Progressive Activation of CyclinB1-Cdk1 Coordinates Entry to Mitosis

Abstract

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.

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

“…This complex formation is triggered by a 10-fold increase in Cyclin B1 expression in the G2 phase. Cyclin B1 expression changes through the cell cycle and cells with a prolonged G2 phase have reduced Cyclin B1 expression when compared to the ordinary expression (Gavet and Pines 2010). While the expression of Cyclin B1 is required for entry into mitosis, its destruction is required to exit from mitosis.…”

Vitexicarpin Induces Apoptosis in Human Prostate Carcinoma PC-3 Cells through G2/M Phase Arrest

“…Different cyclin and CDK combinations determine their downstream targeted proteins. Cyclin B1/CDK1 complex is pivotal in regulating cells from G2 to M phase transition (Giono and Manfredi, 2006;Enomoto et al, 2009;Gavet and Pines, 2010). To elucidate the molecular mechanism underlying this G2/M phase blockage, we measured the expressions of Cyclin B1 and CDK1 using Western blotting analysis.…”

The Renaissance or the cuckoo clock

“…Although centrosomes themselves are not required for some animal cells to enter mitosis [42,43], it is likely that they could facilitate the initial activation of Cyclin B-Cdk by raising the local concentration of Cyclin B-Cdk1 and its activator, Cdc25. Immunofluorescence and live-cell [44,45] analyses show that Cyclin B1 binds to microtubules and centrosomes before mitosis, but fluorescence recovery after photobleaching (FRAP) analysis shows that a Cyclin B1-green fluorescent protein (GFP) marker rapidly diffuses throughout the cell: the Cyclin B1 signal recovers to 100 per cent within 1 s of bleaching the centrosome (J. Richardson & J. Pines 2004, unpublished data). Some Cdc25 can also be observed at the centrosome [46][47][48]; therefore, in a manner similar to Turing-type symmetry breaking, it is likely that the auto-amplification of Cyclin B-Cdk1 and Cdc25 will first occur on centrosomes.…”