Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Mar;26(5):1691-9.
doi: 10.1128/MCB.26.5.1691-1699.2006.

Active role for nibrin in the kinetics of atm activation

Karen Cerosaletti  1 , Jocyndra Wright, Patrick Concannon
Affiliations

Active role for nibrin in the kinetics of atm activation

Karen Cerosaletti et al. Mol Cell Biol. 2006 Mar.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Expression of the Mre11-NLS transgene in NBS cells and interaction with Rad50. (A) Western blot analysis of GM0637 fibroblasts or NBS-ILB1 cells stably transduced with the pLXIN retroviral vector alone (LXIN), wild-type nibrin (NBS1), wild-type Mre11 (Mre11), or an Mre11-NLS transgene with two artificial NLS sequences and an HA tag attached to the Mre11 C terminus. Individual clones of NBS cells expressing the Mre11-NLS transgene were isolated (NLS.8, NLS.11, and NLS.12). A total of 25 μg of total cellular protein per lane was separated on a 7% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The Western blot (WB) was probed with monoclonal antibodies specific for Mre11 or the HA tag. (B) Mre11 was immunoprecipitated (IP) from 250 μg of total cellular protein isolated from the above cell lines using a polyclonal anti-Mre11 antibody or a monoclonal anti-HA antibody. Immunoprecipitated proteins were separated on a 3 to 8% SDS-polyacrylamide gel, and the immunoblot was probed with a polyclonal Rad50 antisera or a monoclonal anti-Mre11 antibody. The arrows indicate the positions of endogenous Mre11 protein and the higher-molecular-weight Mre11-NLS protein.
FIG. 2.
FIG. 2.
Subcellular localization of the Mre11-NLS protein in NBS cells. (A) Cytoplasmic and nuclear proteins were isolated from GM0637 control fibroblasts or NBS-ILB1 cells stably transduced with the pLXIN retroviral vector alone (LXIN), wild-type nibrin (NBS1), wild-type Mre11 (Mre11), or the Mre11-NLS transgene (NLS.8, NLS.11, and NLS.12). A total of 105 cell equivalents of protein per lane were electrophoresed on 7% SDS-polyacrylamide gels. Western blots (WB) were probed with a monoclonal anti-Mre11 antibody, a polyclonal antisera specific for the cytoplasmic heat shock protein (Hsp90), or a monoclonal antibody specific for the telomeric protein, TRF2. The arrows indicate the positions of endogenous Mre11 protein and the higher-molecular-weight Mre11-NLS protein. (B) Immunofluorescence analysis was performed on NBS-ILB1 cells stably transduced with the pLXIN retroviral vector alone, wild-type nibrin (NBS1), or the Mre11-NLS transgene (NLS.8). Fibroblasts were stained for nibrin and Mre11 expression using a polyclonal anti-nibrin antibody (red) and a monoclonal antibody to Mre11 (green). The Mre11-NLS protein was specifically detected using a monoclonal antibody specific for the HA tag (HA, green). Immunofluorescence was detected by confocal microscopy at 488 and 568 nm. Individual fields were Z-planed and stacked. Magnification, ×600.
FIG. 3.
FIG. 3.
Atm activation and function after irradiation in NBS cells expressing the Mre11-NLS protein. (A) Atm autophosphorylation at serine 1981 was assessed in NBS-ILB1 cells stably transduced with the pLXIN vector alone (LXIN), wild-type nibrin (NBS1), nibrin lacking the C-terminal 100 aa (Nb652), a C-terminal 300-aa fragment of nibrin (NbFR5), wild-type Mre11 (Mre11), or the Mre11-NLS transgene (NLS.8, NLS.11, and NLS.12). Cells were unirradiated or exposed to 3 Gy of ionizing radiation (IR), and total cellular lysates were harvested after 15 min. Atm was immunoprecipitated (IP) from 800 μg of protein using a polyclonal anti-Atm antibody and protein was separated on 3 to 8% SDS-polyacrylamide gels. Western blots (WB) were probed with a monoclonal phosphoserine 1981 antibody or a polyclonal Atm antibody. (B) Chk2 threonine 68 phosphorylation and p53 serine 15 phosphorylation by Atm were assessed in the above cells, as well as GM0637 control fibroblasts, 15 min after exposure to 0 or 3 Gy of ionizing radiation. A total of 20 μg of total cellular protein per lane was separated on 7% SDS-polyacrylamide gels. Immunoblots were probed with a phosphothreonine 68 antisera or a monoclonal anti-Chk2 antibody. For p53, the immunoblots were probed with phosphoserine 15 antisera or a monoclonal p53 antibody.
FIG. 4.
FIG. 4.
Atm activation and function after irradiation in ATLD cells expressing the Mre11-NLS protein. (A) Expression of the Mre11 NLS transgene in ATLD cells was assessed by Western blot analysis. Total cellular lysates were isolated from GM0637 control fibroblasts and ATLD D6809 cells stably transduced with the pLXIN retroviral vector (ATLD LXIN), wild-type Mre11 (ATLD Mre11), or the Mre11-NLS transgene (ATLD NLS). Then, 25 μg of protein per lane was electrophoresed on a 7% SDS-polyacrylamide gel and Western blotted (WB). The immunoblot was probed with a monoclonal anti-Mre11 antibody or a polyclonal antibody specific for Hsp90, as a loading control. The arrows indicate the endogenous Mre11 protein and the higher-molecular-weight Mre11-NLS protein. (B) Nibrin phosphorylation in the above cell lines was analyzed 15 