): Cyclin C is a member of the subset of cyclins that promotes progression from G1 to S phase in model systems. Its mechanism of action remains poorly understood. Recently a cyclin-dependent kinase partner has been identified, cdk8, which is homologous to an RNA polymerase II C-terminal domain (CTD) kinase in yeast. Preliminary data suggest that cyclin C may have direct effects on cell cycle progression, and may associate with a novel protein recognized by an PSTAIRE antibody characteristic of a subset of cdks involved in cell cycle control. In addition, cyclin C is overexpressed in many lung cancers. The proposed experiments will test (1) whether cyclin C associates with proteins related to cell cycle control, including a cdk in addition to cdk8; and (2) whether the association of cyclin C with these proteins is essential for cell cycle progression. In addition to providing further insights into the biology of cell cycle control, the proposed experiments may uncover new targets for the design of antineoplastic agents effective against the malignancy responsible for the highest number of cancer-related deaths among men and women in the United States. To isolate cyclin C associated proteins, affinity chromatography will be used to test cell lysates for proteins that specifically bind to an immobilized cyclin C-maltose binding fusion protein. The identity of some of these proteins will be tested by immunoblotting using antibodies specific for cell cycle-related proteins. Other cyclin C-associated proteins will be identified by partial purification, sequencing and cloning. To confirm the associations between cyclin C and these proteins in vivo, immunoprecipitations will be performed with well-characterized antibodies. Cyclin C-associated protein complexes will be tested for kinase activity using several substrates in vitro. Finally, levels of cyclin C-associated proteins will be altered in vivo, and effects on cell cycle progression determined. The work will be performed under the auspices of a division whose members are largely committed to the investigation of cell cycle regulation. The experiments will train the candidate in the techniques required for developing and analyzing immunologic reagents, and will provide new exposure to protein biochemistry and purification. The proposal is designed to satisfy an interest in cell cycle biology, and allow the principal investigator to complete his training in 3 years, as he prepares for independence.
