Proper functioning and regulation of the cell cycle in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Major control over cell cycle progression is exerted by ubiquitin-mediated proteolysis, particularly during the exit from mitosis and in G1. The initiation of anaphase and completion of the cell cycle require the degradation of regulatory proteins such as cyclins, securin (which regulates chromosome separation), and microtubule motor proteins. The ubiquitin ligase for these proteins is the Anaphase Promoting Complex (APC). Substrates are targeted to the APC by two substrate recognition proteins, Cdc20 and Cdh1, active during late mitosis and in G1, respectively. Our long-term goal is to understand how the ubiquitination of APC substrates is regulated at a mechanistic level and how this regulation affects processes ranging from the maintenance of a non-dividing state to the development of cancers. Despite astounding recent advances in structural studies of the APC, there remain a number of important regulatory issues. We will address APC regulation using a combination of yeast molecular genetics and biochemical assays as well as studies in mammalian cells. Since APC structure, function and regulation are highly conserved, insights from yeast should continue to be applicable to humans. To further our understanding of APC regulation, we propose the following Specific Aims: 1) To characterize Cdh1 regulation during vegetative growth. We will determine how Cdh1 ?auto- ubiquitinates,? how Cdh1 recognizes itself for ubiquitination, how this auto-ubiquitination is regulated by phosphorylation of Cdh1, how Cdh1 stability is affected by substrate demand, and the role of Cdh1 up- regulation as cells approach stationary phase. 2) To determine how and why Cdh1 is downregulated during meiosis and sporulation. We will dissect the signaling pathways leading from the absence of glucose in diploid cells that lead to Cdh1 degradation, and determine the function of Cdh1 degradation in this important life cycle stage. 3) To determine whether human melanomas have compromised APCCdh1 activity. We will follow up on our observation that CDH1 is inactivated in many human melanomas by determining whether APCCDH1 function is reduced in melanoma cell lines, by CDH1 mutation or other regulation of CDH1 levels or activity. APC regulation and mis-regulation play important roles in the normal and abnormal growth of cells, in their responses to many anti-cancer agents, and in the mis-segregation of chromosomes leading to aneuploidy and the development of tumors. Our studies will further our understanding of this essential cell cycle regulatory process and, in the longer term, provide targets for therapeutic intervention, directed towards the APC itself or to pathways regulating APC activity.
Proper functioning of the cell cycle requires that certain regulatory proteins be eliminated during or just following cell division; errors in this process can lead to many abnormalities, most notably cancer as a result of the incorrect segregation of chromosomes to daughter cells. We are studying how the enzyme that attaches a tag to proteins to signal that they should be degraded is regulated, both at the basic level, in normal cells, and in cancer cells?specifically melanomas?in which the enzyme often appears to be mutated. We expect that these studies will lead to future efforts to modulate this machinery for therapeutic benefit.
