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Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein
A Lopez-Girona1, B Furnari, O Mondesert
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.
Insights
Rad24, a 14-3-3 protein, controls the location of Cdc25 in fission yeast cells. DNA damage triggers Chk1 and Rad24 to export Cdc25 from the nucleus, enforcing the DNA-damage checkpoint.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- DNA damage triggers cell-cycle checkpoints to allow DNA repair before cell division.
- The Chk1 protein phosphorylates Cdc25, a mitotic inducer, to enforce this checkpoint.
- 14-3-3 proteins bind to phosphorylated Cdc25, but their regulatory role remains unclear.
Purpose of the Study:
- To investigate the role of the 14-3-3 protein Rad24 in the DNA-damage checkpoint.
- To elucidate how 14-3-3 proteins regulate the intracellular distribution of Cdc25.
Main Methods:
- Fission yeast genetics
- Analysis of protein localization (nuclear accumulation/export)
- Checkpoint assays
- Site-directed mutagenesis of a putative nuclear-export signal in Rad24.
Main Results:
- Elimination of Rad24 leads to nuclear accumulation of Cdc25.
- DNA-damage checkpoint activation induces net nuclear export of Cdc25, dependent on Chk1 and Rad24.
- Mutation of a nuclear-export signal in Rad24 disrupts Rad24 localization, Cdc25 export, and the DNA-damage checkpoint.
Conclusions:
- Rad24 regulates the intracellular distribution of Cdc25 in response to DNA damage.
- Rad24 functions as a nuclear-export signal, enhancing Cdc25 export from the nucleus.
- This mechanism is crucial for enforcing the DNA-damage checkpoint in fission yeast.