Active role for nibrin in the kinetics of atm activation.
The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. Previously, we showed that a C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, was sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, we expressed an Mre11 transgene with a C-terminal NLS sequence in NBS fibroblasts. The Mre11-NLS protein complexed with Rad50, localized to the nucleus in NBS fibroblasts, and associated with chromatin. However, Atm autophosphorylation was not stimulated in cells expressing Mre11-NLS, nor were downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, we expressed nibrin transgenes lacking the Atm binding domain in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation was dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction.
Molecular Genetics Program, Benaroya Research Institute, Seattle, WA 98101, USA.
Karen
K
Cerosaletti
Jocyndra
J
Wright
Patrick
P
Concannon
D017766:Acid Anhydride Hydrolases; D021581:Active Transport, Cell Nucleus; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D019457:Chromosome Breakage / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics / Q000473:pathology; D000076228:MRE11 Homologue Protein; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013577:Syndrome; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes