Kinases orchestrate phospho-proteome dynamics during mitotic exit

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin-dependent kinase (Cdk) activity reaches its peak, the Anaphase Promoting Complex (APC) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk-counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3456 phosphosites on 1101 proteins with high temporal resolution as cells progress synchronously through mitosis. This illustrates sequential protein dephosphorylation and reveals that the order arises from successive inactivation of S and M phase Cdks and of the mitotic kinase Polo. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. Our findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases instruct the order of both protein dephosphorylation as well as phosphorylation.