RESUMO
Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the ß-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.
Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Deleção de Sequência , Sequência de Aminoácidos , Animais , Caseína Quinase II/deficiência , Linhagem Celular , Sobrevivência Celular , Camundongos Knockout , Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Estabilidade Proteica , Especificidade por SubstratoRESUMO
OBJECTIVES: In humans, non-obstructive azoospermia (NOA) is a major cause of male infertility. However, the aetiology of NOA is largely unknown. Previous studies reported that protein CK2ß was abundantly and broadly expressed in spermatogenic cells. Here, we investigate whether protein CK2ß participates in spermatogenesis. MATERIALS AND METHODS: In this study, we separated spermatogenic cells using STA-PUT velocity sedimentation, analysed the expression pattern of protein CK2ß by immunoblotting, specifically deleted Ck2ß gene in early-stage spermatogenic cells by crossing Ck2ßfl mice with Stra8-Cre+ mice and validated the knockout efficiency by quantitative RT-PCR and immunoblotting. The phenotypes of Ck2ßfl/Δ ;SCre+ mice were studied by immunohistochemistry and immunofluorescence. The molecular mechanisms of male germ cell development arrest were elucidated by immunoblotting and TUNEL assay. RESULTS: Ablation of Ck2ß gene triggered excessive germ cell apoptosis, germ cell development arrest, azoospermia and male infertility. Inactivation of Ck2ß gene caused distinctly reduced expression of Ck2α' gene and CK2α' protein. CONCLUSIONS: Ck2ß is a vital gene for germ cell survival and male fertility in mice.
Assuntos
Apoptose/genética , Azoospermia , Caseína Quinase II/deficiência , Células Germinativas , Animais , Azoospermia/enzimologia , Azoospermia/genética , Azoospermia/patologia , Caseína Quinase II/metabolismo , Deleção de Genes , Células Germinativas/enzimologia , Células Germinativas/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2ß in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2ß deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2ß-deficient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our findings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF.
Assuntos
Caseína Quinase II/genética , Atresia Folicular/genética , Infertilidade Feminina/genética , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Insuficiência Ovariana Primária/genética , Animais , Caseína Quinase II/deficiência , Sobrevivência Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Feminino , Atresia Folicular/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Camundongos , Camundongos Knockout , Oócitos/patologia , Folículo Ovariano/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Casein kinase II subunit alpha (CK2α) is highly expressed in many malignant tumor tissues, including lymphomas and leukemia. To investigate the role of CK2α in cell proliferation and apoptosis of malignant lymphomas and leukemia, 2 lymphoma cell lines and one leukemia cell line were infected with CK2α shRNA lentivirus or negative control shRNA lentivirus, and stably infected cell lines were established. Real-time PCR and Western blot results showed that the mRNA and protein levels of CK2α were significantly reduced in CK2α knockdown cells. The tetrazolium-based colorimetric (MTT) assay found that down-regulation of CK2α inhibited the proliferation of these cells. Flow cytometry analysis showed that inhibition of CK2α induced cell cycle arrest and apoptosis of lymphoma and leukemia cells. In accordance with these, down-regulation of CK2α also reduced the protein levels of proliferating cell nuclear antigen (PCNA), cyclinD1, and bcl-2, and increased the protein expression of bax, cleaved caspase-3, cleaved caspase-9, and cleaved poly(ADP ribose) polymerase (PARP). Moreover, knockdown of CK2α impeded the growth of xenograft tumors in vivo. In summary, our study revealed that CK2α may contribute to the development of malignant lymphoma and leukemia, and serve as the therapeutic target of these malignant tumors.
