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Molecular and Cellular Biology, August 2001, p. 5214-5222, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.5214-5222.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Chk2 Activation Dependence on Nbs1 after DNA Damage

Giacomo Buscemi,1 Camilla Savio,2 Laura Zannini,1 Francesca Miccichè,1 Debora Masnada,1 Makoto Nakanishi,3 Hiroshi Tauchi,4 Kenshi Komatsu,4 Shuki Mizutani,5 KumKum Khanna,6 Phil Chen,6 Patrick Concannon,7 Luciana Chessa,2 and Domenico Delia1,*

Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan,1 and Department of Experimental Medicine and Pathology, University "La Sapienza," 00161 Rome,2 Italy; Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601,3 Department of Radiation Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553,4 and Department of Pediatrics and Developmental Biology, Postgraduate Medical School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519,5 Japan; The Queensland Institute of Medical Research, Brisbane, Queensland, Australia6; and Molecular Genetics Program, Virginia Mason Research Center and Department of Immunology, University of Washington School of Medicine, Seattle, Washington7

Received 3 November 2000/Returned for modification 8 January 2001/Accepted 3 May 2001

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 gamma - 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.

* Corresponding author. Mailing address: Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. Phone: 39-02-23902641. Fax: 39-02-23902764. E-mail: delia{at}istitutotumori.mi.it.

Molecular and Cellular Biology, August 2001, p. 5214-5222, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.5214-5222.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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