Interactions of protein kinase CK2 subunits

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2 that has Asp 156 mutated to Ala (CK2A156) is able to bind the CK2 subunit and to compete effectively in this binding with wild-type subunits and . The interaction between CK2A156 and CK2 was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2A156 coprecipitated the subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2A156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2A156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2A156 affects the endogenous activity of CK2. Transfection experiments with CK2 and subunits and CK2A156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2 indicated that CK2 is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.     

Identification of protein kinase CK2 as a potent kinase of Epstein-Barr virus latent membrane protein 1

The C-terminus of latent membrane protein 1 (LMP1) can be phosphorylated in vivo. However, the protein kinase responsible for LMP1 phosphorylation has not yet been identified. In this study, GST fusion proteins containing the C-terminus of LMP1 were generated and used as substrates to survey the kinases that phosphorylate LMP1. Among several purified protein kinases tested, only protein kinase CK2 (CK2) could specifically phosphorylate LMP1. Using the in-gel kinase assay in the absence and presence of a selective CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole, CK2 was determined to be the major kinase to phosphorylate LMP1 in lymphoma and epithelial cell lines. This is the first study to show that CK2 is a potent kinase to phosphorylate LMP1 in vitro.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Activation of human topoisomerase I by protein kinase CK2

The enzymatic studies were performed to reveal a mode of activation of human topoisomerase I by a direct interaction with protein kinase CK2. In the absence of ATP CK2 kinase activated DNA relaxation about twofold. CK2 subunit was identified as solely responsible for the stimulation of relaxing activity by CK2 kinase. CK2 activated the relaxation only at the excess of the substrate over topoisomerase I. At the equimolar ratio of the substrate DNA and topoisomerase I the activation was not observed. There was also no effect of CK2 on camptothecin-induced cleavage of DNA by htopo I. These results identify an accelerated movement of topoisomerase I between substrate molecules as a cause of the activation of DNA relaxation by CK2 kinase.     

Expression and localization of epitope-tagged protein kinase CK2

Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2α and/or CK2α′) subunits and two subunits (CK2β) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2α and CK2α′ exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2α and CK2α′ were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., α₂β₂, α′₂β₂) instead of heterotetrameric complexes (i.e., αα′β₂) that are present in many cells. Epitope-tagged CK2α and CK2α′ displayed kinase activity and the ability to form complexes with CK2β. The results of these studies also indicate definitively that CK2α and CK2α′ are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2α and CK2α′ resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2. J. Cell. Biochem. 64: 525–537. © 1997 Wiley-Liss, Inc.     

Intermolecular contact sites in protein kinase CK2

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55-70 of subunit (55-70) and 65-80 of subunit (65-80). This has been supported by crosslinking of subunits with peptides 65-80 and 55-70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-65-80; anti-55-70). Functionally, 55-70 is a negative regulatory region for the kinase activity of subunit . The opposite, stimulatory property of subunit has been assigned to its C-terminal part. Subdivision of peptide 155-181, that has stimulatory effect, into overlapping peptides and assaying for a binding and binding competition revealed a tight physical contact at 162-175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit a as indicated by crosslinking and peptide competition. The crosslinking data also confirm a - contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment.     

Validation of protein kinase CK2 as oncological target

Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits α and/or α′ and two regulatory subunits β whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.     

蛋白激酶CK2的结构及其生理功能研究进展

蛋白激酶CK2是一种常见的、进化保守的、普遍存在的蛋白激酶.近年来,越来越多的研究表明CK2具有多种磷酸化蛋白底物,这些底物在生长发育及各类疾病中都具有重要的作用,因此CK2可以通过调控这些底物的磷酸化参与这些生理过程.文中简要综述了蛋白激酶CK2的结构特征及其在生长发育、免疫、肿瘤等疾病中的生理功能,以期为进一步研究...     

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (α < eqid1 > β₂) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol {23}: 975–987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio–temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.     

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.     

Extracellular protein kinase CK2 is a novel associating protein of Neuropilin-1

Neuropilin-1 (NRP1) is a multifunctional transmembrane protein which has a short cytoplasmic region with no particular functional domain, and is considered to act as a co-receptor for both VEGFs and semaphorins. However, the molecular mechanisms by which NRP1 carries out such versatile functions are still poorly understood. Here we identified protein kinase CK2 holoenzyme as a novel NRP1 binding protein by our combined purification strategy using epitope-tag immunoprecipitation followed by reverse-phase column chromatography. Further we showed that CK2 binds to the extracellular domain of NRP1 which is also phosphorylated by CK2 both in vitro and in vivo. Our findings of novel molecular interactions and modification of NRP1 may provide a new clue to understand the diverse functions of NRP1.     

Phosphorylation of human high mobility group N1 protein by protein kinase CK2

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.     

A multifunctional network of basic residues confers unique properties to protein kinase CK2

Protein kinase CK2 is characterized by a number of features, including substrate specificity, inhibition by polyanionic compounds and intrasteric down-regulation by its -subunit, which denote a special aptitude to interact with negatively charged ligands. This situation may reflect the presence in CK2 catalytic subunits of several basic residues that are not conserved in the majority of other protein kinases. Some of these residues, notably K49 in the Gly rich loop, K74, K75, K76, K77, K79, R80, K83 in the Lys rich segment and R191, R195, K198 in the p+1 loop, have been shown by mutational studies to be implicated to various extents and with distinct roles in substrate recognition, inhibition by heparin and by pseudosubstrate and instrasteric regulation. Molecular modelization based on crystallographic data provide a rationale for the biochemical observations, showing that several of these basic residues are clustered around the active site where they make contact with individual acidic residues of the peptide substrate. They can also mediate the effect of polyanionic inhibitors (e.g. heparin) and of regulatory elements present in the b-subunit, in the N terminal segment of the catalytic subunit and possibly in other proteins interacting with CK2. Our data also disclose a unique mode of binding of the phosphoacceptor substrate which bridges across the catalytic cleft making contacts with both the lower and upper lobes of CK2.     

Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1

The serine/arginine subfamily of protein kinases has been conserved throughout evolution and its members are thought to play important roles in the regulation of multiple cellular processes. Mammalian SRPK1 has been considered as a constitutively active kinase that is predominantly expressed in testis. In the present study, recombinant GST-SRPK1 was used as substrate to identify potential protein kinase(s) in testis extracts, involved in phosphorylating and thereby regulating the activity of this enzyme. Using a panel of chromatography media, inhibition by heparin, immunoblot analysis, and phosphopeptide mapping, CK2 was determined to be the major kinase that phosphorylates SRPK1. Phosphorylation of SRPK1 by CK2 occurred mainly at Ser(51) and Ser(555) in vitro, and resulted in approximately 6-fold activation of the enzyme. These findings suggest that SRPK1 may be an important cellular target for CK2 action.     

Protein kinase CK2 interacts with a multi-protein binding domain of p53

p53 is one of the most powerful negative regulators of growth. To manage this in an efficient way it has to interact with a set of different cellular proteins. Most contacts with the cellular environment occur in the N- or the C-terminal domain of the protein. Since we previously found that p53 binds to the regulatory -subunit of CK2 we now analyzed N- and C-terminal domains of p53 separately for the binding of protein kinase CK2, an enzyme which seems to have a certain importance for proliferation processes. With different overlay assays we could map the binding domain of protein kinase CK2 to a sequence between amino acids 325-344, a region which coincides with the interaction domain of some other p53 binding proteins. We also found that the regulatory -subunit of protein kinase CK2 binds independent of the catalytic -subunit to this C-terminal domain of p53.