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1.
Cell ; 182(3): 685-712.e19, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645325

RESUMO

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Proteômica/métodos , Células A549 , Animais , Antivirais/farmacologia , Células CACO-2 , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Biomed Khim ; 66(2): 130-137, 2020 Feb.
Artigo em Russo | MEDLINE | ID: mdl-32420893

RESUMO

Protein kinase CK2 is an important enzyme in the nervous system. The nuclear forms of CK2 regulate chromatin structure and gene expression, the key processes for long-term memory formation. Memory modulators, the Structural Analogues of Etimizole (SAE), were able to increase or decrease the activity of chromatin-associated CK in the cortex and hippocampus of rat brain in vitro. In vivo memory enhancers from SAE-group (3 mg/kg) stimulated CK2 activity and the transcriptional ability of chromatin in the cortex and hippocampus, starting from 30 min with a peak for 60 min and a duration up to 180 min. At these periods the memory inhibitor from the SAE-group reduced CK2 activity and chromatin transcription. It is assumed that the modulating effect of SAE on CK2 activity and transcription underlies the effects of these compounds on long-term memory.


Assuntos
Caseína Quinase II/metabolismo , Córtex Cerebral/efeitos dos fármacos , Cromatina , Etimizol/análogos & derivados , Etimizol/farmacologia , Hipocampo/efeitos dos fármacos , Animais , Fosforilação , Ratos , Transcrição Genética
3.
Nat Commun ; 11(1): 2289, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385263

RESUMO

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adulto , Idoso , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/patologia , Feminino , Deleção de Genes , Haploinsuficiência/genética , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
4.
PLoS One ; 15(4): e0232019, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343709

RESUMO

Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is α-synuclein (α-syn). Studies have shown that mutations, increased expression or abnormal processing of α-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of α-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2α (CK2α). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2α can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2α enhanced their kinase activity towards the phosphorylation of α-syn at S129. In an A53T α-syn transgenic mouse model of PD, we found that increased GRK6 and CK2α S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 α-syn. Treatment of A53T α-syn transgenic mice with Nω-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2α in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T α-syn transgenic mice reduced the levels of pSer129 α-syn and α-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2α can enhance the phosphorylation of pSer129 α-syn in an animal model of PD.


Assuntos
Caseína Quinase II/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Óxido Nítrico/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Fatores Etários , Animais , Caseína Quinase II/química , Modelos Animais de Doenças , Quinases de Receptores Acoplados a Proteína G/química , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Óxido Nítrico Sintase Tipo I/genética , Nitroarginina/administração & dosagem , Nitroarginina/farmacologia , Estresse Nitrosativo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Fosforilação , Serina/metabolismo , alfa-Sinucleína/química
5.
Anticancer Res ; 40(3): 1419-1426, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132038

RESUMO

BACKGROUND/AIM: Anti-cancer drug resistance restricts the efficacy of chemotherapy in malignant tumors. Casein kinase 2α (CK2α) is highly expressed in 5-fluorouracil (5FU)-resistant colorectal cancer (CRC) cells. We hypothesized that inhibition of CK2α might reduce CRC resistance to 5FU. MATERIALS AND METHODS: To investigate the role of CK2α in 5FU-resistant CRC cells, we assessed cell viability, apoptosis, cyclin-dependent kinase 4 (CDK4) activity, cell-cycle progression, invasion, and sphere formation in 5FU-resistant CRC cells. RESULTS: CK2α levels were significantly increased in 5FU-resistant CRC cells compared to those in wild-type CRC cells. During exposure to 5FU, viability, CDK4 activity, cell-cycle progression, invasion, and sphere formation were enhanced, while apoptosis was decreased in 5FU-resistant CRC cells. These effects were mediated by the inhibiting effects of CK2α on endoplasmic reticulum (ER) stress. Combination of CK2α knockdown with 5FU treatment promoted apoptosis of 5FU-resistant CRC cells by inducing ER stress. CONCLUSION: 5FU treatment in combination with a CK2α inhibitor may exert a synergistic effect against drug-resistant cancer cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Caseína Quinase II/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Quinase 4 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fluoruracila/administração & dosagem , Humanos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia
6.
Nat Commun ; 11(1): 1077, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103024

RESUMO

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.


Assuntos
Caseína Quinase II/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Divisão Celular Assimétrica/fisiologia , Sítios de Ligação/fisiologia , Camelídeos Americanos , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenvolvimento Embrionário/fisiologia , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/ultraestrutura , Fosforilação , Ligação Proteica/fisiologia , Conformação Proteica
7.
Biochem Biophys Res Commun ; 523(3): 639-644, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31941600

RESUMO

CREB3 (Luman) is a family member of ER resident transcription factors, which are cleaved upon the induction of ER stress. Their N-terminal fragments shuttle into the nucleus where they regulate the transcription of target genes. Here, we found that human CREB3 is phosphorylated within its transcription activation domain on serine 46 by protein kinase CK2. Further analyses revealed that the phosphorylation of this site does neither affect the cleavage by S1P/S2P proteases, nor the nuclear localisation nor the transcriptional activity of CREB3. However, phosphorylation at serine 46 reduced the stability of CREB3.


Assuntos
Caseína Quinase II/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sequência de Aminoácidos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Células HEK293 , Humanos , Fosforilação , Estabilidade Proteica
8.
Biochem Biophys Res Commun ; 524(2): 280-287, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-31987502

RESUMO

Activation of the Wnt/ß-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2ß, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/ß-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/ß-catenin signaling pathway in ESCs.


Assuntos
Caseína Quinase II/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Camundongos , Mapas de Interação de Proteínas , Proteólise
9.
Cell Prolif ; 53(1): e12726, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31755150

RESUMO

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 Knockout
10.
Plant Biol (Stuttg) ; 22 Suppl 1: 143-152, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30597713

RESUMO

Plastid casein kinase 2 (CK2), which is a major Ser/Thr-specific enzyme in higher organisms, plays an essential role in plant development and diverse abiotic stresses. CKB1 is a regulatory subunit beta of CK2. To expand our understand of functions of the CKB1 gene in Arabidopsis thaliana, protein changes among wild-type (WT) and CKB1 gain- and loss-of-function mutants were compared. Proteins extracted from the CKB1 knockout mutant and overexpressing mutant were compared with Col-0 plants using 2D-PAGE. Proteins regulated by CKB1 were identified with matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF), and its transcript was verified by qRT-PCR. Bioinformatics analysis, including gene ontology and protein-protein interaction analysis, were employed. The results of mass spectra and bioinformatics analysis suggest that CKB1 may have functions in regulation of the ribosomal protein L10 (RPL10) family and is involved in ultraviolet-B (UV-B) response. Furthermore, qRT-PCR verification showed CKB1 expression was up-regulated by UV-B stress. The expression levels of five genes in the RPL10 family were reduced in the ckb1 T-DNA insertion mutants, whereas they increased in the CKB1 overexpressing mutants under both normal conditions and UV-B treatment. In conclusion, CKB1 has important functions in UV-B radiation stress. Our study implies that CKB1 positively regulates UV-B radiation stress signalling, possibly through modulating expression of the RPL10 family.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Caseína Quinase II , Regulação da Expressão Gênica de Plantas , Proteína Ribossômica L10 , Raios Ultravioleta , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Caseína Quinase II/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteína Ribossômica L10/genética , Estresse Fisiológico/efeitos da radiação
11.
Exp Oncol ; 41(4): 304-311, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31868330

