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Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase
F M Townsley1, A Aristarkhov, S Beck
1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.
Insights
The human UbcH10 protein, a homolog of clam E2-C, is crucial for mitotic cyclin destruction and cell cycle progression. Mutant UbcH10 inhibits cyclin degradation, arresting cells in mitosis by blocking key proteolysis events.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Ubiquitin-dependent proteolysis of mitotic cyclins is essential for cell cycle completion.
- The anaphase promoting complex (APC) and ubiquitin carrier protein E2-C catalyze this process in clam eggs.
Purpose of the Study:
- To clone and characterize the human homolog of clam E2-C.
- To investigate the role of the human E2-C homolog (UbcH10) in cell cycle regulation.
Main Methods:
- Cloning of human UbcH10.
- In vitro ubiquitination and cyclin B destruction assays using clam oocyte extracts.
- Transfection of dominant-negative mutant UbcH10 into mammalian cells.
Main Results:
- Human UbcH10 shares 61% amino acid identity with clam E2-C and can functionally substitute for it in vitro.
- Dominant-negative UbcH10 inhibits cyclin B ubiquitination and destruction in clam extracts.
- Mutant UbcH10 in mammalian cells causes M phase arrest and inhibits anaphase onset by blocking proteolysis.
Conclusions:
- E2-C/UbcH10-mediated ubiquitination is vital for both cdc2 inactivation and sister chromatid separation.
- These processes are coordinated during mitotic exit via ubiquitin-dependent proteolysis.