CDK Activation in Fission Yeast

	[Enlarge Image] Evolution of the CAK-CDK network: conserved components with divergent functions Metazoans and budding yeast each have a single CAK, but they are not homologous to one another. In contrast, fission yeast has two CAKs: the Mcs6 complex, which is homologous to the metazoan CAK (Cdk7); and Csk1, which is homologous to budding yeast Cak1.

Activating phosphorylation of the CDKs that control the cell cycle is a conserved step carried out by divergent enzymes in different species. In metazoans, including mammals and Drosophila, the major CDK-activating kinase (CAK) is itself a CDK (Cdk7), which depends on association with a cyclin (cyclin H) for full activity. In contrast, budding yeast contains a single-subunit CAK (Cak1) encoded by an essential gene. Remarkably, the fission yeast Schizosaccharomyces pombe contains two CAKs — one homologous to Cdk7 of higher eukaryotes and one superficially similar to budding yeast Cak1.

[Enlarge Image] Repression of Sep1-dependent genes in Mcs6-complex mutants In both mcs6 and pmh1 mutants, the genes dependent on the forkhead transcription factor Sep1 are repressed under non-permissive conditions. (Blue is decreased, Yellow is increased, Black is unchanged relative to permissive temperature; Gray indicates no detectable signal, which also probably indicates repression.)

The Mcs6-Mcs2-Pmh1 complex of S. pombe is homologous to mammalian Cdk7-cyclin H-Mat1. mcs6⁺, mcs2⁺ and pmh1⁺ are all essential genes, whereas csk1⁺ can be deleted from the genome without affecting viability or exponential growth rates. The redundancy between the two enzymes in activating the cell-cycle CDK explains why Csk1 is not absolutely required. Why then is the Mcs6 complex essential? An important clue is the decreased Pol II CTD phosphorylation in temperature-sensitive Mcs6-complex mutants at restrictive temperatures, which suggests that the essential function of the complex is in transcription. Surprisingly, however, global gene expression analysis by micorarray hybridization revealed that mcs6 and pmh1 mutants were defective at transcribing only a small subset (~5%) of fission yeast genes. Interestingly, genes transcribed periodically late in mitosis and during G1 under control of the forkhead transcription factor Sep1 were highly enriched among the transcripts repressed by Mcs6-complex impairment. This selective impairment of cell cycle-regulated gene expression could explain the cell division defects of mcs6, mcs2 and pmh1 mutants, and indicates that the Mcs6 complex is influencing cell-cycle progression both directly, as a CAK, and indirectly by facilitating transcription of cell cycle-regulated genes.

The powerful genetic tools available in yeast make S. pombe an attractive system in which to address questions that remain unanswered about the CAK-CDK network in all eukaryotes, including humans. What specialized function(s), if any might depend on Csk1, the nonessential CAK of fission yeast? We are analyzing other potential targets for activation by the fission yeast CAK network, which may help to explain the growth defect and genomic instability we have observed in strains in which csk1⁺ is deleted. We are also investigating the molecular basis of the connection between the Mcs6 complex and the forkhead factor Sep1, which was revealed by a combination of genetic interactions and functional genomics. Because the linkage between CDK activation and regulation of gene expression by Pol II phosphorylation has been maintained in mammalian organisms, understanding that coordination in a genetically tractable organism such as fission yeast may provide insights into how to target the CAK complex in cancer cells.