Precise control of the cell cycle is of utmost importance for the well-being of a cell. Deregulation of cell cycle progression can result in neoplastic growth and aberrant development. One of the key transitions in the cell cycle is called Restriction Point in mammalian cells or Start in yeast. At this point in Gl, cells decide upon internal and external signals to enter a quiescent or G0 state, start a developmental program or commit to a new round of cell cycle. Execution of the G1/S transition is dependent on Gl cyclin dependent kinases (CDKs), which catalyze key steps in G1 and S phase. Gl CDK activity is regulated at many levels, including transcriptional activation and regulated degradation of its catalytic subunits, the Gl cyclins. A highly conserved protein degradation complex termed SCF (for Skp1-Cdc53-F-box protein) is thought to recognize and mark Gl cyclins (as well as a number of other regulatory proteins) for degradation by the ubiquitin-proteasome pathway. A major gap of knowledge in the field concerns the specificity and temporal regulation of the SCF towards its substrates. The work proposed here using molecular genetic and biochemical tools in yeast cells should help to close that gap. We have recently established that phosphorylation of G1 cyclins serves as an essential signal for rapid destruction. We have preliminary results demonstrating that a component of the SCF complex, the F-box protein Grr1, binds to wild type G1 cyclins, but not to a mutant Gl cyclin that is highly stabilized and no longer responds to growth inhibitory and cell cycle intrinsic signals. The goals of this proposal are: 1) What are the determinants of protein instability in G1 cyclins? 2) Which domains in components of the SCF complex confer specificity towards a substrate, and what is the molecular nature of the SCF-substrate interaction? 3) Using genetic and biochemical approaches, we will identify novel components or regulators of the SCF. Given that both the cell cycle machinery and the SCF- mediated protein degradation pathway are highly conserved from yeast to man, we expect that the information gained from our experiments will not only deepen our knowledge about the molecular mechanisms that control cell proliferation but will also help to devise cures for the profound illnesses that are linked to cell proliferation and ubiquitin-mediated proteolysis including cancer, neurodegenerative disorders and ion channel diseases.
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