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Effects of mutant Ran/TC4 proteins on cell cycle progression
M Ren1, E Coutavas, P D'Eustachio
1Department of Cell Biology, NYU Medical Center, New York 10016.
Abstract:
Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.
Insights
The Ran/TC4 protein regulates cell cycle progression. A mutant lacking GTP hydrolysis capability arrests cells in G1 and G2 phases, highlighting the GTPase cycle
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Ran/TC4, a RAS superfamily member, is a nuclear protein involved in GTP binding and hydrolysis.
- Previous studies suggested a role for Ran/TC4 in cell cycle regulation based on DNA replication inhibition by a GTP hydrolysis-deficient mutant.
Purpose of the Study:
- To investigate the role of Ran/TC4 in cell cycle progression.
- To analyze the cellular phenotypes associated with mutant and wild-type Ran/TC4 expression.
Main Methods:
- Utilized an efficient transfection system introducing cDNAs into 293/Tag cells.
- Analyzed cell cycle progression (G1 and G2 phases) following expression of wild-type and mutant Ran/TC4 proteins.
- Investigated the effect of deleting a carboxy-terminal hexapeptide from the Ran/TC4 mutant.
Main Results:
- Expression of a GTP hydrolysis-deficient Ran/TC4 mutant inhibited cell proliferation, causing arrest predominantly in G2 and also in G1 phases.
- Nuclear localization of the Ran/TC4 mutant was unaffected by the deletion of its carboxy-terminal hexapeptide.
- Deletion of the carboxy-terminal hexapeptide abolished the inhibitory effect of the Ran/TC4 mutant on cell cycle progression.
Conclusions:
- Normal cell cycle progression is coupled to the Ran/TC4 GTPase cycle.
- The carboxy-terminal hexapeptide of Ran/TC4 is crucial for its function in cell cycle regulation.
- Nuclear regulators like RCC1 and MPF likely mediate the coupling between the Ran/TC4 GTPase cycle and cell cycle progression.