CK2 activity is modulated by growth rate in Saccharomyces cerevisiae

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k_cat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.     

Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.     

Programmed cell death protein 5 (PDCD5) is phosphorylated by CK2 in vitro and in 293T cells

CK2 is a multifunctional kinase, involved in cell growth, apoptosis, DNA integrity preservation, viral infection, and many other biological processes. Based on an analysis of phosphopeptides database derived from phosphoproteomic studies we previously identified a list of potential new CK2 substrates, including, among others, Programmed Cell Death 5 (PDCD5), a protein involved in cell death and down-regulated in different forms of human tumors. Here we provide experimental evidence that PDCD5 is indeed a bona fide substrate of CK2. PDCD5 is phosphorylated in vitro by both CK2α subunit and by the CK2 holoenzyme at a residue, S118, which is found phosphorylated in vivo. We also show that PDCD5 is phosphorylated by CK2 in 293T cells. Transfection of the non-phosphorylatable mutant (S118A) impairs the PDCD5 acceleration of either doxorubimicin- or UV-induced apoptosis in U2OS cells. Our results suggest a functional link between the CK2 phosphorylation and the apoptotic potential of PDCD5.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

Identification of barley CK2alpha targets by using the protein microarray technology

We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of [gamma-33P]ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin.     

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.     

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.     

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.     

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.     

Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.     

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.     

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.     

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.     

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.     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.     

Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line

Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the β subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10’s regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.     

Genetic interactions among ZDS1,2, CDC37, and protein kinase CK2 in Saccharomyces cerevisiae

We report here the identification of the homologous gene pair ZDS1,2 as multicopy suppressors of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CK2. Overexpression of ZDS1,2 suppressed the temperature sensitivity, geldanamycin (GA) sensitivity, slow growth, and flocculation of multiple cka2 alleles and enhanced CK2 activity in vivo toward a known physiological substrate, Fpr3. Consistent with the existence of a recently described positive feedback loop between CK2 and Cdc37, overexpression of ZDS1,2 also suppressed the temperature sensitivity, abnormal morphology, and GA sensitivity of a CK2 phosphorylation-deficient mutant of CDC37, cdc37-S14A, as well as the GA sensitivity of a cdc37-1 allele. A likely basis for all of these effects is our observation that ZDS1,2 overexpression enhances Cdc37 protein levels. Activation of the positive feedback loop between CK2 and Cdc37 likely contributes to the pleiotropic nature of ZDS1,2, as both CK2 and Cdc37 regulate diverse cellular functions.