RESUMO

AIM: Transforming growth factor ß1 (TGF ß1) is a potent regulator of breast tumorigenesis. It inhibits proliferation of carcinoma cells, but the strength of its inhibitory action varies for cells from benigh, non-metastatic or metastatic tumors. The aim of this work was to generate a proteome profile of TGF ß1 action on non-tumorigenic human breast epithelial cells 184A1, and validate predicted involvement of casein kinase 2α (CK2α), p53 and structure-specific recognition protein-1 (SSRP1). MATERIALS AND METHODS: Two-dimensional gel electrophoresis and mass spectrometry were used to identify TGF ß1-regulated proteins in 184A1 human breast immortalized non-tumorigenic cells. 184A1 cells may serve as a model of benign breast neoplasia. These cells were obtained from normal mammary tissue, were immortalized but are not malignant, and were obtained from the American Type Culture Collection. The systemic analysis was performed by using the Cytoscape tool. Transfection of cells with CK2α construct and small interfering RNAs to CK2α and SSRP1 were used to assess an impact of CK2α and SSRP1 on phosphorylation of the p53 and cell proliferation. RESULTS: Proliferation of 184A1 cells was transiently inhibited by TGF ß1. We identified 100 and 47 unique proteins which changed their expression and/or 35S-incorporation, respectively, upon treatment with TGF ß1 for 2 h, 8 h or 24 h. Cell proliferation, death, migration, and metabolism were among the biological regulatory processes retrieved by the network analysis as affected by the identified proteins. The network analysis suggested that TGF ß1 may affect the phosphorylation of p53 at Ser392 by engaging CK2α. This was confirmed by the immunoblotting and cell proliferation assays. CONCLUSION: We report here the list of 147 TGF ß1-regulated proteins in immortalized non-tumorigenic human breast epithelial cells, and show involvement of CK2α in the regulation of p53 Ser392 phosphorylation.


Assuntos
Células Epiteliais/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caseína Quinase II/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Epiteliais/patologia , Feminino , Humanos , Fosforilação , Mapas de Interação de Proteínas , Proteômica
12.
Int J Mol Sci ; 20(23)2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779225

RESUMO

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (ß) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3ß axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2ß regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.


Assuntos
Caseína Quinase II/genética , Neurônios/citologia , Animais , Caseína Quinase II/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Mutação , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
13.
Mol Cells ; 42(11): 773-782, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31617338

RESUMO

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKTmTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, p21Cip1/WAF1 is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of p21Cip1/WAF1. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of p21Cip1/WAF1, leading to H3K9me3 and SAHFs formation.


Assuntos
Caseína Quinase II/metabolismo , Senescência Celular , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caseína Quinase II/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Células HCT116 , Heterocromatina/genética , Humanos , Lisina/metabolismo , Células MCF-7 , Metilação , Fosforilação , Estabilidade Proteica , Interferência de RNA , Serina/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética
14.
Proc Natl Acad Sci U S A ; 116(45): 22721-22729, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636192

RESUMO

Exposure to microbe-associated molecular patterns (MAMPs) causes dendritic cells (DCs) to undergo a remarkable activation process characterized by changes in key biochemical mechanisms. These enhance antigen processing and presentation, as well as strengthen DC capacity to stimulate naïve T cell proliferation. Here, we show that in response to the MAMPS lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (Poly I:C), RNA polymerase III (Pol lII)-dependent transcription and consequently tRNA gene expression are strongly induced in DCs. This is in part caused by the phosphorylation and nuclear export of MAF1 homolog negative regulator of Poll III (MAF1), via a synergistic casein kinase 2 (CK2)- and mammalian target of rapamycin-dependent signaling cascade downstream of Toll-like receptors (TLRs). De novo tRNA expression is necessary to augment protein synthesis and compensate for tRNA degradation driven by TLR-dependent DC exposure to type-I IFN. Although protein synthesis is not strongly inhibited in absence of RNA Pol III activity, it compromises the translation of key DC mRNAs, like those coding for costimulatory molecules and proinflammatory cytokines, which instead can be stored in stress granules, as shown for CD86 mRNA. TLR-dependent CK2 stimulation and subsequent RNA Pol III activation are therefore key for the acquisition by DCs of their unique T cell immune-stimulatory functions.