Assuntos
Apoptose , Regulação para Baixo , Lentivirus/metabolismo , Leucemia/patologia , Linfoma/patologia , Caseína Quinase II/deficiência , Caseína Quinase II/metabolismo , Proliferação de Células , Humanos , Lentivirus/genética , Leucemia/enzimologia , Linfoma/enzimologiaRESUMO
We have previously shown that casein kinase 2 (CK2) negatively regulates dopamine D1 and adenosine A2A receptor signaling in the striatum. Ablation of CK2 in D1 receptor-positive striatal neurons caused enhanced locomotion and exploration at baseline, whereas CK2 ablation in D2 receptor-positive neurons caused increased locomotion after treatment with A2A antagonist, caffeine. Because both, D1 and A2A receptors, play major roles in the cellular responses to l-DOPA in the striatum, these findings prompted us to examine the impact of CK2 ablation on the effects of l-DOPA treatment in the unilateral 6-OHDA lesioned mouse model of Parkinson's disease. We report here that knock-out of CK2 in striatonigral neurons reduces the severity of l-DOPA-induced dyskinesia (LID), a finding that correlates with lowered pERK but unchanged pPKA substrate levels in D1 medium spiny neurons as well as in cholinergic interneurons. In contrast, lack of CK2 in striatopallidal neurons enhances LID and ERK phosphorylation. Coadministration of caffeine with a low dose of l-DOPA reduces dyskinesia in animals with striatopallidal knock-out to wild-type levels, suggesting a dependence on adenosine receptor activity. We also detect reduced Golf levels in the striatonigral but not in the striatopallidal knock-out in response to l-DOPA treatment.Our work shows, in a rodent model of PD, that treatment-induced dyskinesia and striatal ERK activation are bidirectionally modulated by ablating CK2 in D1- or D2-positive projection neurons, in male and female mice. The results reveal that CK2 regulates signaling events critical to LID in each of the two main populations of striatal neurons.SIGNIFICANCE STATEMENT To date, l-DOPA is the most effective treatment for PD. Over time, however, its efficacy decreases, and side effects including l-DOPA-induced dyskinesia (LID) increase, affecting up to 78% of patients within 10 years of therapy (Hauser et al., 2007). It is understood that supersensitivity of the striatonigral pathway underlies LID, however, D2 agonists were also shown to induce LID (Bezard et al., 2001; Delfino et al., 2004). Our work implicates a novel player in the expression of LID, the kinase CK2: knock-out of CK2 in striatonigral and striatopallidal neurons has opposing effects on LID. The bidirectional modulation of dyskinesia reveals a central role for CK2 in striatal physiology and indicates that both pathways contribute to LID.
Assuntos
Caseína Quinase II/fisiologia , Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Discinesia Induzida por Medicamentos/metabolismo , Receptores de Dopamina D1/biossíntese , Receptores de Dopamina D2/biossíntese , Animais , Caseína Quinase II/deficiência , Corpo Estriado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Discinesia Induzida por Medicamentos/genética , Feminino , Expressão Gênica , Levodopa/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genéticaRESUMO
Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.
Assuntos
Caseína Quinase II/fisiologia , Fragmentos de Peptídeos/fisiologia , Ativação Plaquetária , Trombopoese , Trombose/patologia , Animais , Plaquetas , Sinalização do Cálcio , Caseína Quinase II/deficiência , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/deficiência , Trombose/etiologia , Trombose/metabolismoRESUMO
T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.
Assuntos
Caseína Quinase II/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Interleucina-17 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Fosforilação , Receptores de Interleucina , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Índice de Gravidade de Doença , Transdução de Sinais , Linfócitos T Reguladores/citologia , Células Th17/patologiaRESUMO
The ataxia telangiectasia mutated (ATM) signaling pathway responds rapidly to DNA double-strand breaks (DSBs) and it is characterized by recruitment of sensor, mediator, transducer and repair proteins to sites of DNA damage. Data suggest that CK2 is implicated in the early cellular response to DSBs. We demonstrate that CK2 binds constitutively the adaptor protein 53BP1 through the tandem Tudor domains and that the interaction is disrupted upon induction of DNA damage. Down-regulation of CK2 results in significant reduction of (i) 53BP1 foci formation, (ii) binding to dimethylated histone H4 and (iii) ATM autophosphorylation. Our data suggest that CK2 is required for 53BP1 accumulation at sites of DSBs which is a prerequisite for efficient activation of the ATM-mediated signaling pathway.
