DNA damage activates checkpoint pathways that induce cell-cycle arrest and subsequent DNA repair or cell death. Cyclin-dependent kinase-2 (CDK2) is inhibited by DNA damage, but whether CDK2 has a role in DNA-damage-induced cell death has been unknown. Donald Tindall and colleagues have addressed this question and found that CDK2-mediated phosphorylation of the transcription factor FOXO1 regulates apoptosis following DNA damage.

Given that FOXO transcription factors control a number of cell-death genes, Tindall and co-workers investigated a possible connection between CDK2 and the activity of FOXO proteins. They found that endogenous CDK2 phosphorylates FOXO1 at residue Ser249, and to a lesser degree residue Ser298, in vitro. This effect was abolished by a CDK inhibitor. The authors developed a phosphorylation-specific antibody and provided in vivo evidence for the CDK2-dependent phosphorylation of FOXO1. The antibody recognized wild-type FOXO1, but not a Ser249 to Ala249 (S249A) mutant of FOXO1. Small-interfering RNA (siRNA)-mediated silencing of CDK2 led to a decrease in FOXO1 phosphorylation, whereas FOXO1 phosphorylation was increased in cells that had been transfected with a constitutively active CDK2 mutant.

The transcriptional activity of FOXO1 was decreased in the presence of ectopically expressed CDK2 and its regulator cyclin E. By contrast, its transcriptional activity was increased by co-transfection of the tumour suppressor phosphatase and tensin homologue (PTEN), which promotes a decrease in FOXO1 phosphorylation. The increased transcriptional activity of FOXO1 was abolished in the presence of wild-type CDK2, but not by a kinase-inactive CDK2 mutant. Also, the inhibitory effect of CDK2 was diminished in a FOXO1 (Ser249, Ser298) double mutant. Together, these data indicate that the transcriptional activity of FOXO1 is repressed primarily by the CDK2-mediated phosphorylation of Ser249.

As residue Ser249 of FOXO1 is located adjacent to a nuclear localization motif, and as overexpression of CDK2 causes the cytoplasmic localization of wild-type FOXO1 but not of the phosphorylation-resistant S249A mutant, CDK2 phosphorylation is thought to induce the cytoplasmic localization of FOXO1 from the nucleus and thereby repress its transcriptional activity.

When Tindall and colleagues treated cells with a DNA-damaging agent, camptothecin or γ-irradiation, phosphorylation of FOXO1 at Ser249 was abolished. siRNA-mediated silencing of either the CHK1 or CHK2 kinase of the DNA-damage-mediated checkpoint pathway partially blocked the camptothecin-induced decrease in FOXO1 phosphorylation. These data indicate that DNA-damaging agents regulate FOXO1 by controlling CHK1- and CHK2-dependent checkpoint pathways. FOXO1-mediated cell death in response to DNA damage was observed both in p53-deficient and p53-containing cell lines, which implies that FOXO1 contributes to the apoptotic response to DNA damage independently of p53.

In summary, CDK2-mediated regulation of FOXO1 represents a novel pathway that links DNA damage to apoptosis. Interestingly, in cells in which FOXO1 was silenced, overexpression of FOXO3a or FOXO4 restored DNA-damage-induced apoptosis. It is therefore important to examine the roles of other FOXO proteins in the selective killing of cells in response to DNA damage.