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The Huayu-Qiangshen-Tongbi (HQT) decoction, a Chinese medical formula, has been identified to show a potent therapeutic effect on rheumatoid arthritis (RA). However, the specific molecular mechanism of HQT in RA has not been well studied. In the present study, LPS-treated human rheumatoid fibroblast-like synoviocyte (FLS) MH7A cells and collagen-induced arthritis (CIA) mice were utilized as in vitro and in vivo models. Our results demonstrated that HQT could efficiently inhibit RA-induced inflammation by reducing the production of cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Moreover, HQT significantly upregulated the expression of miR-125b. Besides, analysis of bioinformatics suggested casein kinase 2 (CK2) was a potential target of miR-125b. Luciferase reporter assay was performed and revealed that miR-125b suppressed CK2 expression in MH7A cells. Furthermore, miR-125b inhibited LPS-induced NF-kappa-B (NF-κB) activation, which is a downstream target of CK2. In addition, the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) and NF-kappa-B inhibitor alpha (IkB-α) enhanced the inhibitory effect of miR-125b on the expression of TNF-α, IL-1β, and IL-6. Taken together, our study revealed that HQT could attenuate RA through upregulating miR-125b to suppress NF-κB-induced inflammation by targeting CK2. The findings of this study should facilitate investigating the mechanism of HQT on RA and discovering novel therapeutic targets for RA. [ABSTRACT FROM AUTHOR]

CIGB-300 is an antitumor peptide that inhibits the CK2-mediated phosphorylation by direct targeting of substrates. This paper aims to describe breakthroughs about the CIGB-300 antineoplastic mechanism related to the inhibition of CK2-mediated phosphorylation and the synergism with anticancer drugs. CK2 phosphorylation assays were performed with catalytic CK2α subunit or the CK2 holoenzyme in presence or not of CIGB-300. CIGB-300/CK2 interaction was verified by pull-down experiments, in situ colocalization and phosphoproteomic analysis of CIGB-300-treated lung cancer cells were also performed. Synergism of the peptide with anticancer drugs was evaluated in vitro and for Cisplatin; it was also tested in vivo. Besides, comparative proteomics of CIGB-300 combined with Cisplatin was conducted. CIGB-300 targeted the CK2α subunit and inhi-bited the enzymatic activity of the holoenzyme in different experimental settings. Likewise, phosphoproteomic and Western Blot analysis allowed for knowing the early CK2 inhibition profile elicited by CIGB-300 at 10 and 30 min of treatment. Moreover, CIGB-300 did synergize with chemotherapeutics and EGFR inhibitors, and molecular events that reportedly support such synergistic interactions were also known. CIGB-300 inhibits the CK2-mediated phosphorylation by using an alternative mechanism of direct interaction with the enzyme itself. Besides, CIGB-300 synergizes with chemotherapeutics and EGFR inhibitors by modulating different proteins related to drug resistance. [ABSTRACT FROM AUTHOR]

Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 μM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug's anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug's pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug's ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL. [ABSTRACT FROM AUTHOR]

Aims: Sonic hedgehog subtype medulloblastoma is featured with overactivation of hedgehog pathway and can be targeted by SMO‐specific inhibitors. However, the resistance is frequently developed leading to treatment failure of SMO inhibitors. W535L mutation of SMO (SMOW535L) is thought to be an oncogenic driver for Sonic hedgehog subtype MB and confer resistance to SMO inhibitors. The regulation network of SMOW535L remains to be explored in comparison with wild‐type SMO (SMOWT). Methods: In this study, we profiled transcriptomes, methylomes, and interactomes of MB cells expression SMOWT or SMOW535L in the treatment of DMSO or SMO inhibitor, respectively. Results: Analysis of transcriptomic data indicated that SMO inhibitor disrupted processes of endocytosis and cilium organization in MB cells with SMOWT, which are necessary for SMO activation. In MB cells with SMOW535L, however, SMO inhibitor did not affect the two processes‐related genes, implying resistance of SMOW535L toward SMO inhibitor. Moreover, we noticed that SMO inhibitor significantly inhibited metabolism‐related pathways. Our metabolic analysis indicated that nicotinate and nicotinamide metabolism, glycerolipid metabolism, beta‐alanine metabolism, and synthesis and degradation of ketone bodies might be involved in SMOW535L function maintenance. Interactomic analysis revealed casein kinase II (CK2) as an important SMO‐associated protein. Finally, we linked CK2 and AKT together and found combination of inhibitors targeting CK2 and AKT showed synergetic effects to inhibit the growth of MB cells with SMO constitutive activation mutation. Conclusions: Taken together, our work described SMO‐related transcriptomes, metabolomes, and interactomes under different SMO status and treatment conditions, identifying CK2 and AKT as therapeutic targets for SHH‐subtype MB cells with SMO inhibitor resistance. [ABSTRACT FROM AUTHOR]

Protein kinase CK2, formally known as casein kinase II, is ubiquitously expressed and highly conserved serine/threonine or tyrosine kinase enzyme that regulates diverse signaling pathways responsible for cellular processes (i.e., cell proliferation and apoptosis) via interactions with over 500 known substrates. The enzyme’s physiological interactions and cellular functions have been widely studied, most notably in the blood and solid malignancies. CK2 has intrinsic role in carcinogenesis as overexpression of CK2 subunits (α, α`, and β) and deregulation of its activity have been linked to various forms of cancers. CK2 also has extrinsic role in cancer stroma or in the tumor microenvironment (TME) including the immune cells. However, very few research studies have focused on extrinsic role of CK2 in regulating immune responses as a therapeutic alternative for cancer. The following review discusses CK2’s regulation of key signaling events [Nuclear factor kappa B (NF-κB), Janus kinase/signal transducer and activators of transcription (JAK/STAT), Hypoxia inducible factor–1alpha (HIF-1α), Cyclooygenase-2 (COX-2), Extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), Notch, Protein kinase B/AKT, Ikaros and Wnt] that can influence the development and function of immune cells in cancer. Potential clinical trials using potent CK2 inhibitors will facilitate and improve the treatment of human malignancies.

