Cyclin-dependent kinases (cdks) form a family of enzymes that coordinate the cell division cycle. The periodic activation of cdks requires their association with proteins called cyclins, and their dissociation from inhibitory subunits, called ckis. Ubiquitin-mediated proteolysis is a major mechanism by which the protein levels of both cyclins and ckis are regulated in response to mitogenic and anti-mitogenic stimuli. Alterations in cdk regulation have been shown to result in abnormal cell growth associated with cancer. Recent evidence indicate the abnormal degradation of cell cycle proteins is associated with oncogenic events. Thus, the identification of the enzymes that regulate the degradation of ckis and cyclins will have a large impact on both basic research and cancer biology. We have previously shown that the intracellular level of the cki p27 is mainly regulated by degradation and that the ubiquitin system controls p27 degradation. Similarly, degradation of other G1 regulatory proteins (Cyclin E, Cyclin D1, p21, E2F-1, E2F-4) is controlled by the ubiquitin- pathway. Yet, the specific enzymes involved in the degradation of G1 regulatory proteins have not been identified. With this study we propose to characterize the human homologs of two yeast proteins, namely SKP-1 and CDC53, which are components of the ubiquitin pathway regulating the G1 phase of the yeast cell cycle (Specific Aim 1).
Under Aim 1, we will also clone and characterize three new proteins that we have found associated specifically with Skp-1 in vivo. We will then investigate the in vivo function of human Skp-1, Cdc53, and Skp-1 associated proteins (Specific Aim 2). Finally, we will test whether human Skp-1 and Cdc53 control the levels of known G1 regulators (Specific Aim 3).
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