Catalytic Subunit Of Casein Kinase Research Articles

Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation.

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

Functional analysis of Trichoderma reesei CKIIα2, a catalytic subunit of casein kinase II

Trichoderma reesei is the most important industrial cellulase-producing filamentous fungus. Although its molecular physiology has been investigated, the signal transduction pathways are not fully understood. In particular, the role of casein kinase II (CKII) is not yet clear. In this work, we carried out functional investigations on a catalytic subunit of CKII, CKIIα2. Comparison of the phenotypic features of T. reesei parent and Δck2α2 strains showed significant changes following ck2α2 disruption. T. reesei Δck2α2 form significantly smaller mycelial pellets in glucose-containing liquid minimum media, have shorter and fewer branch hyphae, produce smaller amounts of chitinases, produce more spores, show more robust growth on glucose-containing agar plates, and consume glucose at a significantly higher rate. Suggestions can be made that CKIIα2 governs chitinase expression, and the disruption of ck2α2 results in lower levels of chitinase production, leading to a weaker cell wall disruption capability, further resulting in weaker hyphal branching, which eventually leads to smaller mycelial pellets in liquid media. Further conclusions can be made that CKIIα2 is involved in repression of sporulation and glucose metabolism, which is consistent with the proposal that CKIIα2 represses global metabolism. These observations make the deletion of ck2α2 a potentially beneficial genetic disruption for T. reesei during industrial applications, as smaller mycelial pellets, more spores and more robust glucose metabolism are all desired traits for industrial fermentation. This work reports novel unique functions of a CKII catalytic subunit and is also the first genetic and physiological investigation on CKII in T. reesei.

Site-Directed Mutagenesis and Structure/Function Studies of Casein Kinase II Correlate Stimulation of Activity by the β Subunit with Changes in Conformation and ATP/GTP Utilization

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23,894-23,902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.

CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop.

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. In this study, we show that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a(L) strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt(L) mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ~32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a(L) strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a(L) strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka(RIP) strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka(RIP) strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop.

NS1 Interaction with CKIIα: Novel Protein Complex Mediating Parvovirus-Induced Cytotoxicity

During a productive infection, the prototype strain of the parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations in permissive A9 fibroblasts, culminating in cell lysis at the end of infection. These cytopathic effects (CPE) result from rearrangements and destruction of the cytoskeletal micro- and intermediate filaments, while other structures such as the nuclear lamina and particularly the microtubule network remain protected throughout the infection (J. P. F. Nüesch et al., Virology 331:159-174, 2005). In order to unravel the mechanism(s) by which parvoviruses trigger CPE, we searched for NS1 interaction partners by differential affinity chromatography, using distinct NS1 mutants debilitated specifically for this function. Thereby, we isolated an NS1 partner polypeptide, whose interaction with NS1 correlated with the competence of the viral product for CPE induction, and further identified it by tandem mass spectrometry and Western blotting analyses to consist of the catalytic subunit of casein kinase II, CKIIalpha. This interaction of NS1 with CKIIalpha suggested interference by the viral protein with intracellular signaling. Using permanent cell lines expressing dominant-negative CKIIalpha mutants, we were able to show that this kinase activity was indeed specifically involved in parvoviral CPE and progeny particle release. Furthermore, the NS1/CKIIalpha complex proved to be able to specifically phosphorylate viral capsids, indicating a mediator function of NS1 for CKII activity and specificity, at least in vitro. Altogether our data suggest that parvovirus-induced CPE is mediated by NS1 interference with intracellular CKII signaling.

Regulation of Cdc2p and Cdc13p Is Required for Cell Cycle Arrest Induced by Defective RNA Splicing in Fission Yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.

Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2.

Casein kinase 2 (CK2) is a tetrameric enzyme constitutively expressed in all eukaryotic tissues. The two known isoforms of the catalytic subunit, CK2alpha and CK2alpha', have been reported to have distinct tissue-dependent subcellular distributions. We recently described a third isoform of the catalytic subunit, designated CK2alpha", which is highly expressed in liver. Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as immunofluorescent microscopy revealed that CK2alpha" was exclusively localized to the nucleus and preferentially associated with the nuclear matrix. CK2alpha and CK2alpha' were found in nuclear, membrane, and cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2alpha" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclear retention indicates that the underlying mechanism of CK2alpha" nuclear localization is dependent on the secondary structure of the holoenzyme directed by the carboxy-terminal sequence.

A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity.

The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes. The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment. In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity. During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro. Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein. Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231. Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function. Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha. The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity.

Identification of the in vivo casein kinase II phosphorylation site within the homeodomain of the cardiac tisue-specifying homeobox gene product Csx/Nkx2.5.

Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2. 5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

Analysis and mapping of plastin phosphorylation.

