Autophosphorylation and ATM activation: Additional sites add to the complexity

Kozlov, Sergei V., Graham, Mark E., Jakob, Burkhard, Tobias, Frank, Kijas, Amanda W., Tanuji, Marcel, Chen, Philip, Robinson, Phillip J., Taucher-Scholz, Gisela, Suzuki, Keiji, So, Sairai, Chen, David and Lavin, Martin F. (2011) Autophosphorylation and ATM activation: Additional sites add to the complexity. The Journal of Biological Chemistry, 286 11: 9107-9119. doi:10.1074/jbc.M110.204065

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Author Kozlov, Sergei V.
Graham, Mark E.
Jakob, Burkhard
Tobias, Frank
Kijas, Amanda W.
Tanuji, Marcel
Chen, Philip
Robinson, Phillip J.
Taucher-Scholz, Gisela
Suzuki, Keiji
So, Sairai
Chen, David
Lavin, Martin F.
Title Autophosphorylation and ATM activation: Additional sites add to the complexity
Journal name The Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 2011-03-18
Year available 2010
Sub-type Article (original research)
DOI 10.1074/jbc.M110.204065
Open Access Status File (Publisher version)
Volume 286
Issue 11
Start page 9107
End page 9119
Total pages 13
Place of publication Bethesda, MD, U.S.A.
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Subject 1303 Biochemistry
1312 Molecular Biology
1307 Cell Biology
Abstract The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser(367), Ser(1893), and Ser(1981)) and acetylation on Lys(3016). We now describe the identification of a new ATM phosphorylation site, Thr(P)(1885) and an additional autophosphorylation site, Ser(P)(2996), that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser(367) and Ser(2996), for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)(1981), ATM phosphorylated at Ser(367) and Ser(2996) localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser(367) and Ser(2996) was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.
Formatted abstract
The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser367, Ser1893, and Ser1981) and acetylation on Lys3016. We now describe the identification of a new ATM phosphorylation site, Thr(P)1885 and an additional autophosphorylation site, Ser(P)2996, that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser 367 and Ser2996, for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)1981, ATM phosphorylated at Ser367 and Ser2996 localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser367 and Ser2996 was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Keyword Double-strand breaks
Dna-damage response
Ataxia-telangiectasia
Dna-damage response
Mrn complex
Mre11-rad50-Nbs1 complex
Chromatin retention
Repair proteins
Nuclear foci
N-terminus
In-vivo
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID CA050519
Institutional Status UQ
Additional Notes First Published on December 13, 2010,

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
School of Medicine Publications
 
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Created: Mon, 28 Mar 2011, 19:50:08 EST by Debbie Banks on behalf of School of Medicine