• Synthesis and spectroscopic characterization have been performed. • The nonlinear optical properties have been examined. • Physicochemical and ADMET properties have been examined. • Anticancer activity has been investigated by molecular docking. The geometric, spectroscopic and electronic features of the newly synthesized 3-Hydroxy-2-(4-chlorophenyl)-7-fluoro-4H-chromen-4-one have been analyzed using a combination of single crystal XRD, FT-IR, NMR shifts, and UV–Vis. spectroscopic methods both experimentally and theoretically. The molecule has crystallized in orthorhombic space group Pca21 and it has stabilized with C H···O, O H···O intra-molecular and O H···O inter-molecular interactions. The geometric parameters (bond lengths and bond angles), vibrational wavenumbers, 1H and 13C NMR chemical shifts, UV–Vis. electronic transitions, the HOMO (highest occupied molecular orbital) and the LUMO (lowest unoccupied molecular orbital) analyses, NLO (non-linear optical properties) and the MEP (molecular electrostatic potential) surface have been computed by using the DFT/B3LYP quantum chemical method with 6–311++ G (d,p) level of theory to compare with the experimental findings. Absorption bands of the molecule have computed around 300 and 360 nm, while observed at 244, 312 and 344 nm in the experimental UV–Vis. spectrum. Moreover, the mean polarizability value of the compound has been calculated 6.47 times greater than urea. The objective of the in silico investigation was to ascertain the biological effect profile of the title compound. The ADMETlab 2.0 web service has been employed to analyze the physicochemical, medicinal chemistry, metabolism, distribution, absorption, excretion and toxicity features of the compound. Its protein kinase CK2 inhibitory activity for the target macromolecule 5M4U has been investigated by means of a molecular docking study to investigate the anti-cancer activity of our flavonol derivative compound. The interactions between the 5M4U macromolecule and the ligand compound. [Display omitted] [ABSTRACT FROM AUTHOR]

Quantitative phosphoproteomic data has mostly been reported from experiments comparing relative phosphopeptides intensities in two or more different conditions, while the ideal parameter to compare is phosphopeptides occupancies. This term is scarcely used and therefore barely implemented in phosphoproteomics studies, and this should be of concern for the scientific journals. In order to demonstrate the relevance of this issue, here we show how the method of choice affects the interpretation of the data. The phosphoproteomic profile modulated in two AML cell lines after CK2 inhibition with CIGB-300 or CX-4945 is shown. Following the downstream action of CK2 the phosphosite intensity and occupancy results were compared to validate the best approach for quantitative phosphoproteomic studies. Even when the total number of quantified phosphopeptides was higher by using the intensity calculation, in all the cases the percent of CK2 consensus sequences which were down-regulated in response to CK2 inhibition was higher using the phosphosite occupancy quantification. To note, a high number of CK2 consensus sequences was found down-regulated with at least a 10% or 15% of phosphosite occupancy variation illustrating that low thresholds of occupancy modulation might be indicative of biological effect. Additionally, several biological processes only appear significantly over-represented in the phosphoproteome quantified by occupancy. The functional enrichment analysis per ranges of occupancy variations also illustrated clear differences among AML cell lines subjected to CK2 inhibition by CX-4945. A low overlap between the phosphoproteomes quantified by intensity and occupancy was obtained illustrating that new developments in proteomics techniques are needed to improve the performance of the occupancy approach. Even in such context, results indicate that occupancy quantification performs better than phosphorylation quantification based on intensity reinforcing the importance of such quantification approach to describe phosphoproteomic data. [ABSTRACT FROM AUTHOR]

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α. • CK1α splice variant abundance varies across normal tissues and cancer cell lines. • Cancer cell lines with high levels of select CK1α splice variants express distinct biological pathways. • The S-insert of CK1α promotes cell growth in a cancer cell line. [ABSTRACT FROM AUTHOR]

Casein kinase II (CK2) has recently emerged as a pivotal mediator in the propagation of inflammation across various diseases. Nevertheless, its role in the pathogenesis of sepsis remains unexplored. Here, we investigated the involvement of CK2 in sepsis progression and the potential beneficial effects of silmitasertib, a selective and potent CK2α inhibitor, currently under clinical trials for COVID-19 and cancer. Sepsis was induced by caecal ligation and puncture (CLP) in four-month-old C57BL/6OlaHsd mice. One hour after the CLP/Sham procedure, animals were assigned to receive silmitasertib (50 mg/kg/i.v.) or vehicle. Plasma/organs were collected at 24 h for analysis. A second set of experiments was performed for survival rate over 120 h. Septic mice developed multiorgan failure, including renal dysfunction due to hypoperfusion (reduced renal blood flow) and increased plasma levels of creatinine. Renal derangements were associated with local overactivation of CK2, and downstream activation of the NF-ĸB-iNOS-NO axis, paralleled by a systemic cytokine storm. Interestingly, all markers of injury/inflammation were mitigated following silmitasertib administration. Additionally, when compared to sham-operated mice, sepsis led to vascular hyporesponsiveness due to an aberrant systemic and local release of NO. Silmitasertib restored sepsis-induced vascular abnormalities. Overall, these pharmacological effects of silmitasertib significantly reduced sepsis mortality. Our findings reveal, for the first time, the potential benefits of a selective and potent CK2 inhibitor to counteract sepsis-induced hyperinflammatory storm, vasoplegia, and ultimately prolonging the survival of septic mice, thus suggesting a pivotal role of CK2 in sepsis and silmitasertib as a novel powerful pharmacological tool for drug repurposing in sepsis. [Display omitted] • CK2 is overactivated during CLP-induced sepsis, thus triggering NF-ĸB-iNOS-NO axis. • Silmitasertib attenuates CK2 activation, reducing local and systemic inflammatory storm. • Silmitasertib reduces NO levels, leading to improved renal perfusion and restoring responsiveness to vasoconstrictors. • Silmitasertib mitigates multiorgan dysfunction and enhances the survival of septic mice. [ABSTRACT FROM AUTHOR]

