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1.
Free Radic Biol Med ; 129: 116-126, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30236788

RESUMO

Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40 S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.


Assuntos
Arsenitos/toxicidade , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Receptores de Quinase C Ativada/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Viabilidade Microbiana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/deficiência , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Quinase C Ativada/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Estresse Fisiológico
2.
Oncotarget ; 8(16): 26732-26743, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423643

RESUMO

Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.


Assuntos
Polipose Adenomatosa do Colo/diagnóstico , Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Neoplasias Colorretais/prevenção & controle , DNA Polimerase II/química , DNA Polimerase III/química , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Linhagem , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Domínios Proteicos/genética , Adulto Jovem
3.
PLoS One ; 10(11): e0143037, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26575035

RESUMO

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. RESULTS: Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. CONCLUSIONS: These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role.


Assuntos
Actinas/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/enzimologia , Proteínas de Ciclo Celular/metabolismo , Viabilidade Microbiana , Mitose , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento
4.
Nucleic Acids Res ; 42(15): 9573-87, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25081204

RESUMO

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca(2+)/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca(2+). Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca(2+). Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca(2+). Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca(2+), due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismo , Cálcio , Sinalização do Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/biossíntese , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Pontos de Checagem do Ciclo Celular , Retroalimentação Fisiológica , Deleção de Genes , Pressão Osmótica , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/biossíntese , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética
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