Chk2 activation dependence on Nbs1 after DNA damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progressio n in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)dependent mann er, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an in hibitory site, blocking entry into mitosis, and p53 on a regulatory site, c ausing G, arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenot ypic defects including chromosome fragility, radiosensitivity, and radiores istant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-d ependent increased phosphorylation and induction of catalytic activity agai nst Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and ac tivation 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 compl ex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mut ated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expressi on is unaffected in NBS cells, hence excluding a mislocalization as the cau se of failed Chk2 activation in Nbs1-null cells, interestingly, the impaire d Chk2 function in NBS cells correlates with the inability, unlike normal c ells, to stop entry into mitosis immediately after irradiation, a checkpoin t abnormality that can be corrected by introduction of the wild-type but no t the S343A mutant form of Nbs1, Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest th at checkpoint defects in NBS cells may result from the inability to activat e Chk2.