RESUMEN
The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ras-GRF1/metabolismo , Quinasa de la Caseína I/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Aumento de la Célula , Polaridad Celular/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Genes cdc/fisiología , Mitosis/genética , Fosforilación , Proteína Fosfatasa 2/genética , Proteínas Tirosina Fosfatasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genética , ras-GRF1/genéticaRESUMEN
The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.