Coordination of the timing and order of cell cycle events is Critical for high-fidelity transmission of genetic information. Consequently, various control mechanisms ensure that initiation of particular events are dependent upon the completion of prior events. In mammalian cells, the restriction point (R-point) is thought to reflect a point where further cell cycle progression is not dependent upon mitogenic stimuli and is the primary point of regulation of entry of somatic cells into S-phase. Progression is dependent upon G1 cyclin dependent kinases (Cdks), which catalyze critical events in G1 and S-phases. These kinases are opposed by negative regulators including Cdk inhibitory proteins and pocket proteins typified by Rb. Rb is thought to be a primary mediator of R-point control, at least in part through its interaction with the E2F family of transcription factors, and is hypothesized to be inactivated by Cdk- dependent phosphorylation. Although these pathways are well characterized in outline form, many aspects of the currect model are based on correlative data and the fundamental biochemical mechanisms responsible for regulating passage through the R-point and initiation of DNA synthesis have yet to be elucidated. Our recent research has demonstrated directly: 1) that selective phosphorylation of Rb can inactivate its growth inhibitory function, 2) that cyclin D/Cdk4, but not cyclin E/Cdk2 or cyclin A/Cdk2, can inactivate Rb in vitro without a requirement for hyperphosphorylation of Rb, 3) that Rb hyperphosphorylated by cyclin A/Cdk2 and cyclin E/Cdk2 still retains the ability to block entry into DNA replication, and 4) that cyclin E/Cdk2 is capable of greatly accelerating S-phase entry in the absence of Rb, Suggesting that one role of Rb is to maintain G1 cells in an S-phase non-permissive state. In this proposal, we seek to further characterize how Cdks regulate both the R-point and the initiation of DNA synthesis using the biochemical and functional systems we have developed.
The specific aims are: 1) to establish how phosphorylation regulates Rb's growth suppression function and interaction with associating proteins, 2) to explore the mechanisms by which Cdks can regulate the rate of entry into S-phase, and 3) to use biochemical approaches to begin to identify important substrates for G1 Cdks. Understanding these processes in greater molecular detail will facilitate an ultimate understanding of the more fundamental questions of how S and M phases of the cell cycle are coordinated and how the cell determines whether it is competent or incompetent for S-phase.
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