Casein Kinase II exacerbates rheumatoid arthritis via promoting Th1 and Th17 cell inflammatory responses.

OBJECTIVES: Studies have demonstrated that CK2 is engaged in CD4+ T cell proliferation and activation. We investigated the potential involvement of CK2 in the pathogenesis of rheumatoid arthritis (RA). METHODS: Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of RA patients, as well as splenocytes of collagen-induced arthritis (CIA) mice were treated with different doses of CK2 inhibitor CX4945 in vitro. Then, the Th1, Th2, Th17, and Treg cell responses were analyzed. In addition, CIA mice were administrated with CX4945 via oral gavage. Accordingly, the arthritis scores, bone destruction, tissue damage, and the CD4+ T cell subsets were assessed. RESULTS: The expression of CK2 was upregulated in CD4+ T cells under RA circumstance. In vitro CX4945 treatment significantly inhibited the Th1 and Th17 cell responses, while promoted the Th2 cell responses in RA patient PBMC, SFMC and CIA mouse splenocytes, dampening IFN-γ and IL-17A production. Moreover, administration of CX4945 ameliorated the severity of arthritis in CIA mice, along with decreased Th1 and Th17 cells. However, CX4945 seemed to have minimal effect on RA Treg cells. CONCLUSION: CK2 serves as an important regulator of the Th1 and Th17 cell axes in RA, thus contributing to the disease aggravation.

Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel.

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical "anchor" ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in ß-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a "switch" that favors Ank-3 binding to increase channel activity.

A complex of distal appendage-associated kinases linked to human disease regulates ciliary trafficking and stability.

Cilia biogenesis is a complex, multistep process involving the coordination of multiple cellular trafficking pathways. Despite the importance of ciliogenesis in mediating the cellular response to cues from the microenvironment, we have only a limited understanding of the regulation of cilium assembly. We previously identified Tau tubulin kinase 2 (TTBK2) as a key regulator of ciliogenesis. Here, using CRISPR kinome and biotin identification screening, we identify the CK2 catalytic subunit CSNK2A1 as an important modulator of TTBK2 function in cilia trafficking. Superresolution microscopy reveals that CSNK2A1 is a centrosomal protein concentrated at the mother centriole and associated with the distal appendages. Csnk2a1 mutant cilia are longer than those of control cells, showing instability at the tip associated with ciliary actin cytoskeleton changes. These cilia also abnormally accumulate key cilia assembly and SHH-related proteins. De novo mutations of Csnk2a1 were recently linked to the human genetic disorder Okur-Chung neurodevelopmental syndrome (OCNDS). Consistent with the role of CSNK2A1 in cilium stability, we find that expression of OCNDS-associated Csnk2a1 variants in wild-type cells causes ciliary structural defects. Our findings provide insights into mechanisms involved in ciliary length regulation, trafficking, and stability that in turn shed light on the significance of cilia instability in human disease.

In Silico and In Vitro Studies of Natural Compounds as Human CK2 Inhibitors.

BACKGROUND: Casein Kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase with broad spectrum of substrates. This enzyme is widely expressed in eukaryotic cells and is overexpressed in different human cancers. Thus, the inhibition of CK2 can induce the physiological process of apoptosis leading to tumor cell death. OBJECTIVES: Selecting natural inhibitors toward the target enzyme using database mining. METHODS: With our continuous effort to discover new compounds with CK2 inhibitory effect, several commercial natural databases were searched using molecular modeling approach and the selected compounds were evaluated in vitro. RESULTS: Three compounds were selected as candidates and evaluated in vitro using CK2 holoenzyme, their effect on three cancer cell lines was determined. The selected candidates were weak inhibitors toward the target enzyme, only one compound showed moderate effect on cell viability. CONCLUSION: Several natural databases were screened, compounds were selected and tested in vitro. Despite the unexpected low inhibitory activity of the tested compounds, this study can help in directing the search of potent CK2 inhibitors and better understand the binding requirements of the ATP competitive inhibitors.

Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.

Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α') and 2 regulatory ß subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2ß is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle-specific gene program; and 3) CK2α' activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α' overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.-Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella-Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.

Casein kinase 2 regulates telomere protein complex formation through Rap1 phosphorylation.