Quantitative analysis of Rumex nepalensis var. remotiflorus revealed that its roots contain rich anthraquinones, which has emodin, chrysophanol, and physcion contents of up to 0.30, 0.67, and 0.98 mg/g, respectively. Further phytochemical study led to the isolation and purification of seven undescribed phenolic constituents, including one flavan derivative with a 13-membered ring, polygorumin A (1), two dianthrone glucosides, polygonumnolides F and G (2 , 3), two diphenylmethanones, rumepalens A and B (4 , 5), and a pair of epimeric oxanthrone C -glucosides, rumejaposides K and L (6a , 6b) from the roots of R. nepalensis var. remotiflorus. Furthermore, 1 undescribed natural product, 1- β -D-glucoside-6′-[(2 E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate]-3-hydroxy-5-methylphenyl (19), and 21 known phenolic compounds were obtained from the aforementioned plant for the first time. Their structures were elucidated through extensive spectroscopic data analysis. Notably, compounds 1 , 4 – 5 , and 7 – 9 exhibited inhibitory activity on α -glucosidase with IC 50 values ranging from 1.61 ± 0.17 to 32.41 ± 0.87 μM. In addition, the isolated dianthrone, chrysophanol bianthrone (14), showed obvious cytotoxicity against four human cancer cell lines (HL-60, SMMC-7721, A-549, and MDA-MB-231) with IC 50 values ranging from 3.81 ± 0.17 to 35.15 ± 2.24 μM. In silico target prediction and molecular docking studies demonstrated that the mechanism of the anticancer activity of 14 may be related to the interaction with protein kinase CK2. [Display omitted] • The chemical constituents of R. nepalensis var. remotiflorus were studied firstly. • Emodin, chrysophanol and physcion are major anthraquinones in the roots. • Seven undescribed phenols and one undescribed natural product were obtained. • Some isolates showed obvious inhibition on α -glucosidase and HL-60 cell lines. [ABSTRACT FROM AUTHOR]

Novel treatments for neurodegenerative disorders are in high demand. It is imperative that new protein targets be identified to address this need. Characterization and validation of nascent targets can be accomplished very effectively using highly specific and potent chemical probes. Human induced pluripotent stem cells (hiPSCs) provide a relevant platform for testing new compounds in disease relevant cell types. However, many recent studies utilizing this platform have focused on neuronal cells. In this study, we used hiPSC-derived microglia-like cells (MGLs) to perform side-by-side testing of a selective chemical probe, SGC-CK2-1, compared with an advanced clinical candidate, CX-4945, both targeting casein kinase 2 (CK2), one of the first kinases shown to be dysregulated in Alzheimer's disease (AD). CK2 can mediate neuroinflammation in AD, however, its role in microglia, the innate immune cells of the central nervous system (CNS), has not been defined. We analyzed available RNA-seq data to determine the microglial expression of kinases inhibited by SGC-CK2-1 and CX-4945 with a reported role in mediating inflammation in glial cells. As proof-of-concept for using hiPSC-MGLs as a potential screening platform, we used both wild-type (WT) MGLs and MGLs harboring a mutation in presenilin-1 (PSEN1), which is causative for early-onset, familial AD (FAD). We stimulated these MGLs with pro-inflammatory lipopolysaccharides (LPS) derived from E. coli and observed strong inhibition of the expression and secretion of proinflammatory cytokines by simultaneous treatment with SGC-CK2-1. A direct comparison shows that SGC-CK2-1 was more effective at suppression of proinflammatory cytokines than CX-4945. Together, these results validate a selective chemical probe, SGC-CK2-1, in human microglia as a tool to reduce neuroinflammation. [ABSTRACT FROM AUTHOR]

CSNK2B has recently been identified as the causative gene for Poirier–Bienvenu neurodevelopmental syndrome (POBINDS). POBINDS is a rare neurodevelopmental disorder characterized by early-onset epilepsy, developmental delay, hypotonia, and dysmorphism. Limited by the scarcity of patients, the genotype–phenotype correlations in POBINDS are still unclear. In the present study, we describe the clinical and genetic characteristics of eight individuals with POBINDS, most of whom suffered developmental delay, generalized epilepsy, and hypotonia. Minigene experiments confirmed that two intron variants (c.367+5G>A and c.367+6T>C) resulted in the skipping of exon 5, leading to a premature termination of mRNA transcription. Combining our data with the available literature, the types of POBINDS-causing variants included missense, nonsense, frameshift, and splicing, but the variant types do not reflect the clinical severity. Reduced casein kinase 2 holoenzyme activity may represent a unifying pathogenesis. We also found that individuals with missense variants in the zinc finger domain had manageable seizures (p = 0.009) and milder intellectual disability (p = 0.003) than those with missense variants in other domains of CSNK2B. This is the first study of genotype–phenotype correlations in POBINDS, drawing attention to the pathogenicity of intron variants and expanding the understanding of neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 μM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug's efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α. [ABSTRACT FROM AUTHOR]

