The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G₁ arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

Human DBC1 (deleted in breast cancer-1; KIAA1967) is a nuclear protein that, in response to DNA damage, competitively inhibits the NAD⁺-dependent deacetylase SIRT1, a regulator of p53 apoptotic functions in response to genotoxic stress. DBC1 depletion in human cells increases SIRT1 activity, resulting in the deacetylation of p53 and protection from apoptosis. However, the mechanisms regulating this process have not yet been determined. Here, we report that, in human cell lines, DNA damage triggered the phosphorylation of DBC1 on Thr454 by ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3-related) kinases. Phosphorylated DBC1 bound to and inhibited SIRT1, resulting in the dissociation of the SIRT1–p53 complex and stimulating p53 acetylation and p53-dependent cell death. Indeed, DBC1-mediated genotoxicity, which was shown in knockdown experiments to be dependent on SIRT1 and p53 expression, was defective in cells expressing the phospho-mutant DBC1^T454A. This study describes the first post-translational modification of DBC1 and provides new mechanistic insight linking ATM/ATR to the DBC1–SIRT1–p53 apoptotic axis triggered by DNA damage.

Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-α2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.