The CyclinB1-Cdk1 kinase is the catalytic activity at the heart of mitosis-promoting factor (MPF), yet fundamental questions concerning its role in mitosis remained unresolved. It is not known when and how rapidly CyclinB1-Cdk1 is activated in mammalian cells, nor how its activation coordinates the substantial changes in the cell at mitosis. Here, we have developed a FRET biosensor specific for CyclinB1-Cdk1 that enables us to assay its activity with very high temporal precision in living human cells. We show that CyclinB1-Cdk1 is inactive in G2 phase and activated at a set time before nuclear envelope breakdown, thereby initiating the events of prophase. CyclinB1-Cdk1 levels rise to their maximum extent over the course of approximately 30 min, and we demonstrate that different levels of CyclinB1-Cdk1 kinase activity trigger different mitotic events, thus revealing how the remarkable reorganization of the cell is coordinated at mitotic entry.
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