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Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase
D V Bulavin1, Y Higashimoto, I J Popoff
1Division of Basic Science, National Cancer Intitute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
Insights
p38 kinase initiates the G2/M cell-cycle checkpoint after DNA damage from ultraviolet radiation. This involves p38 phosphorylating Cdc25B, which is crucial for checkpoint initiation and cell-cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Genotoxic stress response involves cell-cycle arrest and DNA repair.
- While checkpoint maintenance is understood, initiation mechanisms remain unclear.
- The G2/M checkpoint prevents entry into mitosis with damaged DNA.
Purpose of the Study:
- Investigate the role of p38 kinase in initiating the G2/M checkpoint after ultraviolet (UV) radiation.
- Elucidate the molecular mechanisms by which p38 influences checkpoint initiation.
Main Methods:
- Utilized human and murine cell lines exposed to UV radiation.
- Employed p38 kinase inhibition and site-directed mutagenesis.
- Performed in vitro and in vivo binding and phosphorylation assays.
- Assessed 14-3-3 protein binding.
Main Results:
- p38 kinase inhibition blocked G2/M checkpoint initiation after UV radiation.
- In vitro, p38 phosphorylated Cdc25B and Cdc25C.
- In vivo, p38 inhibition prevented Cdc25B phosphorylation at Ser309 and subsequent 14-3-3 binding.
- Mutation of Ser309 in Cdc25B inhibited checkpoint initiation.
Conclusions:
- p38 kinase plays a critical role in initiating the G2/M checkpoint following UV-induced DNA damage.
- Regulation of Cdc25B phosphorylation by p38 is a key event for G2/M checkpoint initiation.
- This finding clarifies a crucial step in the cellular response to genotoxic stress.