min after exposure to 0 or 3 Gy of ionizing radiation (IR). Nibrin was immunoprecipitated (IP) from 250 μg of total cellular protein using a polyclonal antinibrin antibody and proteins were separated on a 3 to 8% SDS-polyacrylamide gel. Western blots were probed with a phosphoserine 343 monoclonal antibody or a polyclonal antibody specific for nibrin. To assess Mre11-NLS complex formation with nibrin, the blot was reprobed with a monoclonal anti-Mre11 antibody. (C) Atm autophosphorylation at serine 1981 was assessed in the above cells lines 15 min after exposure to 0 or 3 Gy of irradiation. Next, 25 μg of total cellular protein per lane was electrophoresed on a 3 to 8% SDS-polyacrylamide gel. The Western blot was probed with a monoclonal phosphoserine 1981 antibody or a polyclonal anti-Atm antisera. Chk2 threonine 68 phosphorylation was analyzed by probing a similar immunoblot with phosphothreonine 68 antisera or a monoclonal anti-Chk2 antibody. To detect p53 serine 15 phosphorylation, immunoblots were probed with phosphoserine 15 antisera or a monoclonal p53 antibody. (D) Irradiation-induced foci were examined 6 h after 12 Gy of irradiation in the above ATLD fibroblasts. Mre11 or Mre11-NLS foci were detected with Mre11 antisera (red) and γ-H2AX foci were detected with a monoclonal γ-H2AX antibody (green). Immunofluorescence was collected by using confocal microscopy at 488 and 568 nm. Individual fields were Z-planed and stacked. Magnification, ×1,000.
FIG. 5.
FIG. 5.
Localization of the Mre11-NLS protein to chromatin in NBS cells. NBS-ILB1 cells stably transduced with the pLXIN retroviral vector alone (LXIN), wild-type nibrin (NBS1), or the Mre11-NLS transgene (Mre11-NLS) were unirradiated or exposed to 3 Gy of ionizing radiation. After 30 min cells were harvested and fractionated into cytoplasmic (lanes C), nuclear soluble (lanes NS), nuclear 125 mM NaCl wash (lanes 125), nuclear 250 mM NaCl wash (lanes 250), and the insoluble nuclear pellet (lanes NP). For comparison, whole-cell lysate was included (lanes WC). A total of 105 cell equivalents of protein per lane was separated on 3 to 8% SDS-polyacrylamide gels and Western blotted (WB). (A) Immunoblots of pLXIN cellular fractions were probed with an anti-Mre11 monoclonal antibody to assess the localization of Mre11 in the absence of full-length nibrin. The blot was reprobed with anti-Hsp90 antisera as a control for cytoplasmic proteins or with a monoclonal TRF2 antibody as a control for nuclear proteins. (B) Localization of Mre11 in cellular fractions from LXIN, NBS1, and Mre11-NLS expressing cells was detected by probing immunoblots with a monoclonal Mre11 antibody.
FIG. 6.
FIG. 6.
Expression of nibrin ΔAtm transgenes in NBS cells and Atm activation after irradiation. (A) Expression and MRN complex formation was analyzed in GM0637 control fibroblasts and NBS-ILB1 cells stably transduced with the pLXIN retroviral vector alone (LXIN), wild-type nibrin (NBS1), nibrin lacking the C-terminal Atm binding domain (NBS1 ΔAtm), a 300-aa C-terminal fragment of nibrin (NbFR5), and NbFR5 lacking the C-terminal Atm binding domain (NbFR5 ΔAtm). Nibrin was immunoprecipitated (IP) from 250 μg of total cellular protein using polyclonal nibrin antisera and proteins were separated on a 3 to 8% SDS-polyacrylamide gel. The Western blot (WB) was probed with an anti-Mre11 monoclonal antibody, a polyclonal Rad50 antibody, or a monoclonal nibrin antibody. (B) Atm autophosphorylation at serine 1981 was assessed in the above cells 15 min after treatment with 0 or 3 Gy of ionizing radiation (IR). Atm was immunoprecipitated from 800 μg of total cellular protein using a polyclonal Atm antibody and proteins were separated on 3 to 8% SDS-polyacrylamide gels. Immunoblots were probed with a monoclonal phosphoserine 1981 antibody or a polyclonal Atm antibody. To detect Chk2 and p53 phosphorylation, 20 μg of total cellular protein isolated from the above cells 15 min after 0 or 3 Gy of irradiation was separated on 7% SDS-polyacrylamide gels. Immunoblots were probed with phosphothreonine 68 antisera or a Chk2 monoclonal antibody. For p53, Western blots were probed with phosphoserine 15 antisera or a monoclonal p53 antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bakkenist, C. J., and M. B. Kastan. 2003. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499-506. - PubMed
    1. Banin, S., L. Moyal, S. Shieh, Y. Taya, C. W. Anderson, L. Chessa, N. I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281:1674-1677. - PubMed
    1. Carney, J. P., R. S. Maser, H. Olivares, E. M. Davis, M. Le Beau, J. R. Yates III, L. Hays, W. F. Morgan, and J. H. Petrini. 1998. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93:477-486. - PubMed
    1. Carson, C. T., R. A. Schwartz, T. H. Stracker, C. E. Lilley, D. V. Lee, and M. D. Weitzman. 2003. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J. 22:6610-6620. - PMC - PubMed
    1. Cerosaletti, K., and P. Concannon. 2004. Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm. J. Biol. Chem. 279:38813-38819. - PubMed

Publication types

MeSH terms