Temporally coordinated destruction of key cell cycle regulatory proteins by the ubiquitin-proteasome system represents an important regulatory mechanism to ensure that specific protein functions are turned off at the right time, in the right compartment and in a unidirectional fashion. Proteolysis of many core components of the cell cycle machinery is controlled by two major classes of ubiquitin ligases: the SCF (Skp1-Cul1-F-box protein) complexes and the Anaphase Promoting Complex/Cyclosome (APC/C). In humans there are sixty- eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin conjugating enzyme. Despite the large number of F-box proteins, only three human SCF ubiquitin ligases (containing the F-box proteins betaTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control (e.g., Cdc25A, cyclin E, Emi1, p21, p27, Wee1). We propose a project focused on a new tier of control of the cell cycle networks and its integration with the ubiquitin system. Using a novel screen, we have identified six novel putative SCF substrates, and we will characterize the mechanism, regulation and biological function of the degradation of one of them, namely E2F3, a protein intimately involved in the control of the cell cycle (Aim 1). We will furthermore identify and characterize those biologically significant substrates that are targeted for destruction by the F-box protein Fbw5 to regulate cell cycle progression (Aim 2). Finally, we will study the role of betaTrcp in controlling the degradation of a novel substrate identified using a biochemical screen: Claspin, a protein that is part of the DMA replication surveillance machinery (Aim 3). Given the crucial function of the cell cycle machinery, altered degradation of cell cycle regulatory proteins is clearly a contributing determinant of the unrestrained proliferation typical of cancer cells. As we continue to unravel the mechanisms of how the scheduled degradation of regulatory proteins by the ubiquitin system controls cellular proliferation, we are committed to the integration of our basic research results with an understanding of malignant transformation. It is anticipated that the results of our studies will have an impact on both basic science and cancer biology.