RESUMO
Calcium/calmodulin-dependent protein kinase (CaMK) is required for diverse cellular functions, and similar kinases exist in fungi. Although mammalian CaMK kinase (CaMKK) activates CaMK and also evolutionarily-conserved AMP-activated protein kinase (AMPK), CaMKK is yet to be established in yeast. We here report that the fission yeast Schizosaccharomyces pombe Ssp1 kinase, which controls G2/M transition and response to stress, is the putative CaMKK. Ssp1 has a CaM binding domain (CBD) and associates with 14-3-3 proteins as mammalian CaMKK does. Temperature-sensitive ssp1 mutants isolated are defective in the tolerance to limited glucose, and this tolerance requires the conserved stretch present between the kinase domain and CBD. Sds23, multi-copy suppressor for mutants defective in type 1 phosphatase and APC/cyclosome, also suppresses the ssp1 phenotype, and is required for the tolerance to limited glucose. We demonstrate that Sds23 binds to type 2A protein phosphatases (PP2A) and PP2A-related phosphatase Ppe1, and that Sds23 inhibits Ppe1 phosphatase activity. Ssp1 and Ppe1 thus seem to antagonize in utilizing limited glucose. We also show that Ppk9 and Ssp2 are the catalytic subunits of AMPK and AMPK-related kinases, respectively, which bind to common beta-(Amk2) and gamma-(Cbs2) subunits.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Sequência de Aminoácidos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Dados de Sequência Molecular , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Supressão Genética , TemperaturaRESUMO
Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3-green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.