Telomeres located at the ends of linear chromosomes play important roles in the maintenance of life. Rap1, a component of the shelterin telomere protein complex, interacts with multiple proteins to perform various functions; further, formation of shelterin requires Rap1 binding to other components such as Taz1 and Poz1, and telomere tethering to the nuclear envelope (NE) involves interactions between Rap1 and Bqt4, a nuclear membrane protein. Although Rap1 is a hub for telomere protein complexes, the regulatory mechanisms of its interactions with partner proteins are not fully understood. Here, we show that Rap1 is phosphorylated by casein kinase 2 (CK2) at multiple sites, which promotes interactions with Bqt4 and Poz1. Among the multiple CK2-mediated phosphorylation sites of Rap1, phosphorylation at Ser496 was found to be crucial for both Rap1-Bqt4 and Rap1-Poz1 interactions. These mechanisms mediate proper telomere tethering to the NE and the formation of the silenced chromatin structure at chromosome ends.

Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efficacy of Immunotherapy in Mice.

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.Significance: These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. Cancer Res; 78(19); 5644-55. ©2018 AACR.

CK2 Oppositely Modulates l-DOPA-Induced Dyskinesia via Striatal Projection Neurons Expressing D1 or D2 Receptors.

We have previously shown that casein kinase 2 (CK2) negatively regulates dopamine D1 and adenosine A2A receptor signaling in the striatum. Ablation of CK2 in D1 receptor-positive striatal neurons caused enhanced locomotion and exploration at baseline, whereas CK2 ablation in D2 receptor-positive neurons caused increased locomotion after treatment with A2A antagonist, caffeine. Because both, D1 and A2A receptors, play major roles in the cellular responses to l-DOPA in the striatum, these findings prompted us to examine the impact of CK2 ablation on the effects of l-DOPA treatment in the unilateral 6-OHDA lesioned mouse model of Parkinson's disease. We report here that knock-out of CK2 in striatonigral neurons reduces the severity of l-DOPA-induced dyskinesia (LID), a finding that correlates with lowered pERK but unchanged pPKA substrate levels in D1 medium spiny neurons as well as in cholinergic interneurons. In contrast, lack of CK2 in striatopallidal neurons enhances LID and ERK phosphorylation. Coadministration of caffeine with a low dose of l-DOPA reduces dyskinesia in animals with striatopallidal knock-out to wild-type levels, suggesting a dependence on adenosine receptor activity. We also detect reduced Golf levels in the striatonigral but not in the striatopallidal knock-out in response to l-DOPA treatment.Our work shows, in a rodent model of PD, that treatment-induced dyskinesia and striatal ERK activation are bidirectionally modulated by ablating CK2 in D1- or D2-positive projection neurons, in male and female mice. The results reveal that CK2 regulates signaling events critical to LID in each of the two main populations of striatal neurons.SIGNIFICANCE STATEMENT To date, l-DOPA is the most effective treatment for PD. Over time, however, its efficacy decreases, and side effects including l-DOPA-induced dyskinesia (LID) increase, affecting up to 78% of patients within 10 years of therapy (Hauser et al., 2007). It is understood that supersensitivity of the striatonigral pathway underlies LID, however, D2 agonists were also shown to induce LID (Bezard et al., 2001; Delfino et al., 2004). Our work implicates a novel player in the expression of LID, the kinase CK2: knock-out of CK2 in striatonigral and striatopallidal neurons has opposing effects on LID. The bidirectional modulation of dyskinesia reveals a central role for CK2 in striatal physiology and indicates that both pathways contribute to LID.

CK2ß regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Casein kinase 2 controls the survival of normal thymic and leukemic γδ T cells via promotion of AKT signaling.

The thymus is the major site for normal and leukemic T-cell development. The dissection of the molecular determinants of T-cell survival and differentiation is paramount for the manipulation of healthy or transformed T cells in cancer (immuno)therapy. Casein kinase 2 (CK2) is a serine/threonine protein kinase whose anti-apoptotic functions have been described in various hematological and solid tumors. Here we disclose an unanticipated role of CK2 in healthy human thymocytes that is selective to the γδ T-cell lineage. γδ thymocytes display higher (and T-cell receptor inducible) CK2 activity than their αß counterparts, and are strikingly sensitive to death upon CK2 inhibition. Mechanistically, we show that CK2 regulates the pro-survival AKT signaling pathway in γδ thymocytes and, importantly, also in γδ T-cell acute lymphoblastic leukemia (T-ALL) cells. When compared with healthy thymocytes or leukemic αß T cells, γδ T-ALL cells show upregulated CK2 activity, potentiated by CD27 costimulation, and enhanced apoptosis upon CK2 blockade using the chemical inhibitor CX-4945. Critically, this results in inhibition of tumor growth in a xenograft model of human γδ T-ALL. These data identify CK2 as a novel survival determinant of both healthy and leukemic γδ T cells, and may thus greatly impact their therapeutic manipulation.

Esculentoside A suppresses lipopolysaccharide-induced pro-inflammatory molecule production partially by casein kinase 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Esculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, an herb which has long been used in Traditional Chinese Medicine for various inflammatory diseases. EsA has been reported to have potent anti-inflammatory properties both in vitro and in vivo. AIM OF THE STUDY: The present study focused on the molecular mechanism of EsA for its anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharide (LPS). METHODS AND RESULTS: Enzyme Linked Immunosorbent Assay (ELISA) showed EsA dose dependently inhibited the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide in RAW264.7 cells. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF-α, IL-6 and iNOS gene expression by EsA on a transcriptional level. Moreover, EsA treatment markedly suppressed LPS-stimulated IκB phosphorylation and degradation as well as LPS-stimulated luciferase reporter construct driven by κB response elements in RAW264.7 cells. In addition, EsA significantly reduced LPS-induced stimulation of p38 and JNK, but not ERK1/2, phosphorylation. Furthermore, we used a computational method called "reverse docking" to search the possible binding proteins of EsA from the potential drug target database (PDTD), and focused on CK2 as the primary binding protein of EsA. Afterward, we further tested EsA directly interacts with recombinant CK2 using SPR assay. In CK2 kinase activity assay, EsA inhibited recombinant CK2 holoenzyme activity obviously in a dose-dependent manner. In addition, TBB (4, 5, 6, 7-tetrabromo-2-benzotriazole, a pharmacological inhibitor of CK2) blocked IL-6 release in a dose-dependent manner, whereas co-treatment of cells with EsA and TBB did not have an additive effect. CONCLUSIONS: Taken together, these results indicate that EsA blocks the LPS-induced pro-inflammatory molecules expression, at least in part, by impediment of LPS-triggered activation of NF-κB and p38/JNK MAPK pathways in macrophages. Furthermore, we discovered for the first time EsA as a ligand for CK2, which was involved in the inhibition of EsA to the expression of inflammatory cytokines. These findings extended our understanding on the cellular and molecular mechanisms responsible for the anti-inflammatory activity of EsA.

Optimal interleukin-7 receptor-mediated signaling, cell cycle progression and viability of T-cell acute lymphoblastic leukemia cells rely on casein kinase 2 activity.

Interleukin-7 and interleukin-7 receptor are essential for normal T-cell development and homeostasis, whereas excessive interleukin-7/interleukin-7 receptor-mediated signaling promotes leukemogenesis. The protein kinase, casein kinase 2, is overexpressed and hyperactivated in cancer, including T-cell acute lymphoblastic leukemia. Herein, we show that while interleukin-7 had a minor but significant positive effect on casein kinase 2 activity in leukemia T-cells, casein kinase 2 activity was mandatory for optimal interleukin-7/interleukin-7 receptor-mediated signaling. Casein kinase 2 pharmacological inhibition impaired signal transducer and activator of transcription 5 and phosphoinositide 3-kinase/v-Akt murine thymoma viral oncogene homolog 1 pathway activation triggered by interleukin-7 or by mutational activation of interleukin-7 receptor. By contrast, forced expression of casein kinase 2 augmented interleukin-7 signaling in human embryonic kidney 293T cells reconstituted with the interleukin-7 receptor machinery. Casein kinase 2 inactivation prevented interleukin-7-induced B-cell lymphoma 2 upregulation, maintenance of mitochondrial homeostasis and viability of T-cell acute lymphoblastic leukemia cell lines and primary leukemia cells collected from patients at diagnosis. Casein kinase 2 inhibition further abrogated interleukin-7-mediated cell growth and upregulation of the transferrin receptor, and blocked cyclin A and E upregulation and cell cycle progression. Notably, casein kinase 2 was also required for the viability of mutant interleukin-7 receptor expressing leukemia T-cells. Overall, our study identifies casein kinase 2 as a major player in the effects of interleukin-7 and interleukin-7 receptor in T-cell acute lymphoblastic leukemia. This further highlights the potential relevance of targeting casein kinase 2 in this malignancy.

Protein Kinase CK2 Regulates Leukocyte-Endothelial Cell Interactions during Ischemia and Reperfusion in Striated Skin Muscle.

