Somatic cell cycle progression is orchestrated by the orderly formation, activation and inactivation of a series of cyclin/Cdk complexes. Cyclin E/Cdk2 is essential for the G1/S transition in vertebrates, but little is known about targets, whose phosphorylation triggers this transition. We plan to identify possible CycE/Cdk2 targets using purified CycE/Cdk2 to phosphorylate a lambdaGEX HeLa cDNA expression library, a method we developed and used successfully to find novel physiological substrates for ERK1 MAPK. Selected CycE/Cdk2 targets from this screen will be analyzed to see if they are phosphorylated in vivo in a cell cycle regulated manner and to establish their function in cell cycle progression. We will analyze how CycE/Cdk2 phosphorylation regulates the function of HsCdc6, a protein needed to assemble prereplication complexes at replication origins. Dbf4p/Cdc7p, a second type of protein kinase required for cell cycle progression, is essential for initiation of DNA replication. We have identified human homologues of yeast Dbf4p and Cdc7p; HsCdc7 activity is cell cycle regulated rising at G1/S. We will examine how HsDbf4 expression, association with HsCdc7 and phosphorylation are regulated in the cell cycle. We will study its function in DNA replication and cell cycle progression, using dominant negative HsCdc7/HsDbf4 mutants. We will conduct crystal structure studies on HsDbf4/Cdc7, with the goal of defining how HsDbf4 activates HsCdc7. We will identify physiological HsDbf4/HsCdc7 substrates by testing phosphorylation of DNA replication proteins, and by carrying out a lambdaGEX expression library screen. Pin1 is a conserved parvulin family peptidyl prolyl isomerase that acts as a mitotic regulator. Pin1 inhibits the G2/M transition, and Ess1p, its budding yeast counterpart, is required for M phase progression. The crystal structure of the Pin1 PPIase domain predicts it will select P.Ser/Thr.Pro bonds; Pin1 binds mitotic phosphoproteins, including Cdc25C and Myt1Hu. We will identify the phosphorylation sites in Cdc25C and Myt1Hu that interact with Pin1, and investigate whether Pin1 PPIase activity affects dephosphorylation of these sites in vitro. To learn more about targets and the regulation of Pin1, we will carry out a yeast screen for extragenic suppressors that restore growth of ess1 temperature sensitive mutants at the restrictive temperature. We will determine if Pin1 function is essential in mammalian cells by analyzing the phenotype of Pin1 knockout mice and cells derived from these mice. Perturbation of cell cycle control is a hallmark of cancer cells, and these studies will contribute to our understanding of mechanisms of oncogenesis.
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