Assuntos
Antineoplásicos/farmacologia , Caseína Quinase II/metabolismo , Quebras de DNA de Cadeia Dupla , Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Doses de Radiação , Efeitos da Radiação , Transdução de Sinais , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Cellular senescence is thought to be an important tumor suppression process in vivo. We have previously shown that p53 activation is necessary for CKII inhibition-mediated cellular senescence. Here, CKII inhibition induced acetylation of p53 at K382 in HCT116 and HEK293 cells. This acetylation event was suppressed by SIRT1 activation. CKIIα and CKIIß were co-immunoprecipitated with SIRT1 in a p53-independent manner. Maltose binding protein pull-down and yeast two-hybrid indicated that SIRT1 bound to CKIIß, but not to CKIIα. CKII inhibition reduced SIRT1 activity in cells. CKII phosphorylated and activated human SIRT1 in vitro. Finally, SIRT1 overexpression antagonized CKII inhibition-mediated cellular senescence. These results reveal that CKII downregulation induces p53 stabilization by negatively regulating SIRT1 deacetylase activity during senescence.
Assuntos
Caseína Quinase II/genética , Senescência Celular/genética , Regulação para Baixo/genética , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Caseína Quinase II/deficiência , Caseína Quinase II/metabolismo , Senescência Celular/efeitos dos fármacos , Células HCT116 , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Lisina/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Estabilidade Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Sirtuína 1/antagonistas & inibidores , Proteína Supressora de Tumor p53/químicaRESUMO
The CK2 α and α' catalytic gene products have overlapping biochemical activity, but in vivo, their functions are very different. Deletion of both alleles of CK2α leads to mid-gestational embryonic lethality, while deletion of both alleles of CK2α' does not interfere with viability or development of embryos; however, adult CK2α'-/-males are infertile. To further elucidate developmental roles of CK2, and analyze functional overlap between the two catalytic genes, mice with combined knockouts were bred. Mice bearing any two CK2 catalytic alleles were phenotypically normal. However, inheritance of a single CK2α allele, without either CK2α' allele, resulted in partial embryonic lethality. Such mice that survived through embryogenesis were smaller at birth than littermate controls, and weighed less throughout life. However, their cardiac function and lifespan were normal. Fibroblasts derived from CK2α+/-CK2α'-/- embryos grew poorly in culture. These experiments demonstrate that combined loss of one CK2α allele and both CK2α' alleles leads to unique abnormalities of growth and development.
Assuntos
Caseína Quinase II/deficiência , Caseína Quinase II/genética , Domínio Catalítico/genética , Crescimento e Desenvolvimento , Animais , Peso Corporal/genética , Proliferação de Células , Fibroblastos/enzimologia , Fibroblastos/patologia , Hidronefrose/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Subunidades Proteicas/metabolismo , Tíbia/crescimento & desenvolvimento , Tíbia/patologia , Bexiga Urinária/crescimento & desenvolvimento , Bexiga Urinária/patologiaRESUMO
CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.
Assuntos
Caseína Quinase II/metabolismo , Desenvolvimento Embrionário , Morfogênese , Animais , Apoptose , Caseína Quinase II/deficiência , Proliferação de Células , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Cabeça/embriologia , Botões de Extremidades/embriologia , Camundongos , Fenótipo , Somitos/citologia , Somitos/enzimologia , Cauda/embriologiaRESUMO
Histone deacetylase 2 (HDAC2) mediates the repression of pro-inflammatory genes by deacetylating core histones, RelA/p65 and the glucocorticoid receptor. Reduced level of HDAC2 is associated with steroid resistant inflammation caused by cigarette smoke (CS)-derived oxidants and aldehydes. However, the molecular mechanisms regulating HDAC2 in response to CS and aldehydes is not known. Here, we report that CS extract, and aldehyde acrolein induced phosphorylation of HDAC2 which was abolished by mutations at serine sites S(394), S(411), S(422) and S(424). HDAC2 phosphorylation required direct interaction with serine-phosphorylated protein kinase CK2alpha and involved reduced HDAC2 deacetylase activity. Furthermore, HDAC2 phosphorylation was required for HDAC2 interaction with transcription factors, co-repressor complex formation, CBP recruitment, acetylation on lysine residues and modulates transrepression activity. Thus, phospho-acetylation of HDAC2 negatively regulates its deacetylase activity which has implications in steroid resistance in chronic inflammatory conditions.
