Jonathon Pines PhD FMedSci, Cancer Research UK Director of Research in Cell Division, Member of the Zoology Department.
How do cells control mitosis?
How do cells regulate entry to mitosis? And, once in mitosis, how do cells coordinate chromosome segregation with cell separation to ensure that the two daughter cells receive an equal and identical copy of the genome? The answers to both questions are provided by the interplay between protein kinases, protein phosphatases, and APC/C-mediated proteolysis, and this is the focus of our research. Since mitosis is a highly dynamic process we study living cells by time-lapse fluorescence microscopy, but complement this with biochemical analyses on cells in which we have knocked-out or mutated specific mitotic regulators using somatic cell recombination.
To understand how cells trigger mitosis we are analysing the behaviour of the key mitotic kinases, the Cyclin A- and B-dependent kinases, and their regulation by phosphorylation and dephosphorylation. We developed a FRET biosensor to assay Cyclin B1-Cdk1 activity in vivo and are using this to define the pathways that regulate the timing of mitosis. To identify the proteins responsible for regulating the Cyclin-Cdks, and provide insights into Cyclin-Cdk substrates, we have analysed protein complexes through the cell cycle by SILAC mass spectrometry and are following up some of the exciting results from this screen.
To understand how proteolysis regulates progress through mitosis we complement the analysis of APC/C-dependent degradation in living cells with biochemical analyses of protein complexes and ubiquitination activity. These studies are revealing how the APC/C is activated and how it is able to select a particular protein for destruction at a specific time. Moreover, the intimate coupling of the APC/C with the spindle assembly checkpoint that is essential to the control of chromosome segregation has meant that our recent work has begun to elucidate the key events in the checkpoint pathway.
• Izawa D and Pines J (2012) Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. Journal of Cell Biology 199, 27-37
• Mansfeld J, Collin P, Collins MO, Choudhary J and Pines J (2011) APC15 drives the turnover of MCC-Cdc20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nature Cell Biology 13, 1234-1244.
• Pagliuca F, Collins MO, Lichawska A, Zegerman P, Choudhary JS and Pines J (2011) Quantitative proteomics reveals the basis for the biochemical specificity of the cell cycle machinery. Molecular Cell 43, 406-417.
• Gavet O and Pines J (2010) Progressive activation of Cyclin B1-Cdk1 coordinates entry to mitosis. Developmental Cell, 18, 533-543.
• Nilsson J,Yekezare M, Minshull J and Pines J (2008) The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nature Cell Beiology, 10, 1411- 1420