Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis

Cdk1-mediated phosphorylation of threonine and serine residues on cell cycle regulators needs to be removed after mitosis. Hein et al. show that the known preference of the PP2A–B55 phosphatase for threonine provides temporal regulation of mitotic exit. Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit 1 , 2 , 3 , 4 , 5 . Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains unexplored. Here we demonstrate that the phosphothreonine preference of PP2A–B55 provides an essential regulatory element of mitotic exit. To allow rapid activation of the anaphase-promoting complex/cyclosome (APC/C) co-activator Cdc20, inhibitory phosphorylation sites are conserved as threonines while serine substitutions delay dephosphorylation and Cdc20 activation. Conversely, to ensure timely activation of the interphase APC/C co-activator Cdh1, inhibitory phosphorylation sites are conserved as serines, and threonine substitutions result in premature Cdh1 activation. Furthermore, rapid translocation of the chromosomal passenger complex to the central spindle is prevented by mutation of a single phosphorylated threonine to serine in inner centromere protein (INCENP), leading to failure of cytokinesis. Altogether, the findings of our work reveal that the inherent residue preference of a protein phosphatase can provide temporal regulation in biological processes.