Molecular mimicry of host proteins by pathogens constitutes a strategy to hijack the host pathways. At present, there is no dedicated resource for mimicked domains and motifs in the host–pathogen interactome. In this work, the experimental host–pathogen (HP) and host–host (HH) protein–protein interactions (PPIs) were collated. The domains and motifs of these proteins were annotated using CD Search and ScanProsite, respectively. Host and pathogen proteins with a shared host interactor and similar domain/motif constitute a mimicry pair exhibiting global structural similarity (domain mimicry pair; DMP) or local sequence motif similarity (motif mimicry pair; MMP). Mimicry pairs are likely to be co-expressed and co-localized. 1,97,607 DMPs and 32,67,568 MMPs were identified in 49,265 experimental HP-PPIs and organized in a web-based resource, ImitateDB (http://imitatedb.sblab-nsit.net) that can be easily queried. The results are externally integrated using hyperlinked domain PSSM ID, motif ID, protein ID and PubMed ID. Kinase, UL36, Smc and DEXDc were frequent DMP domains whereas protein kinase C phosphorylation, casein kinase 2 phosphorylation, glycosylation and myristoylation sites were frequent MMP motifs. Novel DMP domains SANT, Tudor, PhoX and MMP motif microbody C-terminal targeting signal, cornichon signature and lipocalin signature were proposed. ImitateDB is a novel resource for identifying mimicry in interacting host and pathogen proteins. [ABSTRACT FROM AUTHOR]

(B) Representative western blot images showed the EpCAM protein level in the small intestines of WT and EpCAM-/- mice at P3 stage. (B) The mRNA expression levels of TSC2 in the small intestines of WT and EpCAM-/- mice at P0 and P3 stage, respectively (P0: WT, n = 6; EpCAM-/-, n = 8. We reported a new role of epithelial cell adhesion molecule (EpCAM) on maintaining the longevity of intestinal epithelial cells (IECs) via regulating the TSC1/mTORC1/Sirtuins pathway. [Extracted from the article]

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication–associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1–Cul1–F-box protein (SCF) ubiquitin ligase complex and regulator of key cellcycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F–SCF complex, represses premature stem cell quiescence exit via ubiquitin–proteasome degradation of these factors. [ABSTRACT FROM AUTHOR]

As a highly conserved serine/threonine kinase with catalytic and regulatory subunits distributed ubiquitously in eukaryotic organisms, casein kinase 2 (CK2) is involved in multiple cellular functions, including immune regulation. In this study, two variants of the catalytic subunit (designated PvCK2α-1 and PvCK2α-2) and the regulatory subunit homologs (designated PvCK2β-1 and PvCK2β-2) in Penaeus vannamei were cloned and characterised. PvCK2α-1 and PvCK2α-2 shared the same genomic sequence consisting of six exons and five introns and encoded the same protein of 350 amino acids with an S_TKc domain, although there was a sequence deletion in 3′-UTR in PvCK2α-2 when compared with PvCK2α-1. Because of the sequence deletion in the ORF, PvCK2β-1 and PvCK2β-2 encoded different proteins with a CK_II_beta domain. The gene structures of PvCK2β-1 and PvCK2β-2 were identical and consisted of four exons and three introns. Semi-quantitative RT-PCR analyses revealed that PvCK2α and PvCK2β were constitutively expressed in all P. vannamei tissues tested, with higher levels detected in the immune-related tissues including hemocytes, hepatopancreas, gills and intestine. In these four tissue types, all variants of PvCK2α and PvCK2β were induced upon challenge with white spot syndrome virus (WSSV), Vibrio parahaemolyticus and Staphyloccocus aureus. The inhibition of PvCK2α, PvCK2β-1 and PvCK2βComb (the amount of PvCK2β-1 and PvCK2β-2) significantly reduced the survival rates of P. vannamei after WSSV infection and significantly increased the WSSV viral loads. Knockdown of PvCK2 by RNAi could distinctly decrease the expression of NF-κB related genes. All of these results suggest that PvCK2 plays an important role in the innate immune response to pathogen challenges in P. vannamei , with a positive role in anti-WSSV response which may be mediated through regulating the expression of NF-κB drived antimicrobial peptide genes. • Two variants of the catalytic (CK2α) and regulatory subunit (CK2β) in P. vannamei were cloned and characterized. • PvCK2α and PvCK2β were constitutively expressed and induced by pathogens challenge in P. vannamei. • Inhibition of PvCK2α and PvCK2β significantly reduced the survival rates of P. vannamei after WSSV infection. • Knockdown of PvCK2α and PvCK2β significantly increased the WSSV viral loads. • Knockdown of PvCK2α and PvCK2β significantly decreased the expression of NF-κB related genes. [ABSTRACT FROM AUTHOR]

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve–glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2′-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA. [ABSTRACT FROM AUTHOR]

The article presents a study which explores the mechanism of stress-induced attacks in an episodic neurologic disorder. It mentions that results suggest that norepinephrine and CK2 are required for the initiation of stress-induced attacks in EA2 and provide previously unidentified targets for therapeutic intervention.

