit of Drosophila melanogaster casein kinase II. The CKA1 gene product appears to be distantly related to other known protein kinases but exhibits highest similarity to the CDC28 gene product and its homolog in other species. Gene replacement techniques have been used to generate a null cka1 mutant allele. Haploid and diploid strains lacking a functional CKA1 gene appear to be phenotypically wild type, presumably because of the presence of the alpha' gene. Interestingly, the CKA1 gene appears to be single copy in the yeast genome; i.e., the alpha' gene, whose existence is known from biochemical studies and protein sequencing, cannot be detected by low-stringency hybridization.