RESUMO
Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.
Assuntos
Neoplasias Encefálicas , Carcinogênese , Proteínas de Ciclo Celular , Glioblastoma , Fatores de Troca do Nucleotídeo Guanina , Mitose/efeitos da radiação , Proteínas de Neoplasias , Proteínas Nucleares , Proteína-Arginina N-Metiltransferases , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/efeitos da radiação , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Mitose/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.
Assuntos
Caseína Quinase II , Cromatina , Proteína HMGA2 , Células-Tronco Hematopoéticas , Camundongos Knockout , Proteína HMGA2/metabolismo , Proteína HMGA2/genética , Animais , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Humanos , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Cromatina/metabolismo , Cromatina/genética , Fator de Necrose Tumoral alfa/metabolismo , Hematopoese , Estresse Fisiológico , Fluoruracila/farmacologia , Regeneração , Fosforilação , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.
Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Humanos , Animais , Camundongos , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Transcrição Gênica , Fosforilação , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Camundongos Knockout , Dano ao DNA , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Cromatina/metabolismo , Ubiquitinação , Reparo por ExcisãoRESUMO
DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development.
Assuntos
Difosfato de Adenosina/metabolismo , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Replicação do DNA , Fosfoglicerato Quinase/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Caseína Quinase II/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , Feminino , Xenoenxertos , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfoglicerato Quinase/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Origem de ReplicaçãoRESUMO
Homologous recombination (HR) plays a key role in maintaining genomic stability, and the efficiency of the HR system is closely associated with tumor response to chemotherapy. Our previous work reported that CK2 kinase phosphorylates HIV Tat-specific factor 1 (HTATSF1) Ser748 to facilitate HTATSF1 interaction with TOPBP1, which in turn, promotes RAD51 recruitment and HR repair. However, the clinical implication of the CK2-HTATSF1-TOPBP1 pathway in tumorigenesis and chemotherapeutic response remains to be elucidated. Here, we report that the CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and renders breast tumors less responsive to chemotherapy. In contrast, deletion mutations of each gene in this axis, which also occur in breast and lung tumor samples, predict higher HR deficiency scores, and tumor cells bearing a loss-of-function mutation of HTATSF1 are vulnerable to poly(ADP-ribose) polymerase inhibitors or platinum drugs. Taken together, our study suggests that the integrity of the CK2-HTATSF1-TOPBP1 axis is closely linked to tumorigenesis and serves as an indicator of tumor HR status and modulates chemotherapy response.
Assuntos
Proteínas de Transporte , Caseína Quinase II , Proteínas de Ligação a DNA , Transdução de Sinais , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Transdução de Sinais/efeitos dos fármacos , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Animais , Feminino , Camundongos , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologiaRESUMO
Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.
Assuntos
Caseína Quinase II , Proteínas do Olho , Animais , Bovinos , Camundongos , Humanos , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Células HEK293 , Proteínas do Olho/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Mamíferos/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismoRESUMO
So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.
Assuntos
Caseína Quinase II , Células Endoteliais , Humanos , Transdução Genética , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Terapia Genética , Transgenes , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genéticaRESUMO
Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.
Assuntos
Caseína Quinase II , DNA , Fator Regulador 1 de Interferon , Viroses , Cromatina , DNA/genética , Fator Regulador 1 de Interferon/genética , Transdução de Sinais/genética , Humanos , Caseína Quinase II/genética , Imunidade Inata , Viroses/genética , Viroses/imunologiaRESUMO
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.
Assuntos
Caseína Quinase II , Células Secretoras de Glucagon , Glucagon , Proteínas de Homeodomínio , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Animais , Glucagon/metabolismo , Camundongos , Humanos , Células Secretoras de Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Transativadores/metabolismo , Transativadores/genética , Masculino , Linhagem Celular , Insulina/metabolismoRESUMO
Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.
