Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2

The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity.     

Interactions between protein kinase CK2 and Pin1. Evidence for phosphorylation-dependent interactions

The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha' subunits together with two regulatory CK2 beta subunits. Our results indicate that Pin1 can interact with CK2 complexes that contain CK2 alpha. Furthermore, Pin1 can interact directly with the C-terminal domain of CK2 alpha that contains residues that are phosphorylated in vitro by p34(Cdc2) and in mitotic cells. Substitution of the phosphorylation sites of CK2 alpha with alanines resulted in decreased interactions between Pin1 and CK2. The other catalytic isoform of CK2, designated CK2 alpha', is not phosphorylated in mitotic cells and does not interact with Pin1, but a chimeric protein consisting of CK2 alpha' with the C terminus of CK2 alpha was phosphorylated in mitotic cells and interacts with Pin1, further implicating the phosphorylation sites in the interaction. In vitro, Pin1 inhibits the phosphorylation of Thr-1342 on human topoisomerase II alpha by CK2. Topoisomerase II alpha also interacts with Pin1 suggesting that the effect of Pin1 on the phosphorylation of Thr-1342 could result from its interactions with CK2 and/or topoisomerase II alpha. As compared with wild-type Pin1, isomerase-deficient and WW domain-deficient mutants of Pin1 are impaired in their ability to interact with CK2 and to inhibit the CK2-catalyzed phosphorylation of topoisomerase II alpha. Collectively, these results indicate that Pin1 and CK2 alpha interact and suggest a possible role for Pin1 in the regulation of topoisomerase II alpha. Furthermore, these results provide new insights into the functional role of the mitotic phosphorylation of CK2 and provide a new mechanism for selectively regulating the ability of CK2 to phosphorylate one of its mitotic targets.     

Binding domain for p21(WAF1) on the polypeptide chain of the protein kinase CK2 beta-subunit

Protein kinase CK2 is a ubiquitous serine/threonine kinase which is involved in many proliferation-related processes in the cell. It is composed of two regulatory beta-subunits and two catalytic alpha-subunits. Its regulation still remains mysterious in spite of many years of intense research. One of its regulators is the cdk inhibitory molecule p21(WAF1)-a protein which is expressed in situations of genotoxic stress. p21(WAF1) binds to the beta-subunit of CK2 and inhibits the activity of CK2. Using deletion mutants of CK2 beta as well as a peptide library consisting of 15-amino-acid-long peptides derived from the polypeptide chain of CK2 beta we mapped the binding region for p21(WAF1) on the polypeptide chain of CK2 beta. We localized an amino-terminal and a carboxy-terminal binding domain. Binding of p21(WAF1) to both regions of the CK2 beta-subunit interferes with the phosphotransferase activity of the CK2 holoenzyme.     

Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.     

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster.

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.     

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.     

The regulatory beta subunit of protein kinase CK2 mediates formation of tetrameric CK2 complexes

Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (alpha and/or alpha') subunits and two regulatory (beta) subunits. Because CK2beta is synthesized in excess of CK2alpha, we hypothesized that formation of CK2beta homodimers precedes the incorporation of the catalytic subunits of CK2 into complexes. To test this hypothesis, we cotransfected cells with two epitope-tagged variants of CK2beta. The results of these cotransfection studies demonstrate that interactions between two CK2beta subunits take place in the absence of CK2alpha. Together with results from previous biosynthetic labeling studies, these results suggest that formation of CK2beta homodimers occurs before incorporation of catalytic subunits of CK2 into CK2 complexes. We also cotransfected Cos-7 cells with a deletion fragment of CK2beta (i.e. Myc-beta1-166) together with full-length hemagglutinin (HA)-tagged CK2beta and/or CK2alpha'. Although complexes between Myc-beta1-166 and HA-beta were readily detected, we obtained no evidence of direct interactions between Myc-beta1-166 and HA-CK2alpha'. These results suggest that residues within the N-terminal 166 amino acids of CK2beta are sufficient for interactions between CK2beta subunits, whereas the C-terminal domain of CK2beta is required for complex formation with the catalytic subunits of CK2. Finally, we observed that expression of full-length HA-beta promotes phosphorylation of Myc-beta1-166 by HA-CK2alpha'.     

Purification and characterization of the CK2alpha'-based holoenzyme, an isozyme of CK2alpha: a comparative analysis

Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.     

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

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.     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

Maize protein kinase CK2: regulation and functionality of three beta regulatory subunits

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.     

Serum-stimulated cell cycle entry of fibroblasts requires undisturbed phosphorylation and non-phosphorylation interactions of the catalytic subunits of protein kinase CK2

Protein kinase CK2 is a pleiotropic Ser/Thr kinase occurring as alpha2beta2, alpha'2beta2, or alphaalpha'beta2 tetramers. A requirement in serum-stimulated cell cycle entry in both the cytoplasm and the nucleus of human fibroblasts for phosphorylation(s) by CK2 has been concluded from stimulation inhibition by microinjected antibodies against the regulatory subunit (beta). We have now examined this idea more directly by microinjection-mediated perturbation of phosphorylation and non-phosphorylation interactions of the catalytic subunits (alpha and alpha'), and by verifying the supposed matching of the cellular partition of CK2 subunits in the fibroblasts employed. While immunostaining and cell fractionation indicate that the partitions of subunits indeed match each other (with their predominant location in the nucleus in both quiescent and serum-stimulated cells), microinjection of substrate or pseudosubstrate peptides competing for the CK2-mediated phosphorylation in vitro resulted in significant inhibition of serum stimulation when placed into the nucleus but not when placed into the cytoplasm. Also inhibitory were nuclear but not cytoplasmic injections of antibodies against alpha and alpha' that affect neither their kinase activity in vitro nor their complexing to beta. The data indicate that the role played by CK2 in serum-stimulated cell cycle entry is predominantly nuclear and more complex than previously assumed, involving not only phosphorylation but also experimentally separable non-phosphorylation interactions by the catalytic subunits.     

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.     

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.