Cells are constantly exposed to DNA damaging agents from endogenous and exogenous sources. These mutagenic events are potentially deleterious and may lead to development of cancer. Cells have evolved mechanisms that detect DNA damage and induce arrest at cell cycle checkpoints to allow time for DNA repair or, if the damage cannot be repaired, induce apoptosis. These mechanisms prevent cells with damaged DNA from dividing thereby maintaining genomic integrity. A key checkpoint pathway activated in response to IR-induced DNA damage is the ATM/ATR-CHK1-CDC25A pathway that induces cell cycle arrest in G2 phase. Both ATM, CHK1 and CDC25A have been found to be dysregulated in cancer cells, and they are also potential targets for cancer therapy. The pathway regulates the cell cycle promoting activity of CDC25A by accelerating its proteolytic degradation. Activated CHK1 stimulates further phosphorylation of CDC25A on critical phosphodegron sites, which is needed for the recognition of CDC25A by ubiquitin ligases. However, the specific kinase responsible for phosphorylation of these phosphodegron sites is currently unknown. Therefore, it is important with further investigation of this critical signaling pathway. Using an RNAi library screen, we identified a new regulator of the IR-induced G2 checkpoint, the NIMA-related kinase NEK11. Depletion of NEK11 with siRNA abrogated the G2 checkpoint to the same extent as ATR depletion. Moreover, NEK11 was required for efficient CDC25A degradation in response to IR. NEK11 phosphorylated key phosphodegron serine residues on CDC25A that are mandatory for its degradation. Given the importance of NEK11 in the IR-induced checkpoint, we wished to explore the upstream regulation of the kinase. We reasoned that CHK1 could control NEK11 activity as well. Using in vitro kinase assays and phosphopeptide mapping, we confirmed 2 serine residues as CHK1 phosphorylation sites in vitro and we are currently investigating the biological effect of the serines 273 and 334 in vivo. In conclusion, we have identified NEK11 as a kinase important for proper control of the IR-induced G2 checkpoint. NEK11 functions by phosphorylating key sites in CDC25A and probably acts downstream of CHK1 in the ATM/ATR-CHK1-CDC25A signaling pathway.