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation. Synopsis: C‐terminal hyperphosphorylation of TDP‐43, a pathological hallmark of several neurodegenerative disorders, enhances the liquidity of TDP‐43 condensates and suppresses TDP‐43 condensation and aggregation, shedding a new light on this TDP‐43 disease‐linked posttranslational modification. TDP‐43 phosphorylation by CK1δ or C‐terminal phosphomimetic mutations suppress TDP‐43 phase separation and aggregation.C‐terminal phosphomimetic mutations enhance liquidity and dynamics of TDP‐43 condensates.Suppression of phase separation is associated with loss of protein‐protein interactions in the C‐terminus and enhanced solvation of negatively charged groups.TDP‐43 bearing C‐terminal phosphomimetic mutations fails to condense into stress‐induced membrane‐less compartments and remains dispersed. [ABSTRACT FROM AUTHOR]

Silmitasertib (CX-4945) is currently being investigated in clinical trials against various types of cancer. The U.S. Food and Drug Administration (FDA) has already granted orphan drug designation to the compound for the treatment of advanced cholangiocarcinoma, medulloblastoma, and biliary tract cancer. Silmitasertib inhibits the serine/threonine protein kinase CK2, which exerts a proliferation-promoting and anti-apoptotic effect on cancer cells. In view of current and future applications, the measurement of silmitasertib levels in plasma is expected to play an important role in the evaluation of therapeutic and toxic concentrations in cancer patients. In the present work, we therefore present an LC–MS/MS method for the quantification of silmitasertib in human plasma. Using a simple liquid–liquid extraction with ethyl acetate and a mixture of n-hexane and ethyl acetate, this method can be performed in any laboratory with mass spectrometry. The validation was carried out according to the FDA guideline. [ABSTRACT FROM AUTHOR]

Casein kinase II (CK2) and cyclin-dependent kinases (CDKs) frequently interact within multiple pathways in pancreatic ductal adenocarcinoma (PDAC). Application of CK2- and CDK-inhibitors have been considered as a therapeutic option, but are currently not part of routine chemotherapy regimens. We investigated ten PDAC cell lines exposed to increasing concentrations of silmitasertib and dinaciclib. Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated, and bioinformatic clustering was used to classify cell lines into sensitive groups based on their response to inhibitors. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) was conducted to assess recurrent mutations and the expression profile of inhibitor targets and genes frequently mutated in PDAC, respectively. Dinaciclib and silmitasertib demonstrated pronounced and limited cell line specific effects in cell death induction, respectively. WES revealed no genomic variants causing changes in the primary structure of the corresponding inhibitor target proteins. RNA-Seq demonstrated that the expression of all inhibitor target genes was higher in the PDAC cell lines compared to non-neoplastic pancreatic tissue. The observed differences in PDAC cell line sensitivity to silmitasertib or dinaciclib did not depend on target gene expression or the identified gene variants. For the PDAC hotspot genes kirsten rat sarcoma virus (KRAS) and tumor protein p53 (TP53), three and eight variants were identified, respectively. In conclusion, both inhibitors demonstrated in vitro efficacy on the PDAC cell lines. However, aberrations and expression of inhibitor target genes did not appear to affect the efficacy of the corresponding inhibitors. In addition, specific aberrations in TP53 and KRAS affected the efficacy of both inhibitors. [ABSTRACT FROM AUTHOR]

[Objectives] The crp gene in Vibrio alginolyticus was studied. [Methods]The crp gene of V. alginolyticus HY9901 was cloned and analyzed by bioinformatics. [Results] The ORF of the crp gene is 633 bp long and the predicted amino acid sequence encompasses 210 amino acid residues. The physicochemical property analysis indicated that the chemical formula of CRP is C1051 H1704N290 O308S10 with a molecular weight of 23. 651 4 kDa, and its theoretical pl is 7. 74. Besides, the protein is stable and hydrophilic. The protein had three protein kinase C phosphorylation site, three casein kinase II phosphorylation site, one N-terminal myristoylation site. The BLAST analysis on the sequence revealed high homology with CRPs in other Vibrio species, and particularly the sequence shares about a homology of 99. 52 % with the CRP in V. parahaemolyticus. The SWlSS-MODEL software simulated the subunit tertiary structural model of the CRP, and the similarity with template 3hif. 1. A was 95. 71 % . [Conclusions ] This study provides a reference for the search for efficient protective antigens against vibrosis. [ABSTRACT FROM AUTHOR]

In order to further indicate whether circRNA Fbxl5 could modulate cardiomyocyte apoptosis via regulating the miR-146a-MED1 axis, we manipulated the expression levels of circRNA Fbxl5 and miR-146a in NMVMs through siRNA-based knockdown and miR-146a inhibitor, respectively. Keywords: myocardial ischemia reperfusion injury; circular RNA; microRNA-146a; cardiomyocyte apoptosis EN myocardial ischemia reperfusion injury circular RNA microRNA-146a cardiomyocyte apoptosis 2539 2550 12 05/17/22 20220401 NES 220401 Introduction Acute myocardial infarction is the leading cause of death worldwide. Our previous study demonstrated that miR-146a was protective against myocardial IRI and attenuated cardiomyocyte apoptosis via directly targeting MED1, which might mediate the apoptosis through regulating p53 signaling pathway.[13] In the present study, we identified circRNA Fbxl5, one of the abundantly expressed circRNAs in the heart, which could function as miR-146a sponge. [Extracted from the article]

