CK2 Substrates Research Articles

Identification and characterization of a novel testis-specific gene CKT2 , which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.

Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2

Previous analyses of the sirtuin family of histone deacetylases and its most prominent member SIRT1 have focused primarily on the identification of cellular targets exploring the underlying molecular mechanisms of its implicated function in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. So far, little is known about the regulation of SIRT1 itself. In the study presented herein, we assigned the main region of SIRT1 in vivo phosphorylation to amino acids 643–691 of the unique carboxy-terminal domain. Furthermore, we demonstrate that SIRT1 is a substrate for protein kinase CK2 both in vitro and in vivo. Both, deletion construct analyses and serine-to-alanine mutations identified SIRT1 Ser-659 and Ser-661 as major CK2 phosphorylation sites that are phosphorylated in vivo as well.

Dephosphorylation of Centrins by Protein Phosphatase 2C and

In the present study, we identified protein phosphatases dephosphorylating centrins previously phosphorylated by protein kinase CK2. The following phosphatases known to be present in the retina were tested: PP1, PP2A, PP2B, PP2C, PP5, and alkaline phosphatase. PP2C α and β were capable of dephosphorylating P-Thr138-centrin1 most efficiently. PP2Cδ was inactive and the other retinal phosphatases also had much less or no effect. Similar results were observed for centrins 2 and 4. Centrin3 was not a substrate for CK2. The results suggest PP2C α and β to play a significant role in regulating the phosphorylation status of centrins in vivo.

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

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.

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2alpha (Km 0.35 microM) and CK2alpha' (Km 0.18 microM) as well as CK2 holoenzyme (Km 1.1 microM). Different Km values argue that CK2beta(beta') subunit has an inhibitory effect on the activity of both CK2alpha and CK2alpha' towards surviving factor Svf1. Reconstitution of alpha'2betabeta' isoform of CK2 holoenzyme shows that beta/beta' subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of alpha2betabeta' isoform, which may be due to interaction between Svf1 and regulatory CK2beta subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.

Protein kinase CK2 interacts with the splicing factor hPrp3p

Numerous signalling pathways in cells are influenced by the ubiquitous Ser/Thr protein kinase CK2. Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. Several of the known CK2 substrates are proteins known to regulate transcriptional events. Here, we describe that protein kinase CK2 interacts with the splicing factor hPrp3p, which is important for the assembly of the spliceosome. In a two-hybrid screen hPrp3p is exclusively bound to the catalytic alpha- or alpha'-subunits of CK2 but not to the regulatory beta-subunit. The interaction was confirmed by coimmunoprecipitation experiments in vitro and in vivo. Moreover, both proteins colocalized in nuclear speckles which is typical for splicing factor compartments within the nucleus. Phosphorylation experiments revealed that hPrp3p is also a substrate of protein kinase CK2. The main phosphorylation site was mapped to C-terminal residues. In vitro and in vivo splicing assays showed that the splicing activity is significantly influenced by the CK2-hPrp3p interaction. Thus, these data showed that CK2 is involved in the regulation of RNA processing.

Systemic administration of a peptide that impairs the protein kinase (CK2) phosphorylation reduces solid tumor growth in mice

The antitumor efficacy of the CK2 inhibitors so far described has not been extensively evaluated in cancer animal models. We have previously demonstrated that a proapoptotic cyclic peptide termed P15 delivered into the cells by the Tat Cell Penetrating Peptide was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice. Here we explored the antitumor effect by systemic administration of P15-Tat in a consecutive 5-day schedule through either intraperitoneal or intravenous route. Importantly, significant delay of tumor growth was observed at 2 mg/kg (p < 0.05), 10 mg/kg (p < 0.01) or 40 mg/kg (p < 0.001) after P15-Tat administration both in syngeneic murine tumors and human tumors xenografted in nude mice. In line with this, the systemic administration of P15-Tat induced apoptosis in the tumor as evidenced by in situ DNA fragmentation. Furthermore, we evidenced that 99mTc-labeled P15-Tat peptide was certainly accumulated on the tumors after administration by both routes. This report becomes the first describing the antitumor effect induced by systemic administration of a peptide that targets the acidic phosphorylation domain for CK2 substrates. Also, our data reinforces the perspectives of P15-Tat for the cancer targeted therapy.

Analysis of the CK2‐dependent phosphorylation of serine 13 in Cdc37 using a phospho‐specific antibody and phospho‐affinity gel electrophoresis

The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829-2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117-38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065-4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg(2+) and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.

In CK2 inactivated cells the cyclin dependent kinase inhibitor Sic1 is involved in cell-cycle arrest before the onset of S phase

Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (α and α′) are encoded by the CKA1, CKA2 genes. cka1Δcka2 ts mutants arrest cell cycle in both G1 and G2/M at 37 °C. Hence, it has been proposed that CK2 plays an important role in cell-cycle progression and several cell-cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5–Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5–Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells.

