Casein Kinase 2 Alpha Research Articles

CK2 expression in cancer and associated clinical characteristics.

e15156 Background: The protein casein kinase 2 (CK2) is a ubiquitously expressed, highly pleiotropic kinase. CK2 may have incredibly varied substrate specificity and catalytic activity, as it functions as both a tetrameric and monomeric enzyme. CK2 participates in many cellular processes such as cellular proliferation, growth, and survival, and has significant potential to assist in oncogenic signaling. We and others have demonstrated CK2 gene and protein overexpression, and sometimes underexpression, in a variety of cancers. Here we investigate how the expression of CK2 subunits in cancer is associated with clinical characteristics in several distinct cancer types. Methods: Data analysis was performed on 32 non-overlapping cancer datasets obtained from The Cancer Genome Atlas (TCGA) and Broad GDAC Firehose Legacy projects, one dataset from Cancer Cell Line Encyclopedia (CCLE), and normal tissue datasets from Gene Expression Omnibus. Statistical analysis was performed using RStudio to explore the relationship between the expression levels of the three CK2 alpha transcripts (CSNK2A1, CSNK2A2, and CSNK2A3) and beta transcripts (CSNK2B) with sample clinical characteristics provided by TCGA/Broad. Relationships between CK2 expression and cancer-type specific characteristics were also explored, such as KRAS mutations in colorectal adenocarcinoma and serum AFP at sample procurement in hepatocellular carcinoma. CCLE data was used to explore correlations between cancer-type dependent CK2 gene expression and half maximal inhibitory concentration (IC50) of over 250 anticancer drugs. Tumor Immune Estimation Resource (TIMER) 2.0 was used to assess the relationship between CK2 subunit expression and tumor immune infiltration. Results: As seen in our previous analysis, CK2 transcripts were upregulated in the majority of cancer types analyzed. We also found that CK2 transcript upregulation correlated with worse survival outcomes in several previously unstudied cancers. CK2 expression differed significantly between cancer subtypes such as in ductal carcinoma and lobular carcinoma in breast cancer. CK2 expression significantly correlated with tumor grade, TNM staging, clinical staging in certain cancer types. CK2 expression significantly correlated with altered response to various anticancer drugs in certain cancers. CK2 expression was associated with both increased and decreased immune infiltration in a variety of cancers. Conclusions: Overall, this study demonstrates that CK2 transcript expression correlates with a variety of cancer-type dependent characteristics and clinical outcomes. As a result, CK2 may be useful as a prognostic biomarker and therapeutic target.

P62SQSTM1 Affects Interacting Substrates MDM2, MDMX and Casein Kinase 1alpha to Yield Protection of Beta-Catenin Along with p53 Silencing for Refractory AML

Interventions targeting individual driver mutations in AML have not overcome resistant disease. Missing has been definition of a signal hub that integrates multiple oncogenic drivers for a composite pathway to resistance, where hub abundance determines signal(s) strength. We hypothesized that AML signal hub is p62SQSTM1, a nonmutant-active signal scaffolding for multiple pathways, including NFkB and MAPK (Moscat, Cell. 2016). Genetic epistasis in AML cell lines validated origin from P62 of such signals. Oxidative stress resistance pathways (OXPHOS) driven by p62SQSTM1 may also join with pathway(s) for leukemic progenitor replication, involving HOXA and beta-catenin within resistant AML's, but this has not been established. We sought merging pathways to OXPHOS-mediated resistance also serving to guard replicative potential through HOXA and beta-catenin. AMLs from patients were tested prior to treatment, and later failure, of induction chemotherapy, when given along with targeted agent (for cases with Flt3 mutation). Resistant AML blasts were found to have tandem overexpression of HOXAs and OXPHOS effectors p62SQSTM1, NRF2, NQO1 (NADPH oxidoreductase), BCL-2 ranging from 2-5-fold log10 relative quantity (RQ-Taqman) above chemo-sensitive core-binding factor AML, which lacks HOXA expression. We hypothesized that disrupting the downstream pathway from p62SQSTM1 to OXPHOS ending on MDM2 and/or MDMX (Todoric, Cancer Cell, 2017) is an approach to affect broad cell extinction. Indeed, besides destabilization/destruction of wild-type p53 affected by MDM2, the paralogue MDMX exists in an induced tripartite complex with CK1alpha (CK1) and p53, whose activity for restraining p53 and protecting beta-catenin from phosphorylation/inactivation is disrupted upon stress-induced CK1 activation (Huang, EMBOJ, 2020; Ueda, Cancer Cell, 2021), which we affected by using a proteasome inhibitor (PI). We found by immunoblotting AML blast lysates following PI treatment in vitro with Ixazomib, CK1 translocates to the nucleus and phosphorylation (S33,S37,T41,S45)/inactivation and cytoplasmic export of beta-catenin ensued, accompanying cell apoptosis, thus implicating MDMX inactivation resulting from PI. Primary p53-wild-type AML blasts or isogenic cell lines were treated in vitro with graded doses of the MDM2 inhibitors Pevonedistat (PEVO) or Siremadlin, either added alone, or in combination with Ixazomib at pharmacokinetically-achievable dose ranges typical in patients. Among high-risk primary AML blasts with Flt3ITD alone, or ITD along with NUP98-NSD1 or mDNMT3A, or Flt3neg with mutant EP300, or with PRDM16-SKI fusion as drivers, synergy/cooperativity was observed between MDM2 inhibitor and PI Ixazomib for inhibition of 3H-thymidine incorporation and visible apoptosis in vitro. Full characterization within PRDM16-SKI blasts by immunoblotting demonstrated expected posttranslational modification (PTM) on beta-catenin by PI, whereas distinct PTM for beta-catenin followed PEVO application, and the combination demonstrated further heterogeneity. Treatment of these blasts with Ixazomib or the combination with PEVO also led to induction of p53 in tandem with the destruction of MDM2/MDMX PTM isoforms and nuclear translocation of CK1. Strong upregulation/nuclear translocation of NRF2 resulted as part of the apoptotic pathway. To further discriminate the requirement for, and the nature of PTMs in the functional outcomes, FLAG-tagged (S33-A, S37-A,T41-A) mutant beta-catenin isoform, also lacking lysine ubiquitin target residues, was transfected in a lentiviral vector into p53-mutant K562 cells as host, and cells were compared to the same cells transfected with non-mutant FLAG-tagged beta-catenin. PEVO/Ixazomib did not induce PTM in mutant FLAG-beta-catenin transfectants, but the expected PTMs occurred in wild-type FLAG-tagged beta-catenin transfectants. In this context, PEVO became a stimulator of cell proliferation of the cells bearing mutant PTM-resistant beta-catenin, thus implicating beta-catenin disruption involving these residues as a requisite for optimal AML cell inhibition with the combination. In addition, a combination of PEVO/Ixazomib was as effective for apoptosis as was Flt3 inhibitor among certain Flt3mutant primary AML blasts. A clinical test of this strategy is warranted in high-risk relapsed/refractory p53WT/p62SQSTM1-expressing AML.

Identification of Phytoconstituents as Potent Inhibitors of Casein Kinase-1 Alpha Using Virtual Screening and Molecular Dynamics Simulations.

Casein kinase-1 alpha (CK1α) is a multifunctional protein kinase that belongs to the serine/threonine kinases of the CK1α family. It is involved in various signaling pathways associated with chromosome segregation, cell metabolism, cell cycle progression, apoptosis, autophagy, etc. It has been known to involve in the progression of many diseases, including cancer, neurodegeneration, obesity, and behavioral disorders. The elevated expression of CK1α in diseased conditions facilitates its selective targeting for therapeutic management. Here, we have performed virtual screening of phytoconstituents from the IMPPAT database seeking potential inhibitors of CK1α. First, a cluster of compounds was retrieved based on physicochemical parameters following Lipinski’s rules and PAINS filter. Further, high-affinity hits against CK1α were obtained based on their binding affinity score. Furthermore, the ADMET, PAINS, and PASS evaluation was carried out to select more potent hits. Finally, following the interaction analysis, we elucidated three phytoconstituents, Semiglabrinol, Curcusone_A, and Liriodenine, posturing considerable affinity and specificity towards the CK1α binding pocket. The result was further evaluated by molecular dynamics (MD) simulations, dynamical cross-correlation matrix (DCCM), and principal components analysis (PCA), which revealed that binding of the selected compounds, especially Semiglabrinol, stabilizes CK1α and leads to fewer conformational fluctuations. The MM-PBSA analysis suggested an appreciable binding affinity of all three compounds toward CK1α.