Assuntos
Células Dendríticas/imunologia , RNA Polimerase III/genética , Linfócitos T/imunologia , Transcrição Genética , Animais , Caseína Quinase II/metabolismo , Células Cultivadas , Ativação Enzimática , Feminino , Camundongos , Fosforilação , RNA Polimerase III/metabolismo , RNA de Transferência/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo
15.
Life Sci ; 236: 116933, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31614146

RESUMO

AIMS: Hepatocellular carcinoma (HCC) pathogenesis involves the interplay of multiple signalling pathways. Notch and Hedgehog (Hh) are two major developmental pathways that act in concert to regulate adult cell repair. CK2α -serine-threonine kinase-down-regulation enhanced apoptotic activity and was proven beneficial for HCC patients. Quercetin is a bioactive flavonoid and has been shown to protect against HCC through its antioxidant activity. This study was carried out to elucidate the antineoplastic effect of quercetin through regulating both Notch and Hh pathways, apoptosis, cell proliferation and CK2α activity. MAIN METHODS: Hepatocellular carcinoma was induced in male Sprague Dawley rats by thioacetamide. Quercetin was administered in both protective and curative doses. Parameters of liver function and oxidative stress were assessed. CK2α, Notch and Hh pathways were evaluated using RT-PCR and ELISA. Apoptosis was investigated by detecting caspase-3, caspase-8 and p53. Proliferative and cell cycle markers as cyclin D1 and Ki-67 were detected immunohistochemically. KEY FINDINGS: Quercetin inhibited CK2α and downregulated mRNA and protein expression of Notch1 and Gli2. Quercetin also suppressed caspase-3 expression but not caspase-8. Quercetin elevated p53 expression whereas proliferative and cell cycle markers cyclin D1 and Ki-67 were downregulated. Markers of hepatic cellular integrity such as AST, ALT, ALP, GGT, albumin and bilirubin were significantly ameliorated. This was confirmed by histological examination. Quercetin also alleviated oxidative stress as shown by SOD, GSH, MDA and NO levels. SIGNIFICANCE: We can conclude that in addition to its antioxidant power, quercetin blocked Notch, Hedgehog, regulated the apoptotic and proliferative pathways and inhibited CK2α in HCC.


Assuntos
Antioxidantes/farmacologia , Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo
16.
Nucleic Acids Res ; 47(20): 10754-10770, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31535131

RESUMO

Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.


Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fosfosserina/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II/metabolismo , Proteína Centromérica A/química , Cromatina/metabolismo , Proteínas de Drosophila/química , Proteínas Mutantes/metabolismo , Fosforilação , Ligação Proteica , Proteólise
17.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500224

RESUMO

Since diabetes is a global epidemic, the development of novel therapeutic strategies for the treatment of this disease is of major clinical interest. Diabetes is differentiated in two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM arises from an autoimmune destruction of insulin-producing ß-cells whereas T2DM is characterized by an insulin resistance, an impaired insulin reaction of the target cells, and/or dysregulated insulin secretion. In the past, a growing number of studies have reported on the important role of the protein kinase CK2 in the regulation of the survival and endocrine function of pancreatic ß-cells. In fact, inhibition of CK2 is capable of reducing cytokine-induced loss of ß-cells and increases insulin expression as well as secretion by various pathways that are regulated by reversible phosphorylation of proteins. Moreover, CK2 inhibition modulates pathways that are involved in the development of diabetes and prevents signal transduction, leading to late complications such as diabetic retinopathy. Hence, targeting CK2 may represent a novel therapeutic strategy for the treatment of diabetes.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos
18.
Cells ; 8(8)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434353