BACKGROUND: Ischemia and reperfusion (I/R) causes tissue injury by inflammatory processes. This involves the upregulation of endothelial surface proteins by phospho-regulated signaling pathways, resulting in enhanced interactions of leukocytes with endothelial cells. Recently, we found that protein kinase CK2 is a crucial regulator of leukocyte-mediated inflammation. Therefore, in this study we investigated the involvement of CK2 in leukocyte-endothelial cell interactions during I/R injury. METHODS: We first analyzed the inhibitory action of (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA) and CX-4945 on CK2 kinase activity and the viability of human dermal microvascular endothelial cells (HDMEC). To mimic I/R conditions in vitro, HDMEC were exposed to hypoxia and reoxygenation and the expression of adhesion molecules was analyzed by flow cytometry. Moreover, we analyzed in vivo the effect of CK2 inhibition on leukocyte-endothelial cell interactions in the dorsal skinfold chamber model of I/R injury by means of repetitive intravital fluorescence microscopy and immunohistochemistry. RESULTS: We found that TBCA and CX-4945 suppressed the activity of CK2 in HDMEC without affecting cell viability. This was associated with a significant downregulation of E-selectin and intercellular adhesion molecule (ICAM)-1 after in vitro hypoxia and reoxygenation. In vivo, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in striated muscle tissue exposed to I/R. CONCLUSION: Our findings indicate that CK2 is involved in the regulation of leukocyte-endothelial cell interactions during I/R by mediating the expression of E-selectin and ICAM-1.

GSK-3 and CK2 Kinases Converge on Timeless to Regulate the Master Clock.

The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, we identify a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2ß phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2ß mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2ß and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair.

A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo.

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.

Genetic variants associated with sleep disorders.

OBJECTIVE: The diagnostic boundaries of sleep disorders are under considerable debate. The main sleep disorders are partly heritable; therefore, defining heritable pathophysiologic mechanisms could delineate diagnoses and suggest treatment. We collected clinical data and DNA from consenting patients scheduled to undergo clinical polysomnograms, to expand our understanding of the polymorphisms associated with the phenotypes of particular sleep disorders. METHODS: Patients at least 21 years of age were recruited to contribute research questionnaires, and to provide access to their medical records, saliva for deoxyribonucleic acid (DNA), and polysomnographic data. From these complex data, 38 partly overlapping phenotypes were derived indicating complaints, subjective and objective sleep timing, and polysomnographic disturbances. A custom chip was used to genotype 768 single-nucleotide polymorphisms (SNPs). Additional assays derived ancestry-informative markers (eg, 751 participants of European ancestry). Linear regressions controlling for age, gender, and ancestry were used to assess the associations of each phenotype with each of the SNPs, highlighting those with Bonferroni-corrected significance. RESULTS: In peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PPARGC1B), rs6888451 was associated with several markers of obstructive sleep apnea. In aryl hydrocarbon receptor nuclear translocator-like (ARNTL), rs10766071 was associated with decreased polysomnographic sleep duration. The association of rs3923809 in BTBD9 with periodic limb movements in sleep was confirmed. SNPs in casein kinase 1 delta (CSNK1D rs11552085), cryptochrome 1 (CRY1 rs4964515), and retinoic acid receptor-related orphan receptor A (RORA rs11071547) were less persuasively associated with sleep latency and time of falling asleep. CONCLUSIONS: SNPs associated with several sleep phenotypes were suggested, but due to risks of false discovery, independent replications are needed before the importance of these associations can be assessed, followed by investigation of molecular mechanisms.

CK2α phosphorylates DBC1 and is involved in the progression of gastric carcinoma and predicts poor survival of gastric carcinoma patients.

CK2α has diverse effects on the tumorigenesis owing to its kinase activity, which phosphorylates various proteins involved in tumorigenesis. We, therefore, investigated the expression and role of CK2α in the phosphorylation of deleted in breast cancer 1 (DBC1) in gastric carcinomas. We used 187 gastric carcinomas and human gastric cancer cells to investigate the roles and relationship between CK2α and DBC1 in gastric carcinomas. Positive expression of CK2α and phospho-DBC1 predicted shorter overall survival and relapse-free survival by univariate analysis. Especially, CK2α expression was an independent prognostic indicator for gastric carcinoma patients. In gastric carcinoma cells, CK2α was bound to DBC1 and phosphorylated DBC1. The phosphorylation of DBC1 by CK2α was evidenced by co-immunoprecipitation of CK2α and DBC1 in a GST pull-down assay, an in vitro kinase assay, and immunofluorescence staining. Inhibition of both CK2α and DBC1 decreased proliferation and invasive activity of cancer cells. Decreased migration and invasive activity was associated with a downregulation of MMP2, MMP9 and the epithelial-mesenchymal transition. A mutation at the phosphorylation site of DBC1 also downregulated the signals related with the epithelial-mesenchymal transition. Our study demonstrated that CK2α is an independent prognostic indicator for gastric carcinoma patients and is involved in tumorigenesis by regulating the phosphorylation of DBC1. In addition, the blocking of CK2α and DBC1 inhibited the proliferation and invasive potential of gastric cancer cells. Therefore, our study suggests that CK2α-DBC1 pathway might be a new therapeutic target for the treatment of gastric carcinoma.