Plastins (fimbrins) are a family of three tissue-specific actin-binding proteins. Both L- and T-plastin have been shown to be involved in cytoskeletal reorganization in signal-transduction pathways. Phosphorylation of the leukocyte-specific L-plastin plays important roles in regulating L-plastin function and leukocyte activation. We created L-plastin mutants, changing each of the Ser-5 and Ser-7 residues to alanine. Expression of either mutant in WI38VA13 fibroblasts did not result in phosphorylation. The transfected wildtype L-plastin was phosphorylated in WI38VA13, but its two-dimensional gel pattern suggested that it was phosphorylated on one residue, whereas the endogenous L-plastin in leukocytes was likely phosphorylated on two residues. The nonleukocyte-specific T-plastin, which has an equivalent Ser-7 residue, was not phosphorylated in T-plastin-expressing fibroblasts or in transfected leukocytes. Expression of CK-IIalpha, the catalytic subunit of casein kinase II, resulted in changes of the protein expression profile in leukocytes but not the phosphorylation status of L- or T-plastin. Phorbol myristate acetate induced L-plastin phosphorylation in both leukocytes and fibroblasts, lending support to the view that protein kinase C is the likely candidate kinase for L-plastin phosphorylation.

Expression and characterization of the recombinant catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica in Escherichia coli.

The alpha catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica has been cloned and overexpressed using the pT7-7 expression vector in Escherichia coli. Casein kinase activity is found in the bacterial extracts. The catalytic subunit is partially expressed in a soluble and active form, which is purified to electrophoretic homogeneity. Most of the enzyme was found in inclusion bodies. In this form, the enzyme, which is almost pure, exhibits a low specific activity. We have focused our efforts on methods to activate the protein from the inclusion bodies. We have studied the renaturation of urea-denaturated CKII catalytic subunit. We have tried different renaturation buffers and found that renaturation by dilution was more efficient than renaturation by dialysis. Treatment of the enzyme found in the inclusion bodies with different nondetergent sulfobetaines (NDSB) led to a time-dependent activation. NDSB195 is a V-type activator of the recombinant catalytic subunit of casein kinase II. The NDSB195-activated enzyme remained active at the room temperature for weeks. Kinetic properties of the recombinant casein kinase II subunit are similar to those of the purified holoenzyme (low Km for ATP and inhibition by heparin). Kinetic study indicates that the beta subunit could interact with the alpha subunit at the level of the catalytic site to enhance activity and to modify the kinetic behavior of the enzyme.

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica.

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form alpha alpha' beta 2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic alpha subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of alpha subunit described in other species, although, uniquely, Y. lipolytica CKII alpha lacks cysteines. We find that the alpha subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKII alpha expression and CKII alpha activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.

Phosphorylation of the C2 subunit of the proteasome in rice ( Oryza sativa L.)

Proteasomes function mainly in the ATP-dependent degradation of proteins that have been conjugated with ubiquitin. To demonstrate the phosphorylation of proteasomes in plants, we conducted an enzymatic dephosphorylation experiment with a crude extract of rice cultured cells. The results indicated that the C2 subunit of the 20S proteasome is phosphorylated in vivo in cultured cells. An in-gel kinase assay and analysis of phosphoamino acids revealed that the C2 subunit is phosphorylated by a 40-kDa serine/threonine protein kinase, the activity of which is inhibited by heparin, a specific inhibitor of casein kinase II. The catalytic subunit of casein kinase II from Arabidopsis was also able to phosphorylate the C2 subunit. These results suggest that the C2 subunit in rice is probably phosphorylated by casein kinase II. Our demonstration of the phosphorylation of proteasomes in plants suggests that phosphorylation might be involved in the general regulation of the functions of proteasomes. © 1997 Federation of European Biochemical Societies.

Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

Site-directed mutagenesis and structure/function studies of casein kinase II correlate stimulation of activity by the beta subunit with changes in conformation and ATP/GTP utilization.

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23,894-23,902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.

Purification and characterization of casein kinase I from broccoli

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

Phosphorylation of Calmodulin by the Catalytic Subunit of Casein Kinase II Is Inhibited by the Regulatory Subunit

Casein kinase II (CKII) is composed of a catalytic subunit (α) and a regulatory subunit (β) that combine to form an α2β2 holoenzyme. The α-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified β subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983, J. Biol. Chem. 258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the a subunit and the holneazyme of Drosophila melanogaster CKII. We demonstrate that the α subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (Rh) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as α-casein and the synthetic peptide RRREEETEEE. The Km of the α subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the α subunit alone. While the Km values of the α subunit and the holoenzyme for calmodulin are similar, the Vmax of the α subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the α subunit contains the necessary determinants for CKII substrate specificity, the β subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the a subunit as well.

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

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.

Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity.

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.

Identification of the catalytic subunit of casein kinase II by affinity labeling with 5'-p-fluorosulfonylbenzoyl adenosine.

Casein kinase II, purified from reticulocytes, was covalently labeled with the ATP affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine. The reaction was monitored by the decrease in enzyme activity and showed saturation kinetics with respect to the sulfonyl compound. This suggested a rapid equilibrium was established between the enzyme and affinity reagent prior to a slower, rate-determining step in the overall inactivation process. The enzyme was protected from the covalent modification by ATP and a series of ATP analogs. Their effectiveness in preventing inactivation by the affinity labeling reagent paralleled their ability to function as inhibitors of the phosphotransferase reaction. When radioactive p-fluorosulfonyl [14C]benzoyl adenosine was used to inactivate the enzyme, the alpha subunit was labeled and incorporation of radioactivity into the alpha subunit was blocked when ADP was included in the reaction mixture. Thus the ATP binding site of casein kinase II was shown to be contained within the domain of the alpha subunit of the alpha 2 beta 2 complex.