Cells respond to changes in their environment by appropriately increasing or decreasing the expression of proteins to obtain an optimal response to the new conditions. This process in which an external stimulus results in changes in gene expression is called signal transduction. It has been recently appreciated that another major mode of regulation of protein level in cells exists and it does not involve transcriptional regulation. It involves changes in proteolysis, either via increases in protein degradation efficiency or by decreasing substrate availability. This mode of regulation of protein levels in cells is rapid and irreversible. The most robust protein degradation system that is responsible for these rapid responses is the ubiquitin-proteolytic pathway. This pathway is characterized by three activities that result in the covalent attachment of a small protein called ubiquitin to lysine side chains of proteins to be degraded. These activities are referred to as the E1 or ubiquitin activating enzyme, the E2 or the ubiquitin conjugating enzyme and the E3 or ubiquitin ligase. Of these three, the E3s or ubiquitin ligases are responsible for the recognition of substrates. We identified Cul3 as being an essential E3 ligase for mammalian cyclin E. We have shown that it is required for mouse embryonic development and normal cell cycle progression in primary fibroblasts. Here we propose to further investigate how Cul1 and Cul3 cooperate to degrade cyclin E. In addition we are investigating the functional significance of mapped binding regions for subunits of the ligase. Lastly, we have identified some novel Cul3 binding proteins using proteomics and are testing a model for a novel mode of substrate recognition. Public Health Relevance: The broad reaching impact of these studies includes most proliferative events especially embryonic development and cancer. Many therapies that utilize stem cells will benefit from our work since such approaches hinge upon understanding how to induce proliferation under some circumstances as well as how to prevent unwanted proliferation. It is clear that the major regulators of these processes are the cullin-based E3 ligases.