Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.     

Identification of Phosphorylation Sites on Human Deoxycytidine Kinase After Overexpression in Eucaryotic Cells

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [³²P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.     

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.     

Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.     

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.     

The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2α belongs to the CMGC family of the eukaryotic protein kinases. CK2 is an important survival and stability factor in eukaryotic cells: its catalytic activity is elevated in a wide variety of tumors while its down-regulation can lead to apoptosis. Thus, CK2 is a valuable target for drug development and for chemical biology approaches of cell biological research, and small organic inhibitors addressing CK2 are of considerable interest. We describe here the complex structure between a C-terminal deletion mutant of human CK2α and the ATP-competitive inhibitor emodin (1,3,8-trihydroxy-6-methylanthraquinone, International Union of Pure and Applied Chemistry name: 1,3,8-trihydroxy-6-methylanthracene-9,10-dione) and compare it with a previously published complex structure of emodin and maize CK2α. With a resolution of 1.5 Å, the human CK2α/emodin structure has a much better resolution than its maize counterpart (2.6 Å). Even more important, in spite of a sequence identity of more than 77% between human and maize CK2α, the two structures deviate significantly in the orientation, in which emodin is trapped by the enzyme, and in the local conformations around the ligand binding site: maize CK2α shows its largest adaptations in the ATP-binding loop, whereas human CK2α shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite different in this respect.     

New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

CK2 is a Ser/Thr protein kinase that regulates the activity of the Drosophila basic-helix-loop-helix (bHLH) repressor M8 encoded by the Enhancer of split Complex (E(spl)C) during neurogenesis. Specifically, phosphorylation appears to elicit a conformational change in an autoinhibited state of M8 to one that is permissive for repression. We describe biochemical and molecular modeling studies that provide new insights into repression by M8. Our studies implicate the phosphorylation domain in autoinhibition, and indicate that binding of the co-repressor Groucho (Gro) is context-dependent. Molecular modeling indicates that the Orange domain, proposed to be a specificity-determinant, may instead play a structural role, and that a conformational rearrangement of this domain may be necessary for repression. This model also provides a structural mechanism for the behavior of mutant alleles of the m8 gene. The insights gained from these studies should be applicable to the conserved metazoan bHLH repressors of the Hairy and Enhancer of Split (HES) family that are related to Drosophila M8.     

Casein kinase 1 delta phosphorylates tau and disrupts its binding to microtubules

Tau hyperphosphorylation precedes neuritic lesion formation in Alzheimer's disease, suggesting it participates in the tau fibrillization reaction pathway. Candidate tau protein kinases include members of the casein kinase 1 (CK1) family of phosphotransferases, which are highly overexpressed in Alzheimer's disease brain and colocalize with neuritic and granulovacuolar lesions. Here we characterized the contribution of one CK1 isoform, Ckidelta, to the phosphorylation of tau at residues Ser202/Thr205 and Ser396/Ser404 in human embryonic kidney 293 cells using immunodetection and fluorescence microscopy. Treatment of cells with membrane permeable CK1 inhibitor 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) lowered occupancy of Ser396/Ser404 phosphorylation sites by >70% at saturation, suggesting that endogenous CK1 was the major source of basal phosphorylation activity at these sites. Overexpression of Ckidelta increased CK1 enzyme activity and further raised tau phosphorylation at residues Ser202/Thr205 and Ser396/Ser404 in situ. Inhibitor IC261 reversed tau hyperphosphorylation induced by Ckidelta overexpression. Co-immunoprecipitation assays showed direct association of tau and Ckidelta in situ, consistent with tau being a Ckidelta substrate. Ckidelta overexpression also produced a decrease in the fraction of bulk tau bound to detergent-insoluble microtubules. These results suggest that Ckidelta phosphorylates tau at sites that modulate tau/microtubule binding, and that the expression pattern of Ckidelta in Alzheimer's disease is consistent with it playing an important role in tau aggregation.     

First Inactive Conformation of CK2α, the Catalytic Subunit of Protein Kinase CK2

The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2β, noncatalytic subunit of CK2). CK2α belongs to the superfamily of eukaryotic protein kinases (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix αC and in the activation segment, leading to a characteristic arrangement of catalytic key elements. For CK2α, however, no strict physiological control of activity is known. Accordingly, CK2α was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N-terminal segment on one side, and helix αC and the activation segment on the other side. We report here the structure of a C-terminally truncated variant of human CK2α in which the enzyme adopts a decidedly inactive conformation for the first time. In this CK2α structure, those regulatory key regions still are in their active positions. Yet the glycine-rich ATP-binding loop, which is normally part of the canonical anti-parallel β-sheet, has collapsed into the ATP-binding site so that ATP is excluded from binding; specifically, the side chain of Arg47 occupies the ribose region of the ATP site and Tyr50, the space required by the triphospho moiety. We discuss some factors that may support or disfavor this inactive conformation, among them coordination of small molecules at a remote cavity at the CK2α/CK2β interaction region and binding of a CK2β dimer. The latter stabilizes the glycine-rich loop in the extended active conformation known from the majority of CK2α structures. Thus, the novel inactive conformation for the first time provides a structural basis for the stimulatory impact of CK2β on CK2α.     

Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.     