HIRIP3 is a nuclear phosphoprotein interacting with and phosphorylated by the serine-threonine kinase CK2

The HIRIP3 protein had been identified from its interaction with the HIRA histone chaperone. Experiments using anti-peptide antisera indicated that this 556-aa protein is nuclear throughout the cell cycle and excluded from condensed chromatin during mitosis. Based on its electrophoretic migration and sensitivity to phosphatase treatment, endogenous HIRIP3 was found to be heavily phosphorylated. HIRIP3 can be phosphorylated in vitro by a recombinant form of the serine-threonine kinase CK2. Moreover, HIRIP3 protein was found to co-purify with a CK2 activity. Together, these data prompt us to propose HIRIP3 as a new member of the growing list of CK2 substrates with a possible role in chromatin metabolism.

Molecular mechanism of human Nrf2 activation and degradation: Role of sequential phosphorylation by protein kinase CK2

Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we identify two phosphorylated forms of endogenous human Nrf2 after chemically induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential roles in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing M r at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 °C) Ca 2+-free media (cold/Ca 2+-free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2-118 formation, the latter of which was prevented by CK2-specific inhibitors. Gel shift assays showed that the Nrf2-118 generated under cold/Ca 2+-free conditions does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response.

Proteomic analysis of SET‐binding proteins

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2-DE and identified by MS using a MALDI-TOF. This experimental approach allowed the recognition of a number of SET-binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2alpha, glycogen phosphorylase (GP), and TCP1-beta) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein-protein interactions.

Protein Kinase CK2 Is Inhibited by Human Nucleolar Phosphoprotein p140 in an Inositol Hexakisphosphate-dependent Manner

Protein kinase CK2 is a ubiquitous protein kinase that can phosphorylate various proteins involved in central cellular processes, such as signal transduction, cell division, and proliferation. We have shown that the human nucleolar phosphoprotein p140 (hNopp140) is able to regulate the catalytic activity of CK2. Unphosphorylated hNopp140 and phospho-hNopp140 bind to the regulatory and catalytic subunits of CK2, respectively, and the interaction between hNopp140 and CK2 was prevented by inositol hexakisphosphate (InsP(6)). Phosphorylation of alpha-casein, genimin, or human phosphatidylcholine transfer protein-like protein by CK2 was inhibited by hNopp140, and InsP(6) recovered the suppressed activity of CK2 by hNopp140. These observations indicated that hNopp140 serves as a negative regulator of CK2 and that InsP(6) stimulates the activity of CK2 by blocking the interaction between hNopp140 and CK2.

Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.

Protein kinase CK2: a newcomer in the ‘druggable kinome’

The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17

Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17

Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.

Variable phosphorylation states of pigment-epithelium–derived factor differentially regulate its function

AbstractThe pigment epithelium–derived factor (PEDF) belongs to the family of noninhibitory serpins. Although originally identified in the eye, PEDF is widely expressed in other body regions including the plasma. This factor can act either as a neurotrophic or as an antiangiogenic factor, and we previously showed that the 2 effects of PEDF are regulated through phosphorylation by PKA and CK2. Here, we studied the interplay between the PKA and CK2 phosphorylation of PEDF, and found that a PEDF mutant mimicking the CK2-phosphorylated PEDF cannot be phosphorylated by PKA, while the mutant mimicking the PKA-phosphorylated PEDF is a good CK2 substrate. Using triple mutants that mimic the PKA- and CK2-phosphorylated and nonphosphorylated PEDF, we found that PEDF can induce several distinct cellular activities dependent on its phosphorylation. The mutant mimicking the accumulative PKA plus CK2 phosphorylation exhibited the strongest antiangiogenic and neurotrophic activities, while the mutants mimicking the individual phosphorylation site mutants had either a reduced activity or only one of these activities. Thus, differential phosphorylation induces variable effects of PEDF, and therefore contributes to the complexity of PEDF action. It is likely that the triple phosphomimetic mutant can be used to generate effective antiangiogenic or neurotrophic drugs.

Fip1 — an Essential Component of the Saccharomyces Cerevisiae Polyadenylation Machinery is Phosophorylated by Protein Kinase CK2

Since Fip1 is phosphoprotein we investigated whether it is a substrate for protein kinase CK2. According to the amino acid sequence Fip1 harbours twenty putative CK2 phosphorylation sites. Here we have report characterization of Fip1 as a substrate for both forms of CK2. Fip1 serves as a substrate for both the recombinant CK2alpha ' (Km 1.28 microM) and holoenzyme (Km 1.4 microM) but not for CK1. By MALDI-MS we identified the two serine residues at positions 73 and 77 as the possible in vitro phosphorylation sites. These data may help to elucidate the role of Fip1 in the mRNA 3'-OH polyadenylation process and the involvement of CK2 mediated phosphorylation in regulation of interactions and activity members of cleavage/polyadenylation factor (CPF) complex.