Assuntos
Alumínio/toxicidade , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Caseína Quinase II/metabolismo , Fosfatos/metabolismo , Alumínio/farmacocinética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatos/farmacologia , Fosforilação , Células Vegetais/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The autosomal dominant Okur-Chung neurodevelopmental syndrome (OCNDS: OMIM #617062) is a rare neurodevelopmental disorder first described in 2016. Features include developmental delay (DD), intellectual disability (ID), behavioral problems, hypotonia, language deficits, congenital heart abnormalities, and non-specific dysmorphic facial features. OCNDS is caused by heterozygous pathogenic variants in CSNK2A1 (OMIM *115440; NM_177559.3). To date, 160 patients have been diagnosed worldwide. The number will likely increase due to the growing use of exome sequencing (ES) and genome sequencing (GS). Here, we describe a novel OCNDS patient carrying a CSNK2A1 variant (NM_177559.3:c.140G>A; NP_808227.1:p.Arg47Gln). Phenotypically, he presented with DD, ID, generalized hypotonia, speech delay, short stature, microcephaly, and dysmorphic features such as low-set ears, hypertelorism, thin upper lip, and a round face. The patient showed several signs not yet described that may extend the phenotypic spectrum of OCNDS. These include prenatal bilateral clubfeet, exotropia, and peg lateral incisors. However, unlike the majority of descriptions, he did not present sleep disturbance, seizures or gait difficulties. A literature review shows phenotypic heterogeneity for OCNDS, whether these patients have the same variant or not. This case report is an opportunity to refine the phenotype of this syndrome and raise the question of the genotype-phenotype correlation.
Assuntos
Caseína Quinase II , Transtornos do Neurodesenvolvimento , Criança , Humanos , Masculino , Caseína Quinase II/genética , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Sequenciamento do Exoma , Predisposição Genética para Doença , Heterozigoto , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Hipotonia Muscular/genética , Hipotonia Muscular/patologia , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Transtornos do Neurodesenvolvimento/diagnóstico , FenótipoRESUMO
Circadian clocks use temperature compensation to keep accurate time over a range of temperatures, thus allowing reliable timekeeping under diverse environmental conditions. Mehra et al. (2009) and Baker et al. (2009) now show that phosphorylation-regulated protein degradation plays a key role in circadian temperature compensation.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Neurospora crassa/fisiologia , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Neurospora crassa/enzimologia , Fosforilação , TemperaturaRESUMO
Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. We identify casein kinase 2 (CK2) as a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the beta1 and alpha subunits of CK2, respectively. Reducing the dose of these subunits, particularly beta1, significantly alters temperature compensation without altering the enzyme's Q(10). By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles effectively phenocopying CK2 mutants.
Assuntos
Caseína Quinase II/fisiologia , Ritmo Circadiano , Neurospora crassa/enzimologia , Neurospora crassa/fisiologia , Caseína Quinase II/química , Caseína Quinase II/genética , Dosagem de Genes , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , TemperaturaRESUMO
Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor-associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor-associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4-infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.
Assuntos
Autoantígenos/metabolismo , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Autoantígenos/genética , Caseína Quinase II/genética , Proteínas de Ciclo Celular/genética , Quimiocinas/metabolismo , Citocinas , Imunidade Inata , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas Fosfatases/genética , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/genética , Receptor 4 Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
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.
Assuntos
Fator 4 Ativador da Transcrição , Caseína Quinase II , Zíper de Leucina , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Fosforilação , Ativação TranscricionalRESUMO
The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.