Drosophila CK2 regulates eye morphogenesis via phosphorylation of E(spl)M8

The Notch effector E(spl)M8 is phosphorylated at Ser159 by CK2, a highly conserved Ser/Thr protein kinase. We have used the Gal4-UAS system to assess the role of M8 phosphorylation during bristle and eye morphogenesis by employing a non-phosphorylatable variant (M8SA) or one predicted to mimic the 'constitutively' phosphorylated protein (M8SD). We find that phosphorylation of M8 does not appear to be critical during bristle morphogenesis. In contrast, only M8SD elicits a severe 'reduced eye' phenotype when it is expressed in the morphogenetic furrow of the eye disc. M8SD elicits neural hypoplasia in eye discs, elicits loss of phase-shifted Atonal-positive cells, i.e. the 'founding' R8 photoreceptors, and consequently leads to apoptosis. The ommatidial phenotype of M8SD is similar to that in Nspl/Y; E(spl)D/+ flies. E(spl)D, an allele of m8, encodes a truncated protein known as M8*, which, unlike wild type M8, displays exacerbated antagonism of Atonal via direct protein-protein interactions. In line with this, we find that the M8SD-Atonal interaction appears indistinguishable from that of M8*-Atonal, whereas interaction of M8 or M8SA appears marginal, at best. These results raise the possibility that phosphorylation of M8 (at Ser159) might be required for its ability to mediate 'lateral inhibition' within proneural clusters in the developing retina. This is the first identification of a dominant allele encoding a phosphorylation-site variant of an E(spl) protein. Our studies uncover a novel functional domain that is conserved amongst a subset of E(spl)/Hes repressors in Drosophila and mammals, and suggests a potential role for CK2 during retinal patterning.     

Interaction between CK2α and CK2β, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.     

Predicting CK2 beta-dependent substrates using linear patterns

CK2 is a constitutively active Ser/Thr protein kinase deregulated in cancer and other pathologies, responsible for about the 20% of the human phosphoproteome. The holoenzyme is a complex composed of two catalytic (α or α´) and two regulatory (β) subunits, with individual subunits also coexisting in the cell. In the holoenzyme, CK2β is a substrate-dependent modulator of kinase activity. Therefore, a comprehensive characterization of CK2 cellular function should firstly address which substrates are phosphorylated exclusively when CK2β is present (class-III or beta-dependent substrates). However, current experimental constrains limit this classification to a few substrates. Here, we took advantage of motif-based prediction and designed four linear patterns for predicting class-III behavior in sets of experimentally determined CK2 substrates. Integrating high-throughput substrate prediction, functional classification and network analysis, our results suggest that beta-dependent phosphorylation might exert particular regulatory roles in viral infection and biological processes/pathways like apoptosis, DNA repair and RNA metabolism. It also pointed, that human beta-dependent substrates are mainly nuclear, a few of them shuttling between nuclear and cytoplasmic compartments.     

The lysine-specific demethylase 1 is a novel substrate of protein kinase CK2

Protein kinase CK2 is a pleiotropic serine/threonine kinase responsible for the generation of a substantial proportion of the human phosphoproteome. CK2 is generally found as a tetramer with two catalytic, α and α′ and two non catalytic β subunits. CK2α C-terminal tail phosphorylation is regulated during the mitotic events and the absence of these phosphosites in α′ suggests an isoform specialization. We used a proteomic approach to identify proteins specifically phosphorylated by a CK2α phosphomimetic mutant, CK2αT344ET360ES362ES370E (CK2α4E), in human neuroblastoma SKNBE cellular extract. One of these proteins is lysine-specific demethylase 1 (LSD1 or KDM1A), an important player of the epigenetic machinery. LSD1 is a FAD-dependent amine oxidase and promotes demethylation of lysine 4 and lysine 9 of mono- and di-methylated histone H3. We found that LSD1 is a new substrate and an interacting partner of protein kinase CK2. Three CK2 phosphosites, (Ser131, Ser137 and Ser166) in the N-terminal region of LSD1 have been identified. This domain is found in all chordates but not in more ancient organisms and it is not essential for LSD1 catalytic event while it could modulate the interaction with CK2 and with other partners in gene repressing and activating complexes. Our data support the view that the phosphorylation of the N-terminal domain by CK2 may represent a mechanism for regulating histone methylation, disclosing a new role for protein kinase CK2 in epigenetics.     

Helicobacter pylori promotes epithelial-to-mesenchymal transition by downregulating CK2β in gastric cancer cells

Strains of Helicobacter pylori that are positive for the oncoprotein CagA (cytotoxin-associated gene A) are associated with gastric cancer and might be related to the epithelial-to-mesenchymal transition (EMT). Casein kinase 2 (CK2) is a serine/threonine protein kinase that plays a major role in tumorigenesis through signaling pathways related to the EMT. However, the role played by the interaction between CagA and CK2 in gastric carcinogenesis is poorly understood. Although CK2α protein expression remained unchanged during H. pylori infection, we found that CK2α kinase activity was increased in gastric epithelial cells. We also found that the CK2β protein level decreased in H. pylori-infected gastric cancer cells in CagA-dependent manner and demonstrated that CagA induced CK2β degradation via HDM2 (human double minute 2; its murine equivalent is MDM2). We observed that CagA induced HDM2 protein phosphorylation and that p53 levels were decreased in H. pylori-infected gastric cancer cells. In addition, downregulation of CK2β induced AKT Ser473 phosphorylation and decreased the AKT Ser129 phosphorylation level in gastric cancer cells. We also found that the downregulation of CK2β triggered the upregulation of Snail levels in gastric cancer cells. Furthermore, our in vivo experiments and functional assays of migration and colony formation suggest that CK2β downregulation is a major factor responsible for the EMT in gastric cancer. Therefore, CK2 could be a key mediator of the EMT in H. pylori-infected gastric cancer and could serve as a molecular target for gastric cancer treatment.     

The p23 co-chaperone protein is a novel substrate of CK2 in Arabidopsis

The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses.     

Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences

Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine.