The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic α- or α′-subunits and two regulatory β-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2α′ in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2α/CK2β and CK2α′/CK2β as well as by CK2α′ alone but not by CK2α alone.     

Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation.     

Role of plant-specific N-terminal domain of maize CK2β1 subunit in CK2β functions and holoenzyme regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.     

Predominance of CK2α over CK2α′ in the mammalian brain

CK2 is a heterotetrameric ubiquitous kinase consisting of two catalytic subunits and two regulatory subunits. The two catalytic subunits, α and α', are highly homologous but differ in their C-terminal regions. It is not known whether CK2α and α' have distinctive substrate specificity, since no α- or α'-specific substrate has been identified. Thus, it is assumed that the two kinase isoforms overlap in their substrate specificity. CK2 protein levels and activity were found to be elevated in the brain when compared to other organs. Here we have studied the protein levels of CK2α and α' isoforms in nine major brain regions. We found that both, CK2α and α', are expressed in all brain regions tested. Whereas CK2α levels do not vary strongly across the regions, CK2α' levels are slightly higher in the cortex and hippocampus than in other regions. Furthermore, we show that CK2α protein levels in the striatum are relatively high when compared to CK2α'. The approximate stoichiometry ratio of CK2α:CK2α' is 8:1. Therefore, one can consider that CK2α levels are predominant in comparison to CK2α' levels throughout the mammalian brain.     

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.     

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.     

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.     

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.     

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2?? and its paralogous isoform CK2????

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open β4/β5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2β. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.     

The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.     

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.     

The regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.     

HIV-1 Rev transactivator: A β-subunit directed substrate and effector of protein kinase CK2

The phosphorylation of HIV-1 Rev by protein kinase CK2 is strictly dependent on the regulatory subunit of the kinase and is deeply affected by conformational changes of the substrate outside the phosphorylation site [12]. Here we show that Rev modulates a variety of CK2 properties, including autophosphorylation, catalytic activity toward calmodulin, and susceptibility to polycationic effectors, whose common denominator is the involvement of the subunit. Rev's two major CK2 sites are located at its N-terminus, immediately adjacent to a helix-loop-helix motif. By comparing the behaviour of full-size Rev with that of synthetic peptides reproducing, with suitable modifications, its N-terminal 26 amino acids including the phosphoacceptor site (Ser 5, Ser 8) and amphipathic helix-1, it appears that the functional interaction of the N-terminal portion of Rev with the N-terminal domain of the subunit must rely on both electrostatic and hydrophobic interactions. The former mainly involve Rev's arginine-rich domain (residues 35–50) in helix-2, while the latter are mostly mediated by residues 12–24 of helix-1. These data disclose the possibility that, besides displaying protective, regulatory and targeting properties with respect to the catalytic subunit, the CK2 subunit also plays a role as a docking site for a subset of CK2 substrates.     

Downregulation of JMJD2a and LSD1 is involved in CK2 inhibition-mediated cellular senescence through the p53-SUV39h1 pathway

Lysine methylation is one of the most important histone modifications that modulate chromatin structure. In the present study, the roles of the histone lysine demethylases JMJD2a and LSD1 in CK2 downregulation-mediated senescence were investigated. The ectopic expression of JMJD2a and LSD1 suppressed the induction of senescence-associated β-galactosidase activity and heterochromatin foci formation as well as the reduction of colony-forming and cell migration ability mediated by CK2 knockdown. CK2 downregulation inhibited JMJD2a and LSD1 expression by activating the mammalian target of rapamycin (mTOR)-ribosomal p70 S6 kinase (p70S6K) pathway. In addition, the down-regulation of JMJD2a and LSD1 was involved in activating the p53-p21^Cip1/WAF1-SUV39h1-trimethylation of the histone H3 Lys9 (H3K9me3) pathway in CK2-downregulated cells. Further, CK2 downregulation-mediated JMJD2a and LSD1 reduction was found to stimulate the dimethylation of Lys370 on p53 (p53K370me2) and nuclear import of SUV39h1. Therefore, this study indicated that CK2 downregulation reduces JMJD2a and LSD1 expression by activating mTOR, resulting in H3K9me3 induction by increasing the p53K370me2-dependent nuclear import of SUV39h1. These results suggest that CK2 is a potential therapeutic target for age-related diseases.     

Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis

Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.