Casein Kinase-1-Alpha Inhibitor (D4476) Sensitizes Microsatellite Instable Colorectal Cancer Cells to 5-Fluorouracil via Authophagy Flux Inhibition

Adjuvant chemotherapy with 5-fluorouracil (5-FU) does not improve survival of patients suffering from a form of colorectal cancer (CRC) characterized by high level of microsatellite instability (MSI-H). Given the importance of autophagy and multi-drug-resistant (MDR) proteins in chemotherapy resistance, as well as the role of casein kinase 1-alpha (CK1α) in the regulation of autophagy, we tested the combined effect of 5-FU and CK1α inhibitor (D4476) on HCT116 cells as a model of MSI-H colorectal cancer. To achieve this goal, the gene expression of Beclin1 and MDR genes, ABCG2 and ABCC3 were analyzed using quantitative real-time polymerase chain reaction. We used immunoblotting to measure autophagy flux (LC3, p62) and flow cytometry to detect apoptosis. Our findings showed that combination treatment with 5-FU and D4476 inhibited autophagy flux. Moreover, 5-FU and D4476 combination therapy induced G2, S and G1 phase arrests and it depleted mRNA of both cell proliferation-related genes and MDR-related genes (ABCG2, cyclin D1 and c-myc). Hence, our data indicates that targeting of CK1α may increase the sensitivity of HCT116 cells to 5-FU. To our knowledge, this is the first description of sensitization of CRC cells to 5-FU chemotherapy by CK1α inhibitor.Graphic abstract

Retraction: miR-199a-3p knockdown inhibits dedifferentiated liposarcoma (DDLPS) cell viability and enhances apoptosis through targeting casein kinase-1 alpha (CK1α).

Retraction of ‘miR-199a-3p knockdown inhibits dedifferentiated liposarcoma (DDLPS) cell viability and enhances apoptosis through targeting casein kinase-1 alpha (CK1α)’ by Ye Cao et al., RSC Adv., 2019, 9, 22755–22763, DOI: 10.1039/C9RA01491H.

The combined effect of oleonuezhenide and wedelolactone on proliferation and osteoblastogenesis of bone marrow mesenchymal stem cells

BackgroundRegulation of the survival and differentiation of bone marrow mesenchymal stem cells is an essential consideration in the development of targeted drugs for treatment of osteoporosis. PurposeThe present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide, two compounds from Chinese formula Er-Zhi-Wan, on osteoblastogenesis and the underlying molecular mechanisms. MethodsMTT assay was taken to evaluate cell proliferation. The alkaline phosphatase (ALP) activity assay was used to determine the activity of ALP. Alizarin red S (ARS) staining was taken to indicate the intensity of the calcium deposits. Quantitative real-time PCR and Western blot were performed to the levels of Runx2, Osteocalcin, and Osterix expression in mouse bone marrow mesenchymal stem cells (BMSCs). Ovariectomized mouse model and bone histomorphometric analysis were also used to research the effects of wedelolactone and oleonuezhenide on bone loss caused by ovariectomy. ResultsWedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization. Osteoblastogenesis-related marker genes including osteocalcin, Runx2, and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide. At the molecular level, oleonuezhenide did not affect GSK-3β phosphorylation induced by wedelolactone, but elevated casein kinase 2-alpha (CK2α) expression, resulting in β-catenin and Runx2 nuclear translocation. In addition, 30 µM wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 µM oleonuezhenide. These cells were protected by oleonuezhenide and maintained osteoblastic activity. Oleonuezhenide increased Wnt5a and CK2α expression. Wedelolactone-reduced extracellular signal-regulated kinase (ERK) phosphorylation was reversed by oleonuezhenide. In ovariectomized mice, administration of wedelolactone and oleonuezhenide prevented ovariectomy-induced bone loss by enhancing osteoblastic activity. ConclusionThese results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis. These two compounds could be developed as a combined therapeutic agent for osteoporosis.

Retracted Article: miR-199a-3p knockdown inhibits dedifferentiated liposarcoma (DDLPS) cell viability and enhances apoptosis through targeting casein kinase-1 alpha (CK1α).

Dedifferentiated liposarcoma (DDLPS) is an aggressive tumor with high mortality. More insight into the biology of DDLPS tumorigenesis is needed to devise novel therapeutic approaches. Previous data showed that miRNA-199a-3p (miR-199a-3p) was strongly upregulated in DDLPS tissues. However, the biological role of miR-199a-3p in DDLPS remains unknown. In this study, we detected miR-199a-3p expression using RT-qPCR and observed that miR-199a-3p was more highly expressed in DDLPS tissues and cell lines (SW872 and LPS141). Functionally, MTT assay, flow cytometry and western blot results demonstrated that knockdown of miR-199a-3p inhibited DDLPS cell viability, enhanced apoptosis rate, and decreased expression of apoptosis-related genes Bax and cleaved caspase 3, as well as increased Bcl-2 expression in vitro. Moreover, xenograft tumors were generated and miR-199a-3p knockdown could suppress DDLPS xenograft tumor growth accompanying decreased proliferating cell nuclear antigen (PCNA) level and increased cleaved caspase 3 level in vivo. Mechanically, luciferase reporter assay and RNA immunoprecipitation (RIP) identified that CK1α was targeted and downregulated by miR-199a-3p. Expression of CK1α was lower in DDLPS tissues. Besides, there was a negative linear correlation between expressions of miR-199a-3p and CK1α in DDLPS tissues. Rescue experiments indicated that CK1α silencing could abolish the effect of miR-199a-3p knockdown on cell viability and apoptosis in DDLPS cells in vitro. In conclusion, knockdown of miR-199a-3p inhibits DDLPS cell viability and enhances apoptosis through targeting CK1α in vitro and in vivo. Our results suggest miR-199a-3p/CK1α axis may be a novel pathogen of DDLPS.

Synthesis of polybrominated benzimidazole and benzotriazole derivatives containing a tetrazole ring and their cytotoxic activity.

A series of new benzimidazole and benzotriazole derivatives containing a tetrazole moiety was synthesized by N-alkylation of 5-aryltetrazole with 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzimidazole and 4,5,6,7-tetrabromo-2-(3-chloropropyl)-2H-benzotriazole. The reaction was regioselective and mostly 2,5-disubstituted tetrazole derivatives were obtained. The effect of all synthesized compounds on human recombinant casein kinase 2alpha subunit (rhCK2α) and cytotoxicity against human T-cell lymphoblast (CCRF-CEM) and breast adenocarcinoma (MCF-7) cell lines were evaluated. The results have shown that many of the synthesized compounds exhibit significant cytotoxicity at micromolar concentration.Graphical abstract

Distinct Signaling Requirements for the Establishment of ESC Pluripotency in Late-Stage EpiSCs.

SummaryIt has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.

Arabidopsis casein kinase 2 α4 subunit regulates various developmental pathways in a functionally overlapping manner

Casein kinase 2 (CK2) is an essential and well-conserved Ser/Thr kinase that regulates proteins in a posttranslational manner. CK2 has been shown to affect a large number of developmental processes across eukaryotes. It is a tetrameric protein composed of a dimer of alpha (catalytic) and beta (regulatory) subunit each. In our previous study we showed that three of the four CK2 α subunits in Arabidopsis act in a functionally redundant manner to regulate various developmental pathways. In this study we constructed two independent CK2 α4 RNAi lines in the CK2 alpha triple mutant background. Through functional characterization of these RNAi lines we show that the fourth α subunit in Arabidopsis also functions redundantly in regulating ABA response, lateral root formation and flowering time. CK2 α4-GFP localizes to the chloroplast in transgenic Arabidopsis seedlings, consistent with the presence of a chloroplast localization signal at the amino-terminus of CK2 α4 subunit. Taken together, our results suggest a functionally overlapping role for the CK2 α4 subunit in regulating various developmental processes in plants.

Casein Kinase 1 Alpha (CK1a) Promotes Nuclear Entry of PERIOD and Represses CLOCK transcriptional activity in the Drosophila melanogaster Circadian Clock

Circadian clocks endow organisms with the ability to temporally coordinate and synchronize their physiology with the surrounding environment, thereby conferring an adaptive advantage. Although the specific clock genes are not highly conserved, the basic design and regulation of the circadian oscillator is remarkably similar across species. In Drosophila melanogaster, the central oscillator exists as interconnected transcriptional translational feedback loops that drive and sustain rhythm. Essential to this regulatory network are the transcriptional activator and repressor: CLOCK (CLK) and PERIOD (PER), respectively, as mutations in these proteins result in arrhythmicity. Functional PER and CLK, however, undergo daily cycle of phosphorylation that regulates both the abundance and the timing for which they act as regulators of the circadian transcriptome. To comprehensively identify proteins responsible for these phosphorylation events, we performed proteomics studies using affinity purification (AP) and label‐free quantitative mass spectrometry (MS) using Drosophila S2 cells. We annotate our result by incorporating published PPIs, knowledge about common AP‐MS contaminants, as well as protein coevolution data. We identified Casein Kinase 1 alpha (CK1α) as a clock kinase that phosphorylates CLK to promote CLK clearance at the per promoter and thereby terminating CLK transcriptional activity. Furthermore, CK1α regulates the timing of PER nuclear entry in the cytoplasm, whereas in the nucleus, CK1α phosphorylates and destabilizes PER.

RNA interference (RNAi) mediated stable knockdown of protein casein kinase 2-alpha (CK2α) inhibits migration and invasion and enhances cisplatin-induced apoptosis in HEp-2 laryngeal carcinoma cells

Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial–mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α.

The tumor suppressor APC differentially regulates multiple beta-catenins through the function of Axin and casein kinase 1alpha during C. elegans asymmetric stem cell divisions

The APC tumor suppressor regulates diverse stem cell processes including gene regulation through Wnt-β-catenin signaling and chromosome stability through microtubule interactions, but how the disparate functions of APC are controlled is not well understood. Acting as part of a Wnt-β-catenin pathway that controls asymmetric cell division, Caenorhabditis elegans APC, APR-1, promotes asymmetric nuclear export of the β-catenin WRM-1 by asymmetrically stabilizing microtubules. Wnt function also depends on a second β-catenin, SYS-1, which binds to the C. elegans TCF POP-1 to activate gene expression. Here, we show that APR-1 regulates SYS-1 levels in asymmetric stem cell division, in addition to its known role in lowering nuclear levels of WRM-1. We demonstrate that SYS-1 is also negatively regulated by the C. elegans homolog of casein kinase 1α (CKIα), KIN-19. We show that KIN-19 restricts APR-1 localization, thereby regulating nuclear WRM-1. Finally, the polarity of APR-1 cortical localization is controlled by PRY-1 (C. elegans Axin), such that PRY-1 controls the polarity of both SYS-1 and WRM-1 asymmetries. We propose a model whereby Wnt signaling, through CKIα, regulates the function of two distinct pools of APC - one APC pool negatively regulates SYS-1, whereas the second pool stabilizes microtubules and promotes WRM-1 nuclear export.

Immunohistochemistry on decalcified rat nasal cavity: trials and successes

Immunohistochemical (IHC) staining for Casein Kinase 2 alpha (CK2a), cyclin-dependent kinase inhibitor 1B (p27Kip1), and heat shock protein 90 (HSP90) was performed on four levels of decalcified rat nasal cavity from four male and three female controls following nose-only exposure to ambient air for 5 days. Successful staining with negligible background artifact was obtained on multiple epithelial and neuroepithelial soft tissues, without antigen retrieval. Independent optimization was needed for each of the three antibodies. A major challenge in this project was maintaining tissue adherence to the slides during tissue incubations. This report documents immunolocalization of CK2a, p27Kip1, and HSP90 in respiratory and olfactory tissue of the rat nose.

Two distinct phosphorylation events govern the function of muscle FHOD3

Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.

Thiazolidinediones Prevent PDGF-BB-induced CREB Depletion in Pulmonary Artery Smooth Muscle Cells by Preventing Upregulation of Casein Kinase 2 α′ Catalytic Subunit

The transcription factor CREB is diminished in smooth muscle cells (SMCs) in remodeled, hypertensive pulmonary arteries (PAs) in animals exposed to chronic hypoxia. Forced depletion of cyclic adenosine monophosphate response element binding protein (CREB) in PA SMCs stimulates their proliferation and migration in vitro. Platelet-derived growth factor (PDGF) produced in the hypoxic PA wall promotes CREB proteasomal degradation in SMCs via phosphatidylinositol-3-kinase/Akt signaling, which promotes phosphorylation of CREB at 2 casein kinase 2 (CK2) sites. Here we tested whether thiazolidinediones, agents that inhibit hypoxia-induced PA remodeling, attenuate SMC CREB loss. Depletion of CREB and changes in casein kinase 2 catalytic subunit expression and activity were measured in PA SMC treated with PDGF. PA remodeling and changes in medial PA CREB and casein kinase 2 levels were evaluated in lung sections from rats exposed to hypoxia for 21 days. We found that the thiazolidinedione rosiglitazone prevented PA remodeling and SMC CREB loss in rats exposed to chronic hypoxia. Likewise, the thiazolidinedione troglitazone blocked PA SMC proliferation and CREB depletion induced by PDGF in vitro. Thiazolidinediones did not repress Akt activation by hypoxia in vivo or by PDGF in vitro. However, PDGF-induced CK2 alpha' catalytic subunit expression and activity in PA SMCs, and depletion of CK2 alpha' subunit prevented PDGF-stimulated CREB loss. Troglitazone inhibited PDGF-induced CK2 alpha' subunit expression in vitro and rosiglitazone blocked induction of CK2 catalytic subunit expression by hypoxia in PA SMCs in vivo. We conclude that thiazolidinediones prevent PA remodeling in part by suppressing upregulation of CK2 and loss of CREB in PA SMCs.

