Characterization of mitotic regulators Acm1 and Cdc14

Michael Melesse, Purdue University

Abstract

Mitotic exit and cytokinesis are driven by a complex set of processes that serve to reset the eukaryotic cell cycle and allow entry into the subsequent cycle. These steps are driven by a combination of molecular events that lead to the termination of high cyclin dependent kinase (Cdk) activity and reversal of Cdk mediated phosphorylation. Proteasomal degradation of cyclins is essential for terminating Cdk activity and active dephosphorylation of Cdk mediated phosphorylation is important for the appropriate completion of the cell cycle. The anaphase promoting complex (APC) is a ubiquitin ligase responsible for promoting events in late mitosis by polyubiquitinating, among other substrates, cyclins and targeting them for degradation. Cdh1 is a highly conserved activator of the APC and its activity is regulated by, among other mechanisms, a pseudosubstrate inhibitor, Acm1. I have demonstrated that Acm1 degradation in G1 is mediated by a non-canonical proteasomal degradation mechanism that does not require polyubiquitin conjugation. I also show that expression of an Acm1 mutant resistant to this degradation mechanism (Acm1N Δ52) leads to reduced cell fitness. The activity of mitotic phosphatases is essential for the reversal of Cdk phosphorylation at the end of the cell cycle. Cdc14 activity is essential in budding yeast. However, it is only recently that its role in ordering late mitotic events has begun to be appreciated. By characterizing Cdc14 substrate selectivity, using phosphopeptide substrates, it has become clear that Cdc14 preferentially dephosphorylates a subset of Cdk phosphorylation sites. Here I show that Cdc14 substrate selectivity is conserved across diverse species and this selectivity can be used to gain insight into Cdc14 substrates in these organisms, where there is limited understanding of Cdc14 function. The substrate selectivity observed for Cdc14 enzymes holds the potential for the development of selective inhibitors. I was able to show that unphosphorylated peptide containing an optimal substrate sequence is able to inhibit Cdc14 activity. This observation points to the possibility of developing inhibitors that incorporate substrate like characteristics and can take advantage of the active site architecture. I have also developed a high throughput screening strategy and utilized it to screen 50,000 small molecules for the ability to inhibit Cdc14 activity. Specific Cdc14 inhibitors hold the potential to study the effects of Cdc14 loss in organisms that have multiple paralogs of the enzyme or in which genetic manipulations are a challenge.

Degree

Ph.D.

Advisors

Hall, Purdue University.

Subject Area

Molecular biology|Genetics|Cellular biology|Biochemistry

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS