The events of the cell cycle occur in a temporally conserved sequence. In particular, DNA replication or S-phase always precedes mitosis. For a somatic cell to enter mitosis prior to completing DNA replication would be either highly mutagenic or lethal. In order to ensure that this does not occur, the cell has developed a feedback or check point control pathway which actively suppresses initiation of mitosis until DNA replication is complete. At the biochemical level we now know that the replication-dependent feedback pathway inhibits mitosis by suppressing the activity of a kinase, which is essential for initiating mitosis, cdc2 kinase. Both genetic and biochemical studies have demonstrated that this kinase is negatively regulated by phosphorylation at two sites, tyrosine 15 and threonine 14. Moreover, some of the proteins which regulate phosphorylation of these sites have been identify. In particular, a tyrosine 15 kinase (wee1) and a tyrosine 15-threonine 14 phosphatase (cdc25) have been shown to regulate cdc2 activity. Recently, we have identified a second kinase which phosphorylates cdc2 on both tyrosine 15 and threonine 14. We have also shown that the feedback pathway which inhibits mitosis during DNA replication increases the activity of one or both of the two identified kinases which inhibit cdc2. The long term objective of this proposal is to develop a detailed mechanistic understanding of both the proteins involved in the feedback pathway and how these proteins interact to maintain cell cycle fidelity. The primary experimental system which we will use for these investigations is cell-free system derived from Xenopus eggs. In the absence of feedback controls this in vitro system spontaneously oscillates between S-phase and mitosis with a regular periodicity. We have shown that inhibition of DNA replication blocks this spontaneous oscillation and causes the cycle to arrest in S-phase. In this proposal we intend to further use this system to: 1) Isolate a novel new membrane- associated kinase which phosphorylates cdc2 on both tyrosine 15 and threonine 14. 2) Determine how the activity of this kinase is regulated by the replication-dependent feedback system. 3) Develop a precise quantitative understanding about how interactions between regulatory proteins composing the feedback pathway either increase or decrease the fidelity of the system. 4) To identify the location of the membrane- associated kinase within the cell. 5) To determine the distribution of the wee1 and cdc25 proteins between the cytoplasm and nuclear compartments and to determine how compartmentalization of the components of the feedback system contributes to the function of the system. 6) To determine if the regulated transport of cdc2-cyclin complexes into nuclei is an important component of the feedback system. 7) To determine whether the cdc2-related kinase cdk2 plays an active role in the feedback system. These investigations should provide valuable information both about how DNA replication and mitosis are temporally coordinated during normal cell division, as well as how small changes in the feedback system could generate a highly mutagenic state.