Lung cancer remains a leading cause of global cancer‑related deaths, therefore the identification of prognostic factors for lung cancer is critical. Casein kinase 2 alpha (CK2α) is one of the driver kinases in various cancers, and it was previously demonstrated that CK2α localization was associated with a poor prognosis in invasive breast cancer. In the present study, the importance of CK2α in the nucleolus was explored as a potential prognostic marker for surgically resected early‑stage lung adenocarcinoma. The present study included 118 patients who underwent pulmonary lobectomy between 2014 and 2018 in Fukushima Medical University Hospital (Fukushima, Japan), and in whom CK2α localization in tumor samples was assessed by immunohistochemistry. Patient and tumor characteristics, including pathological stage, histological type and histological grade, were analyzed. Recurrence‑free survival (RFS) and overall survival were evaluated in relation to nucleolar CK2α staining. CK2α staining in the nucleoli was observed in 50.8% of lung adenocarcinoma tumors. Positive nucleolar CK2α staining was independent of pathological stage, histological type and histological grade. Patients with positive nucleolar CK2α staining exhibited significantly worse RFS compared with patients with negative staining. Multivariate analysis identified nucleolar CK2α staining and lymph node metastasis as independent poor prognostic factors. The results of the present study suggested that nucleolar CK2α staining is a novel and independent prognostic factor in surgically resected early‑stage lung adenocarcinoma. These findings indicated the potential of nucleolar CK2α as a predictive biomarker for future recurrence, and a guide to treatment decisions. Further research is required, particularly in understanding the molecular mechanisms linking nucleolar CK2α to recurrence.

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

Specific de novo mutations in the CSNK2A1 gene, which encodes CK2α, the catalytic subunit of protein kinase CK2, are considered as causative for the Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS is a rare congenital disease with a high phenotypic diversity ranging from neurodevelopmental disabilities to multi-systemic problems and characteristic facial features. A frequent OCNDS mutation is the exchange of Lys198 to Arg at the center of CK2α′s P+1 loop, a key element of substrate recognition. According to preliminary data recently made available, this mutation causes a significant shift of the substrate specificity of the enzyme. We expressed the CK2αLys198Arg recombinantly and characterized it biophysically and structurally. Using isothermal titration calorimetry (ITC), fluorescence quenching and differential scanning fluorimetry (Thermofluor), we found that the mutation does not affect the interaction with CK2β, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2αLys198Arg crystal structure and its comparison with wild-type structures revealed a significant shift of the anion binding site harboured by the P+1 loop. This observation supports the notion that the Lys198Arg mutation causes an alteration of substrate specificity which we underpinned here with enzymological data.

Glioblastoma multiforme (GBM) remains the deadliest brain tumor in adults. GBM tumors are also notorious for drug and radiation resistance. To inhibit GBMs more effectively, polymalic acid-based blood-brain barrier crossing nanobioconjugates were synthesized that are delivered to the cytoplasm of cancer cells and specifically inhibit the master regulator serine/threonine protein kinase CK2 and the wild-type/mutated epidermal growth factor receptor (EGFR/EGFRvIII), which are overexpressed in gliomas according to The Cancer Genome Atlas (TCGA) GBM database. Two xenogeneic mouse models bearing intracranial human GBMs from cell lines LN229 and U87MG that expressed both CK2 and EGFR at different levels were used. Simultaneous knockdown of CK2α and EGFR/EGFRvIII suppressed their downstream prosurvival signaling. Treatment also markedly reduced the expression of programmed death-ligand 1 (PD-L1), a negative regulator of cytotoxic lymphocytes. Downregulation of CK2 and EGFR also caused deactivation of heat shock protein 90 (Hsp90) co-chaperone Cdc37, which may suppress the activity of key cellular kinases. Inhibition of either target was associated with downregulation of the other target as well, which may underlie the increased efficacy of the dual nanobioconjugate that is directed against both CK2 and EGFR. Importantly, the single nanodrugs, and especially the dual nanodrug, markedly suppressed the expression of the cancer stem cell markers c-Myc, CD133, and nestin, which could contribute to the efficacy of the treatments. In both tumor models, the nanobioconjugates significantly increased (up to 2-fold) animal survival compared with the PBS-treated control group. The versatile nanobioconjugates developed in this study, with the abilities of anti-cancer drug delivery across biobarriers and the inhibition of key tumor regulators, offer a promising nanotherapeutic approach to treat GBMs, and to potentially prevent drug resistance and retard the recurrence of brain tumors.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform–specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform–specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders. [ABSTRACT FROM AUTHOR]

The Poirier–Bienvenu neurodevelopmental syndrome is an autosomal dominant disorder characterized by intellectual disability and epilepsy. The disease is caused by mutations in the CSNK2B gene, which encodes the beta subunit of casein kinase II, and it has important roles in neuron development and synaptic transmission. In this study, five Chinese patients were diagnosed with Poirier–Bienvenu neurodevelopmental syndrome caused by CSNK2B mutations by whole exome sequencing. We detected four different de novo variants of the CSNK2B gene in these five unrelated Chinese patients: two novel mutations, namely, c.100delT (p.Phe34fs*16) and c.158_159insA (p.Asp55fs*4), and two recurrent mutations, namely, c.1A>G (p.Met1?) and c.332 G >C (p.R111P). All five patients showed mild-to-profound intellectual disabilities/or learning disabilities and developmental delays, with or without seizures. Although intellectual disability/developmental delay and epilepsy are the most common manifestations of CSNK2B deficiency, the clinical phenotypes of probands are highly variable, and there is no significant correlation between genotype and phenotype. An abnormal stature may be another common manifestation of CSNK2B deficiency. Here, we report the effects of growth hormone (GH) therapy on the patients' linear height. In conclusion, Poirier–Bienvenu neurodevelopmental syndrome is a highly heterogeneous disease caused by mutations in the CSNK2B gene. The phenotype was highly variable, and no significant correlation of genotype and phenotype was found. Patients with short-stature and CSNK2B deficiency may benefit from GH therapy. The identification and characterization of these novel variants will expand the genotypic and phenotypic spectrum of Poirier–Bienvenu neurodevelopmental syndrome. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder that exhibits pathological changes in both tau and synaptic function. AD patients display increases in hyperphosphorylated tau and synaptic activity. Previous studies have individually identified the role of NR2B subunit-containing NMDA receptors in AD related synaptic dysfunction and aggregated tau without reconciling the conflicting differences and implications of NR2B expression. Inhibition of extrasynaptically located NR2B mitigates tau pathology in AD models, whereas the inhibition of synaptic NR2B replicates tau-associated hyperactivity. This suggests that a simultaneous increase in extrasynaptic NR2B and decrease in synaptic NR2B may be responsible for tau pathology and synaptic dysfunction, respectively. The synaptic location of NR2B is regulated by casein kinase 2 (CK2), which is highly expressed in AD patients. Here, we used patient brains diagnosed with AD, corticobasal degeneration, progressive supranuclear palsy or Pick's disease to characterize CK2 expression across these diverse tauopathies. Human derived material was also utilized in conjunction with cultured hippocampal neurons in order to investigate AD-induced changes in NR2B location. We further assessed the therapeutic effect of CK2 inhibition on NR2B synaptic distribution and tau pathology. We found that aberrant expression of CK2, and synaptically translocated NR2B, is unique to AD patients compared to other tauopathies. Increased CK2 was also observed in AD-tau treated neurons in addition to the mislocalization of NR2B receptors. Tau burden was alleviated in vitro by correcting synaptic:extrasynaptic NR2B function. Restoring NR2B physiological expression patterns with CK2 inhibition and inhibiting the function of excessive extrasynaptic NR2B with Memantine both mitigated tau accumulation in vitro. However, the combined pharmacological treatment promoted the aggregation of tau. Our data suggests that the synaptic:extrasynaptic balance of NR2B function regulates AD-tau pathogenesis, and that the inhibition of CK2, and concomitant prevention of NR2B mislocalization, may be a useful therapeutic tool for AD patients. [ABSTRACT FROM AUTHOR]

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections. [ABSTRACT FROM AUTHOR]

Most transcription factors possess at least one long intrinsically disordered transactivation domain that binds to a variety of coactivators and corepressors and plays a key role in modulating the transcriptional activity. Despite the crucial importance of these domains, the structural and functional basis of transactivation remains poorly understood. Here, we focused on activating transcription factor 4 (ATF4)/cAMP response element-binding protein-2, an essential transcription factor for cellular stress adaptation. Bioinformatic sequence analysis of the ATF4 transactivation domain sequence revealed that the first 125 amino acids have noticeably less propensity for structural disorder than the rest of the domain. Using solution nuclear magnetic resonance spectroscopy complemented by a range of biophysical methods, we found that the isolated transactivation domain is predominantly yet not fully disordered in solution. We also observed that a short motif at the N-terminus of the transactivation domain has a high helical propensity. Importantly, we found that the N-terminal region of the transactivation domain is involved in transient long-range interactions with the basic-leucine zipper domain involved in DNA binding. Finally, in vitro phosphorylation assays with the casein kinase 2 show that the presence of the basic-leucine zipper domain is required for phosphorylation of the transactivation domain. This study uncovers the intricate coupling existing between the transactivation and basic-leucine zipper domains of ATF4, highlighting its potential regulatory significance. [ABSTRACT FROM AUTHOR]

(J) ChIP-qPCR assay in 293T cells that ectopically overexpress Flag-tagged HMGA1-WT or HMGA1-S3A, and with siSP1 in these Flag-HMGA1 expressing cells. (B) Western blotting for pan-phospho-serine levels in 293T cells transduced with MOCK, Flag-tagged HMGA1-WT, HMGA1-S99A, HMGA1-S102A, HMGA1-S103A or HMGA1-S3A. ChIP analysis using anti-flag antibodies or control normal mouse IgG was performed in four groups: HMGA1-WT/siCtrl, HMGA1-WT/siSP1, HMGA1-S3A/siCtrl and HMGA1-S3A/siSP1. [Extracted from the article]

Extensive inflammation causes epithelial cell hyperplasia in the airways and Bcl‐2‐interacting killer (Bik) reduces epithelial cell and mucous cell hyperplasia without affecting resting cells to restore homeostasis. These observations suggest that Bik induces apoptosis in a cell cycle‐specific manner, but the mechanisms are not understood. Mice were exposed to an allergen for 3, 14, or 30 days and Bik expression was induced in airway epithelia of transgenic mice. Bik reduced epithelial and mucous cell hyperplasia when mice were exposed to an allergen for 3 or 14 days, but not when exposure lasted for 30 days, and Ki67‐positivity was reduced. In culture, Bik expression killed proliferating cells but not quiescent cells. To capture the stage of the cell cycle when Bik induces cell death, airway cells that express fluorescent ubiquitin cell cycle indicators were generated that fluoresce red or green during the G0/G1 and S/G2/M phases of the cells cycle, respectively. Regardless of the cell cycle stage, Bik expression eliminated green‐fluorescent cells. Also, Bik, when tagged with a blue‐fluorescent protein, was only detected in green cells. Bik phosphorylation mutants at threonine 33 or serine 35 demonstrated that phosphorylation activated Bik to induce death even in quiescent cells. Immunoprecipitation and proteomic approaches identified casein kinase IIα to be responsible for phosphorylating and activating Bik to kill cells in S/G2/M. As casein kinase 2 alpha (CKIIα) is expressed only during the G2/M phase, we conclude that Bik activation in airway epithelial cells selectively targets hyperplastic epithelial cells, while leaving resting airway cells unaffected. [ABSTRACT FROM AUTHOR]