Assuntos
Caseína Quinase II , Peptídeos e Proteínas de Sinalização Intracelular/genética , PTEN Fosfo-Hidrolase , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proliferação de Células , Eutérios/metabolismo , Feminino , Camundongos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Placenta/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
As highly expressed in several human cancers, Casein Kinase 2 (CK2) is involved in chemotherapy-induced resistance. As a new potent CK2 inhibitor, DN701 is used to overcome chemoresistance through its synergistic antitumor effect with 5-fluorouracil (5-FU). Translesion DNA synthesis (TLS) has drawn our attention because it is associated with the development of chemo-resistance and tumor recurrence. The in vitro biological properties of 5-FU-resistant colon cancer cells revealed that DN701 combined with 5-FU could overcome chemo-resistance via blocking CK2-mediated aryl hydrocarbon receptor (AhR) and TLS-induced DNA damage repair (DDR). Moreover, pharmacologic and genetic inhibitions of AhR potently reduced TLS-promoted genomic instability. The mechanistic studies showed that combined DN701 with 5-FU was investigated to inhibit CK2 expression level and AhR-TLS-REV1 pathway. Meanwhile, DN701 combined with 5-FU could reduce CK2-AhR-TLS genomic instability, thus leading to superior in vivo antitumor effect. The insights provide a rationale for combining DN701 with 5-FU as a therapeutic strategy for patients with colon cancer.
Assuntos
Caseína Quinase II , Neoplasias do Colo , Humanos , Caseína Quinase II/genética , Caseína Quinase II/farmacologia , Caseína Quinase II/uso terapêutico , Receptores de Hidrocarboneto Arílico/genética , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , DNA , Resistencia a Medicamentos AntineoplásicosRESUMO
BCL-2 family proteins are frequently aberrantly expressed in mantle cell lymphoma (MCL). Recently, the BCL-2-specific inhibitor venetoclax has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In MCL, venetoclax has shown promising efficacy in early clinical trials; however, a significant subset of patients is resistant. By conducting a kinome-centered CRISPR-Cas9 knockout sensitizer screen, we identified casein kinase 2 (CK2) as a major regulator of venetoclax resistance in MCL. Interestingly, CK2 is over-expressed in MCL and high CK2 expression is associated with poor patient survival. Targeting of CK2, either by inducible short hairpin RNA (shRNA)-mediated knockdown of CK2 or by the CK2-inhibitor silmitasertib, did not affect cell viability by itself, but strongly synergized with venetoclax in both MCL cell lines and primary samples, also if combined with ibrutinib. Furthermore, targeting of CK2 reduced MCL-1 levels, which involved impaired MCL-1 translation by inhibition of eIF4F complex assembly, without affecting BCL-2 and BCL-XL expression. Combined, this results in enhanced BCL-2 dependence and, consequently, venetoclax sensitization. In cocultures, targeting of CK2 overcame stroma-mediated venetoclax resistance of MCL cells. Taken together, our findings indicate that targeting of CK2 sensitizes MCL cells to venetoclax through downregulation of MCL-1. These novel insights provide a strong rationale for combining venetoclax with CK2 inhibition as therapeutic strategy for MCL patients.
Assuntos
Antineoplásicos , Linfoma de Célula do Manto , Humanos , Adulto , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Regulação para Baixo , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-bcl-2 , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêuticoRESUMO
Protein kinase CK2 (also known as Casein Kinase 2) is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory CK2ß subunits. CK2 is overexpressed and overactive in B cell acute lymphoblastic leukemia and diffuse large B cell lymphomas, leading to inappropriate activation of the NF-κB, JAK/STAT, and PI3K/AKT/mTOR signaling pathways and tumor growth. However, whether CK2 regulates normal B cell development and differentiation is not known. We generated mice lacking CK2α specifically in B cells (using CD19-driven Cre recombinase). These mice exhibited cell-intrinsic expansion of marginal zone B cells at the expense of transitional B cells, without changes in follicular B cells. Transitional B cells required CK2α to maintain adequate BCR signaling. In the absence of CK2α, reduced BCR signaling and elevated Notch2 signaling activation increased marginal zone B cell differentiation. Our results identify a previously unrecognized function for CK2α in B cell development and differentiation.
Assuntos
Linfócitos B/imunologia , Caseína Quinase II/metabolismo , Células Precursoras de Linfócitos B/imunologia , Animais , Antígenos CD19/metabolismo , Caseína Quinase II/genética , Diferenciação Celular , Células Cultivadas , Integrases/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year.