Mitotic　anaphase　onset　is　a　key　cellular　process　tightly　regulated　by　multiple　kinases.　The　involvement　of　mitogen-activated　protein　kinases　(MAPKs)　in　this　process　has　been　established　in　Xenopus　egg　extracts.　However,　the　detailed　regulatory　cascade　remains　elusive,　and　it　is　also　unknown　whether　the　MAPK-dependent　mitotic　regulation　is　evolutionarily　conserved　in　the　single-cell　eukaryotic　organisms　such　as　fission　yeast　(Schizosaccharomyces　pombe).　Here,　we　show　that　two　MAPKs　in　S.　pombe　indeed　act　in　concert　to　restrain　anaphase-promoting　complex/cyclosome　(APC/C)　activity　upon　activation　of　the　spindle　assembly　checkpoint　(SAC).　One　MAPK,　Pmk1,　binds　to　and　phosphorylates　Slp1Cdc20,　the　co-activator　of　APC/C.　Phosphorylation　of　Slp1Cdc20　by　Pmk1,　but　not　by　Cdk1,　promotes　its　subsequent　ubiquitylation　and　degradation.　Intriguingly,　Pmk1-mediated　phosphorylation　event　is　also　required　to　sustain　SAC　under　environmental　stress.　Thus,　our　study　establishes　a　new　underlying　molecular　mechanism　of　negative　regulation　of　APC/C　by　MAPK　upon　stress　stimuli,　and　provides　a　previously　unappreciated　framework　for　regulation　of　anaphase　entry　in　eukaryotic　cells.　©　2024,　Sun,　Chen,　Song　et　al.