RESUMO

The protein kinase Csnk2/CK2 is important for cell proliferation, differentiation, and survival. Previously, we showed that CK2 binds distinctive proteins at neuromuscular junctions (NMJs) of mice and phosphorylates some of them. CK2 likely stabilizes clustered nicotinic acetylcholine receptors (AChRs). In the absence of the ß-subunit of CK2 in skeletal muscle fibers, mice develop an age-dependent decrease of grip strength accompanied by NMJ fragmentation and impairments of neuromuscular transmission. However, the precise role of CK2ß regarding the clustering of AChRs and downstream signaling at NMJs is unknown. Here, we compared conditional CK2ß-deficient mice with controls and found in the mutants (1) a lower decrement of endplate potentials after repetitive stimulation and decrements of nerve-evoked compound muscle action potentials decayed more rapidly after synaptic transmission was partially blocked, (2) that their muscle weakness was partially rescued by administration of an acetylcholine esterase inhibitor, (3) fragmented NMJs and impaired AChR clustering was detected in muscles and cultured muscle cells, (4) enlarged myonuclei, (5) impaired synaptic gene expression, and (6) a high turnover rate of their AChR clusters in vivo. Altogether, our data demonstrate a role for CK2 at the NMJ by maintaining a high density of AChRs and ensuring physiological synaptic gene expression.


Assuntos
Caseína Quinase II/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Expressão Gênica , Camundongos , Transmissão Sináptica
19.
Plant Cell Physiol ; 60(12): 2785-2796, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31424513

RESUMO

Phosphorus is one of the most important macronutrients required for plant growth and development. The importance of phosphorylation modification in regulating phosphate (Pi) homeostasis in plants is emerging. We performed phosphoproteomic profiling to characterize proteins whose degree of phosphorylation is altered in response to Pi starvation in rice root. A subset of 554 proteins, including 546 down-phosphorylated and eight up-phosphorylated proteins, exhibited differential phosphorylation in response to Pi starvation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the differentially phosphorylated proteins indicated that RNA processing, transport, splicing and translation and carbon metabolism played critical roles in response to Pi starvation in rice. Levels of phosphorylation of four mitogen-activated protein kinases (MAPKs), including OsMAPK6, five calcium-dependent protein kinases (CDPKs) and OsCK2ß3 decreased in response to Pi starvation. The decreased phosphorylation level of OsMAPK6 was confirmed by Western blotting. Mutation of OsMAPK6 led to Pi accumulation under Pi-sufficient conditions. Motif analysis indicated that the putative MAPK, casein kinase 2 (CK2) and CDPK substrates represented about 54.4%, 21.5% and 4.7%, respectively, of the proteins exhibiting differential phosphorylation. Based on the motif analysis, 191, 151 and 46 candidate substrates for MAPK, CK2 and CDPK were identified. These results indicate that modification of phosphorylation profiles provides complementary information on Pi-starvation-induced processes, with CK2, MAPK and CDPK protein kinase families playing key roles in these processes in rice.


Assuntos
Caseína Quinase II/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Caseína Quinase II/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Oryza/fisiologia , Fosfatos/deficiência , Proteínas de Plantas/genética
20.
Eur J Med Chem ; 181: 111581, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400711

RESUMO

Casein kinase (CK) is a type of conserved serine/threonine protein kinase that phosphorylates many important proteins in body. Researchers found that CK is involved in a variety of signaling pathways, and also plays an important role in inflammation, cancer, and nervous system diseases. Thus, it is considered to be a promising target for the treatment of related diseases. Many CK small molecule inhibitors have been reported so far, and most are ATP competitive inhibitors. However, these CK inhibitors lack the basic properties required for in vivo use, such as selectivity, cell permeability, metabolic stability, correct pharmacokinetic characteristics, and cellular environment. But small molecule inhibitors still have an advantage in drug research due to their controllable pharmacological and pharmacokinetic properties. CX-4945 discovered by Cylene Pharmaceutical is the only one CK2 inhibitor entering into Phase II clinical trials till now. In recent years, significant advances have been made in the design of non-competitive inhibitors of CK and in the application of multi-target inhibition strategies. Here, we review the published CK inhibitors and analyze their structure-activity relationships (SAR). We also summarized the eutectic structure with identified hot spots to provide a reference for future drug discovery.


Assuntos
Caseína Quinase II/antagonistas & inibidores , Caseína Quinase I/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular
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