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Dunphy's Home Page
dunphy@caltech.edu
Ph.D., 1985, Stanford University
Regulation of Mitosis Promoting Factor and the Cell Cycle
In dividing eukaryotic cells, a diverse family of cyclin-dependent kinases (Cdks) directs the progression of the cell cycle by regulating the accurate replication of the genome during S-phase and the faithful segregation of the chromosomes at mitosis (M-phase). The M-phase of the cell cycle is controlled by maturation-promoting factor (MPF), which contains a B-type cyclin and the Cdc2 protein kinase. MPF acts by phosphorylating a wide variety of substrates that collaborate in the execution of mitotic processes such as chromosome condensation, nuclear envelope breakdown, and spindle assembly. Our laboratory is interested in the molecular mechanisms underlying the regulation of MPF during the cell cycle. With this information, we hope ultimately to obtain a sophisticated understanding of how cell cycle control mechanisms contribute to carcinogenesis, terminal differentiation, and senescence.
The action of MPF must be controlled both temporally and spatially in a very stringent manner. This strict regulation is imparted by a number of checkpoint mechanisms that operate during the preparation for mitosis. A key question in the cell cycle field is how these checkpoint mechanisms control the activation and/or action of Cdks such as MPF so that cell cycle transitions occur only when all essential prerequisites have been fulfilled. In the case of M-phase, these requirements include the absence of unreplicated or damaged DNA, the attainment of an adequate cell size for division, and the accumulation of the various proteins necessary for spindle assembly and other mitotic processes.
The activation of MPF is controlled by an intricate series of phosphorylation reactions on the Cdc2 subunit. Specifically, phosphorylation of threonine residue 161 (T161) is absolutely required for catalytic activity, but the effect of this phosphorylation is overridden during interphase by concomitant inhibitory phosphorylations on tyrosine 15 (Y15) and threonine (T14). The tyrosine phosphorylation and subsequent dephosphorylation of Cdc2 are mediated by the Wee1 and Cdc25 proteins, respectively.
The major focus of our laboratory is on how the activation of Cdc2 is controlled both during the normal cell cycle and when the cell cycle has been perturbed by activation of a checkpoint. To this end, we have been engaged in a comprehensive analysis of Cdc2-specific regulatory factors in Xenopus egg extracts. The Xenopus egg system offers many advantages for the study of cell cycle regulatory mechanisms: the progression of the cell cycle can be reconstituted in cytoplasmic extracts from these eggs, and the replication checkpoint can be triggered in vitro by addition of DNA synthesis inhibitors such as aphidicolin. To facilitate these studies, we make extensive use of recombinant DNA technology to overproduce cell cycle proteins in either bacteria or baculovirus-infected insects cells. Moreover, in conjunction with our biochemical studies, we are taking advantage of the fission yeast system to exploit genetic approaches to identify novel Xenopus regulators of the cell cycle.
