A key event in the progression through and exit from mitosis is the ubiquitin-dependent degradation of cyclins and other key proteins by the Anaphase Promoting Complex (APC; also called the cyclosome). APC substrates contain degradation motifs such as the Destruction Box and the KEN Box that are required for their ubiquitination and for their binding to the APC activators, Cdc20 and Cdh1. Both motifs are required for efficient degradation, but either one alone suffices for binding to Cdh1p. We have proposed a pathway for the assembly of the APC-Cdh1p-substrate complex in which APC-free Cdh1p first binds a substrate. Engagement of the substrate Destruction Box by Cdh1p stimulates the binding of Cdh1p to the APC, presumably via a conformation change in Cdh1p. The current studies are aimed at furthering our understanding of APC-mediated proteolysis in budding yeast. In particular, we wish to identify novel APC substrates, to understand how APC-Cdh1p/Cdc20p-substrate complexes are assembled and disassembled, and to understand how the spindle assembly checkpoint makes use of a KEN box to turn off APC-mediated proteolysis when chromosomes are not properly attached to the mitotic spindle. To achieve these goals, I propose the following Specific Aims: 1) To Identify Novel APC Substrates. 2) To Assess the Generality of the APC-Cdh1p-Substrate Assembly Pathway. 3) To Determine How D-Box Engagement Stimulates Cdh1p Binding to the APC: 4) To Determine the Roles of Conserved Cdh1p Motifs and of Phosphorylation in APC-Cdh1p-Substrate Assembly. 5) To Determine Whether Disassembly of the APC-Cdh1p-Substrate Complex is an Active Process. 6) To Determine the Function of the MadSp KEN Box in the Spindle Assembly Checkpoint. Degradation of key proteins is essential for cell division. Understanding this degradation is thus important for understanding normal cell proliferation, and how this process goes awry in disease states, particularly cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM076200-01
Application #
7017968
Study Section
Cellular Signaling and Dynamics Study Section (CSD)
Program Officer
Zatz, Marion M
Project Start
2006-01-01
Project End
2009-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
1
Fiscal Year
2006
Total Cost
$351,525
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wang, Ruiwen; Solomon, Mark J (2012) Identification of She3 as an SCF(Grr1) substrate in budding yeast. PLoS One 7:e48020
Ostapenko, Denis; Burton, Janet L; Solomon, Mark J (2012) Identification of anaphase promoting complex substrates in S. cerevisiae. PLoS One 7:e45895
Ostapenko, Denis; Solomon, Mark J (2011) Anaphase promoting complex-dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae. Mol Biol Cell 22:2175-84
Ostapenko, Denis; Burton, Janet L; Wang, Ruiwen et al. (2008) Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation. Mol Cell Biol 28:4653-64
Ko, Nolan; Nishihama, Ryuichi; Tully, Gregory H et al. (2007) Identification of yeast IQGAP (Iqg1p) as an anaphase-promoting-complex substrate and its role in actomyosin-ring-independent cytokinesis. Mol Biol Cell 18:5139-53