Subunit Of CK2 Research Articles

Missense mutation in the activation segment of the kinase CK2 models Okur-Chung neurodevelopmental disorder and alters the hippocampal glutamatergic synapse.

Exome sequencing has enabled the identification of causative genes of monogenic forms of autism, amongst them, in 2016, CSNK2A1, the gene encoding the catalytic subunit of the kinase CK2, linking this kinase to Okur-Chung Neurodevelopmental Syndrome (OCNDS), a newly described neurodevelopmental condition with many symptoms resembling those of autism spectrum disorder. Thus far, no preclinical model of this condition exists. Here we describe a knock-in mouse model that harbors the K198R mutation in the activation segment of the α subunit ofCK2. This region is a mutational hotspot, representing one-third of patients. These mice exhibit behavioral phenotypes that mirror patient symptoms. Homozygous knock-in mice die mid-gestation while heterozygous knock-in mice are born at half of the expected mendelian ratio and are smaller in weight and size than wildtype littermates. Heterozygous knock-in mice showed alterations in cognition and memory-assessing paradigms, enhanced stereotypies, altered circadian activity patterns, and nesting behavior. Phosphoproteome analysis from brain tissue revealed alterations in the phosphorylation status of major pre- and postsynaptic proteins of heterozygous knock-in mice. In congruence, we detect reduced synaptic maturation in hippocampal neurons and attenuated long-term potentiation in the hippocampus of knock-in mice. Taken together, heterozygous knock-in mice (CK2αK198R/+) exhibit significant face validity, presenting ASD-relevant phenotypes, synaptic deficits, and alterations in synaptic plasticity, all of which strongly validate this line as a mouse model of OCNDS.

Distinctive expression and cellular localisation of zinc homeostasis-related proteins in breast and prostate cancer cells

BackgroundZinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. MethodsQuantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. ResultsIn response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2β was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. ConclusionThe findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

Silmitasertib (CX-4945) Disrupts ERα/HSP90 Interaction and Drives Proteolysis through the Disruption of CK2β Function in Breast Cancer Cells.

Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa.

Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapy.

In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (β) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies. Biochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2β compared to normal B lymphocytes. Mechanistically, CK2β was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin. Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2β has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.

Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2

Histidine residues contribute to numerous molecular interactions, owing to their structure with the ionizable aromatic side chain with pKa close to the physiological pH. Herein, we studied how the two histidine residues, His115 and His160 of the catalytic subunit of human protein kinase CK2, affect the binding of the halogenated heterocyclic ligands at the ATP-binding site. Thermodynamic studies on the interaction between five variants of hCK2α (WT protein and four histidine mutants) and three ionizable bromo-benzotriazoles and their conditionally non-ionizable benzimidazole counterparts were performed with nanoDSF, MST, and ITC. The results allowed us to identify the contribution of interactions involving the particular histidine residues to ligand binding. We showed that despite the well-documented hydrogen bonding/salt bridge formation dragging the anionic ligands towards Lys68, the protonated His160 also contributes to the binding of such ligands by long-range electrostatic interactions. Simultaneously, His 115 indirectly affects ligand binding, placing the hinge region in open/closed conformations.

Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

CK2β Regulates Hematopoietic Stem Cell Biology and Erythropoiesis.

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the β regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2β knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2β were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.

Discovery and Exploration of Protein Kinase CK2 Binding Sites Using CK2α′Cys336Ser as an Exquisite Crystallographic Tool

The structural knowledge about protein kinase CK2 is dominated by crystal structures of human CK2α, the catalytic subunit of human CK2, and the product of the CSNK2A1 gene. In contrast, far fewer structures of CK2α′, its paralogous isoform and the product of the CSNK2A2 gene, have been published. However, according to a PDB survey, CK2α′ is the superior alternative for crystallographic studies because of the inherent potential of the single mutant CK2α′Cys336Ser to provide crystal structures with atomic resolution. In particular, a triclinic crystal form of CK2α′Cys336Ser is a robust tool to determine high-quality enzyme-ligand complex structures via soaking. In this work, further high-resolution CK2α′Cys336Ser structures in complex with selected ligands emphasizing this trend are described. In one of these structures, the “N-terminal segment site”, a small-molecule binding region never found in any eukaryotic protein kinase and holding the potential for the development of highly selective substrate-competitive CK2 inhibitors, was discovered. In order to also address the binding site for the non-catalytic subunit CK2β, which is inaccessible in these triclinic CK2α′Cys336Ser crystals for small molecules, a reliable path to a promising monoclinic crystal form of CK2α′Cys336Ser is presented. In summary, the quality of CK2α′Cys336Ser as an exquisite crystallographic tool is solidified.

Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image β-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to β-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

Characterization of RAGE and CK2 Expressions in Human Fetal Membranes.

At the feto-maternal interface, fetal membranes (FM) play a crucial role throughout pregnancy. FM rupture at term implicates different sterile inflammation mechanisms including pathways activated by the transmembrane glycoprotein receptor for advanced glycation end-products (RAGE) belonging to the immunoglobulin superfamily. As the protein kinase CK2 is also implicated in the inflammation process, we aimed to characterize the expressions of RAGE and the protein kinase CK2 as a candidate regulator of RAGE expression. The amnion and choriodecidua were collected from FM explants and/or primary amniotic epithelial cells throughout pregnancy and at term in spontaneous labor (TIL) or term without labor (TNL). The mRNA and protein expressions of RAGE and the CK2α, CK2α', and CK2β subunits were investigated using reverse transcription quantitative polymerase chain reaction and Western blot assays. Their cellular localizations were determined with microscopic analyses, and the CK2 activity level was measured. RAGE and the CK2α, CK2α', and CK2β subunits were expressed in both FM layers throughout pregnancy. At term, RAGE was overexpressed in the amnion from the TNL samples, whereas the CK2 subunits were expressed at the same level in the different groups (amnion/choriodecidua/amniocytes, TIL/TNL), without modification of the CK2 activity level and immunolocalization. This work paves the way for future experiments regarding the regulation of RAGE expression by CK2 phosphorylation.

CK2β-regulated signaling controls B cell differentiation and function.

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the β regulatory subunit of CK2. CK2βKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2βKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2βKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2β have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2βKO mice suggesting the importance of the β subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

Kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

Protein phosphorylation plays a critical role in cell cycle progression. In Caenorhabditis elegans , Casein Kinase II (CK2) negatively regulates centrosome assembly, and Protein Phosphatase 2A (PP2A) SUR-6 /B55 acts as a positive regulator of centrosome duplication, suggesting CK2 and PP2A SUR-6 /B55 play opposing roles in centrosome assembly. Here, we examined the genetic interaction between kin-3 , encoding the catalytic subunit of CK2, and sur-6 , encoding the PP2Aregulatory subunit SUR-6 /B55, in Caenorhabditis elegans embryos. Our results show that kin-3 (RNAi) partially restores normal centrosome duplication and embryonic viability to hypomorphic sur-6 mutants, suggesting that kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Transcriptome Analysis of Protein Kinase MoCK2, which Affects Acetyl-CoA Metabolism and Import of CK2-Interacting Mitochondrial Proteins into Mitochondria in the Rice Blast Fungus Magnaporthe oryzae.