• Mn activates YY1 through posttranslational phosphorylation in astrocytes. • Mn phosphorylates YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2). • Mn reduces EAAT2 expression via YY1 phosphorylation and subsequent YY1 nuclear translocation. Impairment of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with neurological disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and manganism, a neurological disorder caused by overexposure to manganese (Mn) which shares the features of sporadic PD. Mechanisms of Mn-induced neurotoxicity include dysregulation of EAAT2 following activation of the transcription factor Yin Yang 1 (YY1) by transcriptional upregulation, but the posttranslational mechanisms by which YY1 is activated to repress EAAT2 remain to be elucidated. In the present study, we tested if Mn activates YY1 through posttranslational phosphorylation in cultured H4 human astrocytes, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2), leading to YY1 nuclear translocation, YY1/HDAC interactions, binding to the EAAT2 promoter, and consequent decreases in EAAT2 promoter activity and mRNA/protein levels. Although further studies are warranted to fully elucidate the mechanisms of Mn-induced YY1 phosphorylation and resultant EAAT2 impairment, our findings indicate that serine phosphorylation of YY1 via AurkB and CK2 is critical, at least in part, to its activation and transcriptional repression of EAAT2. [ABSTRACT FROM AUTHOR]

Highly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small‐molecule inhibitor has reached clinical trials—CX‐4945. In this study, we present the indeno[1,2‐b]indole derivative 5‐isopropyl‐4‐methoxy‐7‐methyl‐5,6,7,8‐tetrahydroindeno[1,2‐b]indole‐9,10‐dione (5a‐2) that decreased the intracellular CK2 activity in A431, A549, and LNCaP tumor cell lines analogous to CX‐4945 (> 75% inhibition at 20 µm) and similarly blocked CK2‐specific Akt phosphorylation in LNCaP cells. Cellular uptake analysis demonstrated higher intracellular concentrations of 5a‐2 (408.3 nm) compared with CX‐4945 (119.3 nm). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC50 = 25 nm (5a‐2) and 3.7 nm (CX‐4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live‐cell imaging revealed notable differences. Whereas CX‐4945 showed a stronger pro‐apoptotic effect on tumor cells, 5a‐2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX‐4945 was localized in the nuclear fraction, whereas 71% of 5a‐2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provides a possible explanation for the different cellular effects. Our study indicates that investigating CK2 inhibition‐mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.

Protein kinase CK2 is a pleiotropic protein kinase, which phosphorylates a number of cellular and viral proteins. Thereby, this kinase is implicated in the regulation of cellular signaling, controlling of cell proliferation, apoptosis, angiogenesis, immune response, migration and invasion. In general, viruses use host signaling mechanisms for the replication of their genome as well as for cell transformation leading to cancer. Therefore, it is not surprising that CK2 also plays a role in controlling viral infection and the generation of cancer cells. Epstein–Barr virus (EBV) lytically infects epithelial cells of the oropharynx and B cells. These latently infected B cells subsequently become resting memory B cells when passing the germinal center. Importantly, EBV is responsible for the generation of tumors such as Burkitt’s lymphoma. EBV was one of the first human viruses, which was connected to CK2 in the early nineties of the last century. The present review shows that protein kinase CK2 phosphorylates EBV encoded proteins as well as cellular proteins, which are implicated in the lytic and persistent infection and in EBV-induced neoplastic transformation. EBV-encoded and CK2-phosphorylated proteins together with CK2-phosphorylated cellular signaling proteins have the potential to provide efficient virus replication and cell transformation. Since there are powerful inhibitors known for CK2 kinase activity, CK2 might become an attractive target for the inhibition of EBV replication and cell transformation.

Aims/hypothesis: Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells., Methods: We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition., Results: We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans., Conclusions/interpretation: These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs., (© 2024. The Author(s).)

The WNT-β-catenin signaling pathway has a major role in regulating cell proliferation and differentiation. Aberrant activation of the pathway contributes to various human cancer types. Because casein kinase CK1α-initiated phosphorylation of β-catenin is a key first step to restrain WNT signaling, effective restoration of CK1α activity represents an innovative strategy to combat WNT-driven cancer. A recent study in JBC reveals the anthelmintic pyrvinium directly binds to CK1α as an activator and also stabilizes CK1α protein, doubling against WNT-driven cancer activity. [ABSTRACT FROM AUTHOR]

An attempted synthesis of 5-aryl-6,7-dihydrodibenzo[b,j][1,10]phenanthroline derivatives from 2,3-dihydroacridin-4(1H)-ones with 2-aminocarboxylic acid derivatives in presence of phosphorus oxychloride at 130˚C yielded a novel class of quin[1,2-b]acridine derivatives. The newly synthesized compounds showed a better cytotoxic activity against HeLa and MCF-7 cell lines during the structure–activity relationship (SAR) studies. To discover with the interactions of these molecules, we carried out molecular docking studies using the human protein kinase CK2 inhibitors. The molecular interaction results provided a number of elegant information for the outlook design of more potent inhibitors. [ABSTRACT FROM AUTHOR]