Cell cycle dependent degradation of Bora and Cdc20

This dissertation is divided in two parts, each characterizing the ubiquitin-proteasome-dependent degradation of a crucial cellular regulator: Bora in mitosis and Cdc20 during the spindle assembly checkpoint.;SCFbeta-Trcp regulates the stability of many proteins involved in cell cycle regulation, signal transduction, and checkpoint establishment. The first part of this dissertation details the identification of novel substrates of human SCFbeta-Trcp via the tandem affinity purification of the beta-Trcp complex coupled with mass spectrometric analysis. With this unbiased approach, we identified not only many known beta-Trcp substrates, but also a list of potentially new substrates. Candidate beta-Trcp substrates were first scored using the consensus degron motif for SCFbeta-Trcp (DpSGXXpS/T). From this initial list, I confirmed that Bora, the ortholog of Drosophila Bora that has been shown to activate the mitotic kinase Aurora A1, is a bona fide substrate of beta-Trcp. Using a similar purification method, I found that Bora binds predominantly to Polo-like kinase 1 (Plk1), a key regulator of mitosis. Therefore, I propose that Bora is phosphorylated by Plk1 to induce its mitotic degradation mediated by SCFbeta-Trcp.;In the second part of this dissertation, I focus on the Anaphase Promoting Complex/Cyclosome (APC/C)- and Mad2-mediated degradation of Cdc20 during the activation of spindle assembly checkpoint (SAC). SAC is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of APC/C, is the major target for inhibition by SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. I found that the SAC also negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once SAC is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. I propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/CCdc20 in response to SAC activation.