The rice pathogen Magnaporthe oryzae causes severe losses to rice production. Previous studies have shown that the protein kinase MoCK2 is essential for pathogenesis, and this ubiquitous eukaryotic protein kinase might affect several processes in the fungus that are needed for infection. To better understand which cellular processes are affected by MoCK2 activity, we performed a detailed transcriptome sequencing analysis of deletions of the MoCK2 b1 and b2 components in relation to the background strain Ku80 and connected this analysis with the abundance of substrates for proteins in a previous pulldown of the essential CKa subunit of CK2 to estimate the effects on proteins directly interacting with CK2. The results showed that MoCK2 seriously affected carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, and the related transporters and reduced acetyl-CoA production. CK2 phosphorylation can affect the folding of proteins and especially the effective formation of protein complexes by intrinsically disordered or mitochondrial import by destabilizing soluble alpha helices. The upregulated genes found in the pulldown of the b1 and b2 mutants indicate that proteins directly interacting with CK2 are compensatorily upregulated depending on their pulldown. A similar correlation was found for mitochondrial proteins. Taken together, the classes of proteins and the changes in regulation in the b1 and b2 mutants suggest that CK2 has a central role in mitochondrial metabolism, secondary metabolism, and reactive oxygen species (ROS) resistance, in addition to its previously suggested role in the formation of new ribosomes, all of which are processes central to efficient nonself responses as innate immunity. IMPORTANCE The protein kinase CK2 is highly expressed and essential for plants, animals, and fungi, affecting fatty acid-related metabolism. In addition, it directly affects the import of essential mitochondrial proteins into mitochondria. These effects mean that CK2 is essential for lipid metabolism and mitochondrial function and, as shown previously, is crucial for making new translation machinery proteins. Taken together, our new results combined with previously reported results indicate that CK2 is an essential protein necessary for the capacities to launch efficient innate immunity responses and withstand the negative effects of such responses necessary for general resistance against invading bacteria and viruses as well as to interact with plants, withstand plant immunity responses, and kill plant cells.

The Catalytic Subunit of Schizosaccharomyces pombe CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4).

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well.

CK2β Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/αʹ catalytic subunits and two CK2β regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2β compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2β in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2β loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2β as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Comparing Two Neurodevelopmental Disorders Linked to CK2: Okur-Chung Neurodevelopmental Syndrome and Poirier-Bienvenu Neurodevelopmental Syndrome-Two Sides of the Same Coin?

In recent years, variants in the catalytic and regulatory subunits of the kinase CK2 have been found to underlie two different, yet symptomatically overlapping neurodevelopmental disorders, termed Okur-Chung neurodevelopmental syndrome (OCNDS) and Poirier-Bienvenu neurodevelopmental syndrome (POBINDS). Both conditions are predominantly caused by de novo missense or nonsense mono-allelic variants. They are characterized by a generalized developmental delay, intellectual disability, behavioral problems (hyperactivity, repetitive movements and social interaction deficits), hypotonia, motricity and verbalization deficits. One of the main features of POBINDS is epilepsies, which are present with much lower prevalence in patients with OCNDS. While a role for CK2 in brain functioning and development is well acknowledged, these findings for the first time clearly link CK2 to defined brain disorders. Our review will bring together patient data for both syndromes, aiming to link symptoms with genotypes, and to rationalize the symptoms through known cellular functions of CK2 that have been identified in preclinical and biochemical contexts. We will also compare the symptomatology and elaborate the specificities that distinguish the two syndromes.

The Okur-Chung Neurodevelopmental Syndrome Mutation CK2K198R Leads to a Rewiring of Kinase Specificity.

Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of protein kinase CK2. The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a complete loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.

Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2αLys198Arg.

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.

Expanding Phenotype of Poirier-Bienvenu Syndrome: New Evidence from an Italian Multicentrical Cohort of Patients.

Background: Poirier–Bienvenu Neurodevelopmental Syndrome (POBINDS) is a rare disease linked to mutations of the CSNK2B gene, which encodes for a subunit of caseinkinase CK2 involved in neuronal growth and synaptic transmission. Its main features include early-onset epilepsy and intellectual disability. Despite the lack of cases described, it appears that POBINDS could manifest with a wide range of phenotypes, possibly related to the different mutations of CSNK2B. Methods: Our multicentric, retrospective study recruited nine patients with POBINDS, detected using next-generation sequencing panels and whole-exome sequencing. Clinical, laboratory, and neuroimaging data were reported for each patient in order to assess the severity of phenotype, and eventually, a correlation with the type of CSNK2B mutation. Results: We reported nine unrelated patients with heterozygous de novo mutations of the CSNK2B gene. All cases presented epilepsy, and eight patients were associated with a different degree of intellectual disability. Other features detected included endocrinological and vascular abnormalities and dysmorphisms. Genetic analysis revealed six new variants of CSNK2B that have not been reported previously. Conclusion: Although it was not possible to assess a genotype–phenotype correlation in our patients, our research further expands the phenotype spectrum of POBINDS patients, identifying new mutations occurring in the CSNK2B gene.