This proposal focuses on the cyclin-dependent protein kinases or Cdks, a family of enzymes that controls the timing and coordination of eukaryotic cell cycle events. The proposed experiments are directed toward two goals. First, collaborative studies of Cdk crystal structure will be carried out with S.H. Kim (U.C. Berkeley). These studies will focus primarily on the Cdk7-cyclin H complex, a poorly understood enzyme with complex mechanisms of assembly. This work will reveal the conformational changes and protein-protein interactions underlying Cdk activation. The major portion of the proposed experiments will address the regulation of the human cyclin-dependent kinase Cdc2. Activation of a complex of Cdc2 and cyclin B1 is required for entry into mitosis. Interestingly, cyclin B1 is cytoplasmic during interphase and then imported into the nucleus in late prophase, bringing the active Cdc2- cyclin B1 complex to its key substrates in the nucleus. The proposed studies will explore the intriguing possibility that cyclin import is a key feature of mitotic control. A permeabilized-cell system, in which cyclin B1 nuclear import can be studied in vitro, will be used to determine: (1) the mechanism of nuclear import, (2) the mechanism that anchors cyclin B1 in the cytoplasm during interphase, and (3) the mechanism by which import is abruptly triggered in late prophase. In addition, adenovirus-based transient expression of dominant cyclin B1 mutants will be used to analyze the role of nuclear import in the timing of mitosis in normal cells and in cells delayed in G2 by DNA damage. At the end of mitosis, cyclin B destruction is thought to be the key mechanism leading to Cdc2 deactivation and mitotic exit. The proposed studies will explore: (1) the role of other mechanisms, such as inhibitory phosphorylation of Cdc2, in the exit from mitosis, and (2) the molecular machinery responsible for cyclin destruction. This work will focus on the purification and identification of the human ubiquitin-protein ligase enzyme (E3) that controls cyclin ubiquitination and destruction in late mitosis. The goal in this work will be to achieve a detailed biochemical understanding of the mechanisms by which this complex enzyme is assembled and regulated during mitosis.