Structural basis for decreased affinity of Emodin binding to Val66-mutated human CK2α as determined by molecular dynamics

Protein kinase CK2 (casein kinase 2) is a multifunctional serine/threonine kinase that is involved in a broad range of physiological events. The decreased affinity of Emodin binding to human CK2 alpha resulting from single-point mutation of Val66 to Ala (V66A) has been demonstrated by experimental mutagenesis. Molecular dynamics (MD) simulations and energy analysis were performed on wild type (WT) and V66A mutant CK2 alpha-Emodin complexes to investigate the subtle influences of amino acid replacement on the structure of the complex. The structure of CK2 alpha and the orientation of Emodin undergo changes to different degrees in V66A mutant. The affected positions in CK2 alpha are mainly distributed over the glycine-rich loop (G-loop), the alpha-helix and the loop located at the portion between G-loop and alpha-helix (C-loop). Based on the coupling among these segments, an allosteric mechanism among the C-loop, the G-loop and the deviated Emodin is proposed. Additionally, an estimated energy calculation and residue-based energy decomposition also indicate the lower instability of V66A mutant in contrast to WT, as well as the unfavorable energetic influences on critical residue contributions.

Identification of genes expressed as a result of lindane exposure in Oreochromis niloticus using differential display

In order to assess the effect of lindane exposure on gene expression in tilapia ( Oreochromis niloticus), twenty male fish were individually weighted and injected intraperitoneally with a single dose of lindane (19.09 mg/kg bw) using corn oil as a carrier vehicle, while a second group of twenty male fish (controls) was only injected with the carrier vehicle. Groups of four fish each were then sacrificed at 3, 6, 12, 18 and 24 h after treatment application and total RNA was extracted from liver tissue. The differential display (DD) technique was then used to identify differentially expressed cDNA fragments between treatment and control fish. A total of fifty cDNA fragments were isolated and sequenced, from which only four showed homology with genes previously described in other fish species, namely the immunoglobulin heavy chain (IgH), coagulation factor V (FV), casein kinase 2 alpha (CK2a), and the receptor protein-tyrosine-like phosphatase (RPT-LP). The expression of such genes was confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). Results showed that lindane exposure triggered the differential expression of these genes during the first 6, 18 and 24 h subsequent to treatment application, suggesting that lindane exposure can trigger a rapid immune system response in tilapias.

Phosphorylation of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression

Fully mature DC and, to a lesser extent, activated T and B cells express CD83, a surface molecule that appears to fulfil an important role in efficient T-cell activation. Recently, it has been shown that CD83 mRNA is transported from the nucleus to the cytoplasm by an uncommon route, involving the cellular RNA-binding protein HuR and the nuclear export receptor CRM1. Moreover, the shuttle phosphoprotein APRIL (ANP32B) has been shown to be required for HuR-mediated nucleocytoplasmic translocation of the CD83 mRNA by acting as an adaptor that links HuR and CRM1. Here, we are able to report that casein kinase 2 (CK2) phosphorylates APRIL on residue threonine244 (Thr(244)) and demonstrate that the CK2-specific inhibitor 4,5,6,7-tetrabromo-2-azabenzimidazole abolishes CD83 expression in activated Jurkat T cells by interfering with the nucleocytoplasmic translocation of CD83 mRNA. Depletion and knockdown studies demonstrate that the CK2 alpha' subunit is necessary for this regulation, whereas the CK2 alpha subunit seems to be dispensable. Taken together, the data presented significantly extend our knowledge of the complex regulation of CD83 mRNA processing and provides a novel strategy to interfere with CD83 expression.

Casein kinase 1α governs antigen-receptor-induced NF-κB activation and human lymphoma cell survival

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.