Cell cycle transitions are regulated by the activation and inactivation of cyclin dependent kinases (Cdks) through association with positive (cyclins) and negative (Cdk inhibitors, CKI) regulators, and by phosphorylation. The balance of these factors control Cdk activity and coordinate cell cycle transitions. Cyclin and CKI levels are controlled transcriptionally and post-translationally, the latter by ubiquitin (Ub)- dependent proteolysis. Protein degradation is required for three transitions in yeast; 5-phase entry, separation of sister chromatids, and exit from mitosis. While several known genes are involved in Ub-mediated proteolysis, their functions and regulation are poorly understood. Moreover, it is unclear how substrates are recognized. We seek to more fully understand the mechanisms regulating this proteolysis pathway through the analysis of SKPI and its associated F-box proteins. SKP1 a newly discovered component of this pathway required for G1 cyclin and CKI destruction in yeast and is also a component of the kinetochore. In yeast, SKP1 works together with CDC53, CDC4, and CDC34, an E2 Ub-conjugating enzyme, to carry out these functions. Human Skp1p directly binds to cyclin F and indirectly binds cyclin A through association with Skp2p. These are unstable cyclins and this association may regulate their stability. Skp1 has been found to associate with a number of proteins through a motif known as an F-box. F-box proteins are hypothesized to recruit substrates to the ubiquitination machinery or to be substrates themselves. We propose to investigate the role of SKP1 and F-box proteins in cell cycle control using biochemical and genetic analyses in yeast and human cells. Our broad objectives are to understand how Skp1p and the destruction machinery function to determine the timing of cyclin and CKI destruction. In addition, we wish to understand how F-box proteins are regulated and how they recognize their targets. Moreover, we wish to identify other components of the Skp1p pathway through continued gene discovery. In particular, we are interested in the identification of additional mammalian F-box proteins and their targets, which may include cell cycle regulators. Dissection of the Skp1/F-box network of interacting genes has important implications both for understanding normal cell cycle transitions and for understanding how alterations in these pathways contribute to the unregulated cell proliferation observed in cancer.
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