Assuntos
Aldeídos/farmacologia , Caseína Quinase II/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas Repressoras/metabolismo , Fumaça , Acetilação , Proteína de Ligação a CREB/metabolismo , Caseína Quinase II/química , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Domínio Catalítico , Linhagem Celular , Misturas Complexas/farmacologia , Inativação Gênica , Histona Desacetilase 2/química , Humanos , Espaço Intracelular/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico , Serina/metabolismo , NicotianaRESUMO
OBJECTIVE: To investigate the effect of protein kinase CK2 gene silencing on the radiosensitization in human nasopharyngeal carcinoma (NPC) cells and its possible mechanism. METHODS: RNA interference (RNAi) technique was used to down-regulate the protein kinase CK2alpha expression in 5-8F cells, and clonogenic assay was employed to observe the changes in the radiosensitivity of the cells. DNA double-strand break was assessed by immunofluorescence staining of gamma-H2AX foci, and the cell apoptosis was examined using Annexin V-FITC/PI double-staining flow cytometry. RESULTS: CK2alpha protein was successfully silenced by siRNA. CK2alpha knockdown significantly decreased the clonogenic activity and increased the radiosensitivity of the NPC cells. After a 15-min exposure of the cells to 1 Gy radiation, significant difference occurred in the gamma-H2AX foci between CK2alpha knockdown cells and the control cells (P<0.01). CK2alpha silencing significantly increased the cell apoptosis after the exposure (P<0.01). CONCLUSIONS: Protein kinase CK2 plays an important role in the radiosensitivity of the NPC cells, and suppression of its expression might be a potential therapeutic approach of cancer.
Assuntos
Caseína Quinase II/deficiência , Caseína Quinase II/genética , Neoplasias Nasofaríngeas/patologia , Interferência de RNA , Tolerância a Radiação/genética , Animais , Anexina A5/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Humanos , Neoplasias Nasofaríngeas/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.
Assuntos
Caseína Quinase II/genética , Domínio Catalítico/genética , Marcação de Genes , Alelos , Animais , Caseína Quinase II/deficiência , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/enzimologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Defeitos do Tubo Neural/enzimologia , Cauda/anormalidades , Cauda/embriologiaRESUMO
Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2alpha and/or CK2alpha') and two CK2beta regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2beta gene is required for early embryonic development, while the CK2alpha' subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2alpha gene in the mouse germ line. Embryos lacking CK2alpha have a marked reduction in CK2 activity in spite of the presence of the CK2alpha' subunit. CK2alpha(-/-) embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2alpha appears to play an essential and uncompensated role in mammalian development.
Assuntos
Caseína Quinase II/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Biomarcadores , Caseína Quinase II/química , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Domínio Catalítico , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Marcação de Genes , Coração/embriologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-gamma and IL-17 and, to a lesser extent, cells that express IFN-gamma or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-gamma(+)/IL-17(+) infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.
Assuntos
Antígenos CD5/metabolismo , Caseína Quinase II/metabolismo , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Interleucina-17 , Linfócitos T/imunologia , Animais , Caseína Quinase II/deficiência , Proliferação de Células , Interferon gama , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Linfócitos T/citologiaRESUMO
Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits alpha and/or alpha' and two regulatory subunits beta whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.
Assuntos
Caseína Quinase II/metabolismo , Neoplasias/enzimologia , Apoptose/efeitos dos fármacos , Caseína Quinase II/biossíntese , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Domínio Catalítico , Transformação Celular Neoplásica , Citometria de Fluxo , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Nocodazol/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Reprodutibilidade dos TestesRESUMO
Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.