RESUMO
Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.
Assuntos
Frutosedifosfatos/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Divisão Celular , Regulação Fúngica da Expressão Gênica , Mutação/genética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The mechanisms by which environmental stress regulates cell cycle progression are poorly understood. In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase. Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely. We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry. Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm. However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Schizosaccharomyces/enzimologia , Fosfatases cdc25/antagonistas & inibidores , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas Fúngicas , Genes Fúngicos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Mitose , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Tirosina/química , Fosfatases cdc25/química , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Dano ao DNA , Primers do DNA , Replicação do DNA , Fase G2 , Genes Fúngicos , Microscopia de Fluorescência , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Tirosina/metabolismoRESUMO
Cmk2, a fission yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, is essential for oxidative stress response. Cells lacking cmk2 gene were specifically sensitive to oxidative stress conditions. Upon stress, Cmk2 was phosphorylated in vivo, and this phosphorylation was dependent on the stress-activated MAPK Sty1/Spc1. Co-precipitation assays demonstrated that Cmk2 binds Sty1. Furthermore, in vivo or in vitro activated Sty1 was able to phosphorylate Cmk2, and the phosphorylation occurred at the C-terminal regulatory domain at Thr-411. Cell lethality caused by overexpression of Wis1 MAPK kinase was abolished by deletion of cmk2 or by mutation of Thr-411 of Cmk2. Taken together, our data suggest that Cmk2 acts downstream of Sty1 and is an essential kinase for oxidative stress responses.
