The AMPK kinase Sak1 activates separase and promotes its nuclear localization during mitosis.

Accurate chromosome segregation during mitosis requires the precise regulation of separase, a protease that cleaves cohesin to trigger the separation of sister chromatids. Here, we identify the AMPK-activating kinase Sak1 as a novel regulator of separase in Saccharomyces cerevisiae. Using a suppressor screen, we discovered a hypermorphic SAK1-E50K allele that restored separase activity and nuclear localization in esp1-2 and pds1Δ mutants. We demonstrated that Sak1 phosphorylates separase independently of Snf1 activation, enhancing its enzymatic function and promoting nuclear accumulation. Loss of Sak1 delays Cdc14 activation and phenocopies FEAR pathway defects, establishing Sak1 as a mitotic regulator. Extending these findings to humans, we demonstrated that human separase and the tumor suppressor kinase LKB1, which is homologous to Sak1, physically associate. This finding reveals an evolutionarily conserved regulatory link between LKB1 and separase, highlighting the kinase-protease interplay as a critical mechanism for ensuring faithful mitotic progression.