RESUMO
The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mob1p is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Proteínas de Ligação a DNA , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Ativação Enzimática , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência , Transdução de Sinais/fisiologiaRESUMO
The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Polaridade Celular/genética , Fosfoproteínas/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Genes Letais/genética , Mitose/genética , Mutação/genética , Miosinas/genética , Miosinas/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMO
Faithful actomyosin ring assembly is pivotal for successful cell division. The mechanisms by which the actomyosin ring is assembled at the correct time and place remain unclear. Recent studies in fission yeast have shown that a myosin II-containing spot may be a novel progenitor structure essential for actomyosin ring assembly.