Ubiquitin-dependent proteolysis of mitotic cyclins and other proteins triggers sister chromosome segregation, cdc2 inactivation and exit from mitosis into G1 of the next cell cycle. The components responsible for this process include a novel ubiquitin carrier protein called E2-C and a 20S complex called the cyclosome (or APC, anaphase promoting complex). E2-C contains a highly conserved N-terminal extension that is, surprisingly, not needed for catalytic activity in vitro. A dominant negative E2-C (dnE2-C) blocks approximately 50 percent of cells in mitosis. As the only component of the cyclin destruction machinery for which a dominant negative currently exists, we will use dnE2-C in biochemical and genetic approaches to investigate the role of this machinery in mitosis and elsewhere in the cell cycle, and to identify its target proteins. We will also try to determine the role played by cdc20, a WD40 protein required for cyclin destruction and G2/M checkpoint control. This proposal is designed to answer the following questions about regulation of the cell cycle, using both biochemical and genetic approaches to study the cell cycles of both embryos and somatic cells. Information from these studies should help understand what molecular mechanisms control cell division in normal development and differentiation, and how defects in these mechanisms contribute to aneuploidy and the formation of cancer cells. Does E2-C function only in mitosis or, as recent evidence suggests, at other points in the cell cycle also? What is the function of the N- terminal extension? Does is act in a checkpoint pathway in G2 or mitosis? What is the role of cdc20 and its associated kinase during cell cycle progression leading to cyclin destruction? What turns off mitotic cyclin destruction at the end of G1 in mammalian cells? Why is the cyclin destruction machinery active in GO-arrested somatic cells?

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD023696-13
Application #
2888954
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Tasca, Richard J
Project Start
1989-09-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
13
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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Crane, Richard; Kloepfer, Angela; Ruderman, Joan V (2004) Requirements for the destruction of human Aurora-A. J Cell Sci 117:5975-83
Tsai, Ming-Ying; Wiese, Christiane; Cao, Kan et al. (2003) A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly. Nat Cell Biol 5:242-8
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Farruggio, D C; Townsley, F M; Ruderman, J V (1999) Cdc20 associates with the kinase aurora2/Aik. Proc Natl Acad Sci U S A 96:7306-11

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