RESUMO
The fission yeast Mcs6-Mcs2-Pmh1 complex, homologous to metazoan Cdk7-cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. We analyzed conditional mutants of mcs6 and pmh1, which activate Cdc2 normally but cannot complete cell division at restrictive temperature and arrest with decreased CTD phosphorylation. Transcriptional profiling by microarray hybridization revealed only modest effects on global gene expression: a one-third reduction in a severe mcs6 mutant after prolonged incubation at 36 degrees C. In contrast, a small subset of transcripts ( approximately 5%) decreased by more than twofold after Mcs6 complex function was compromised. The signature of repressed genes overlapped significantly with those of cell separation mutants sep10 and sep15. Sep10, a component of the Pol II Mediator complex, becomes essential in mcs6 or pmh1 mutant backgrounds. Moreover, transcripts dependent on the forkhead transcription factor Sep1, which are expressed coordinately during mitosis, were repressed in Mcs6 complex mutants, and Mcs6 also interacts genetically with Sep1. Thus, the Mcs6 complex, a direct activator of Cdc2, also influences the cell cycle transcriptional program, possibly through its TFIIH-associated kinase function.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Ativação Enzimática , Perfilação da Expressão Gênica , Genes Fúngicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Temperatura , Transcrição Gênica , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cyclin-dependent kinases (CDKs) involved in cell cycle control require activation by phosphorylation, but CDK-activating kinase (CAK) has diverged between metazoans and budding yeast. Fission yeast has two CAKs: the essential Mcs6 complex, homologous to the metazoan CDK7 complex implicated in cell cycle control and transcription; and Csk1, a nonessential ortholog of budding yeast Cak1. Both can activate the major CDK, Cdc2, but Csk1 can also activate Mcs6, so it was unclear whether the pathway is a linear cascade or a network. Here, we show that a mutation, mcs6-13, which selectively abrogates CDK activation, blocks both G1/S and G2/M transitions, but only when csk1(+) is absent. In contrast, gradual depletion or rapid inactivation of Mcs6 in csk1(+) cells causes cell separation defects or growth arrest, respectively, accompanied by decreased phosphorylation of RNA polymerase II (RNAP II), but not of Cdc2. Finally, neither cell cycle arrest nor CAK failure is recapitulated by a second mutation in mcs6-13 that prevents Mcs6 activation by Csk1, indicating that Csk1 activates Cdc2 directly in vivo. Thus, Mcs6 acts in concert with Csk1 to activate Cdc2 and independently to support transcription and facilitate cell separation. Csk1 likewise has multiple physiologic targets, including Mcs6 and Cdc2.