The CDK Pef1 and Protein Phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes

Abstract

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wildtype background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4 based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.3 DNA capture may be achieved by modulating the catalytic activity of the loader and concerted transcriptional responses may involve a local and temporal control of loading and unloading activities. How cells orchestrate cohesin functions is largely unknown. Intriguingly, the kleisin subunit of cohesin is targeted by multiple phosphorylation events. In fission yeast, Rad21 shows multiple phospho-isoforms whose relative abundance fluctuates along the cell cycle [39]. Our recent work showed that Protein Phosphatase 4 (PP4) controls the phosphorylation status of Rad21 and modulates Wpl1 activity [40], leading to the idea that cohesin functions could be spatially and temporally fine-tuned by altering the balance between kinase and phosphatase activities.Here we report on the control of cohesin deposition by the opposite activities of the Pef1 CDK and PP4. Pef1 was first described as a PSTAIRE-related protein in fission yeast [41]. The CDK has three known cyclin partners called Pas1, Psl1 and Clg1 and was reported to facilitate the G1 to S phase transition and to regulate life span [42; 43]. Its closest human homolog, CDK5, is involved in a myriad of cellular functions and pathologies, from neurodegenerative diseases to multiple solid and hematological cancers [44]. We identified pef1 in a genetic screen for mutants able to rescue the cohesin loader mutant mis4-367. Pef1 ablation or inhibition of its kinase activity increased cohesin deposition and rescued sister-chromatid cohesion defects of the mis4 mutant. In otherwise wild-type cells Pef1 ablation increased the binding of both cohesin and its loader to their regular sites along chromosomes. Genetic analyses indicated that Pef1 acts through the phosphorylation of multiple targets. We identified one of these within the kleisin Rad21. Specifically, the Pef1/Psl1 complex phosphorylates Rad21 on T262 and preventing this phosphorylation event recapitulates in part th...