Portal de Periódicos da CAPES

Threonine 68 of Chk2 Is Phosphorylated at Sites of DNA Strand Breaks

2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês

10.1074/jbc.c100587200

1083-351X

Irene M. Ward, Xianglin Wu, Junjie Chen,

Cancer-related Molecular Pathways

The protein kinase Chk2 has been implicated in signaling DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 becomes rapidly phosphorylated at threonine 68 by ataxia-telangiectasia mutated (ATM). Here we show that the Thr68-phosphorylated form of Chk2 forms distinct nuclear foci in response to ionizing radiation. Only this activated form of Chk2 localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decline correlating with the predicted value of slowly re-joining double strand breaks. These results suggest that Chk2 is regulated at the sites of DNA strand breaks in response to ionizing radiation. The protein kinase Chk2 has been implicated in signaling DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 becomes rapidly phosphorylated at threonine 68 by ataxia-telangiectasia mutated (ATM). Here we show that the Thr68-phosphorylated form of Chk2 forms distinct nuclear foci in response to ionizing radiation. Only this activated form of Chk2 localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decline correlating with the predicted value of slowly re-joining double strand breaks. These results suggest that Chk2 is regulated at the sites of DNA strand breaks in response to ionizing radiation. ionizing radiation ataxia-telangiectasia-mutated hydroxyurea glutathione S-transferase polyacrylamide gel electrophoresis hemagglutinin gray camptothecin Chk2 (Cds1), an evolutionary conserved protein kinase, is an important component of the DNA damage response pathway. Chk2−/− ES cells are defective in maintaining ionizing radiation (IR)1-induced G2arrest, and Chk2 null thymocytes fail to stabilize p53 and to induce G1 arrest and apoptosis (1Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1042) Google Scholar). Chk2 has also been reported to directly phosphorylate BRCA1 and to regulate its response to DNA damage (2Lee J.S. Collins K.M. Brown A.L. Lee C.H. Chung J.H. Nature. 2000; 404: 201-204Crossref PubMed Scopus (459) Google Scholar). Moreover, heterozygous mutations in the Chk2 gene have been identified in a subset of patients with Li-Fraumeni syndrome (3Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M. Li F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (749) Google Scholar), suggesting that Chk2 acts as a tumor suppressor.The activation of Chk2 in response to DNA damage requires phosphorylation at threonine 68 (Thr68) (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Chk2T68 mutants show reduced Chk2 kinase activation and a diminished induction of the p53-dependent G1 arrest in response to ionizing radiation (5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar). The phosphorylation of Chk2 at Thr68 in response to IR is ATM (ataxia-telangiectasia-mutated)-dependent, although an ATM-independent pathway exists in response to ultraviolet radiation (UV) and hydroxyurea (HU) treatment (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, little is known how Chk2 activation is initiated or how the activity of Chk2 is down-regulated following DNA repair. Given the physiological impact of the DNA damage pathway in genome stability and cancer prevention, it is necessary to gain a better understanding of the mechanisms underlying DNA damage signal transduction. Here we show that the Thr68-phosphorylated form of Chk2 (Chk2T68P) forms distinct nuclear foci in response to ionizing radiation. Only this activated form localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decrease with time. Together, our findings suggest that Chk2 activity is initiated and regulated at the sites of DNA strand breaks in response to ionizing radiation.DISCUSSIONOur data show that phosphorylation and activation of Chk2 by ATM correlate with the severity of DNA damage in vivo. The activated form of Chk2 localizes in distinct foci at the sites of DNA strand breaks within minutes following ionizing radiation. The disappearance of these foci coincides with that of γ-H2AX and 53BP1 and correlates with the reported time course of DNA repair (8Kodym R. Horth E. Int. J. Radiat. Biol. 1995; 68: 133-139Crossref PubMed Scopus (19) Google Scholar, 9Nunez M.I. Villalobos M. Olea N. Valenzuela M.T. Pedraza V. McMillan T.J. Ruiz de Almodovar J.M. Br. J. Cancer. 1995; 71: 311-316Crossref PubMed Scopus (72) Google Scholar, 11Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (1948) Google Scholar,12Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (399) Google Scholar). The phosphorylation and activation of Chk2 by ATM occurs most likely at the sites of DNA breaks. First, ATM can be directly activated by DNA breaks in vitro (14Smith G.C. Cary R.B. Lakin N.D. Hann B.C. Teo S.H. Chen D.J. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11134-11139Crossref PubMed Scopus (146) Google Scholar, 15Chan D.W. Son S.C. Block W. Ye R. Khanna K.K. Wold M.S. Douglas P. Goodarzi A.A. Pelley J. Taya Y. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2000; 275: 7803-7810Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Second, following DNA damage, ATM co-localizes with γ-H2AX foci in vivo (16Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). It is not yet known whether ATM directly phosphorylates Chk2 or this phosphorylation event requires a mediator. In budding yeast, phosphorylation and activation of the Chk2 homologue scRad53 by Mec1, an ATM homologue, require scRad9, a protein with C-terminal tandem BRCT motifs (BRCA1 C-terminus). It is intriguing that phosphorylated Chk2 co-localizes with 53BP1, a protein also containing C-terminal tandem BRCT motifs. It remains to be determined whether the phosphorylation and activation of Chk2 requires 53BP1 in mammals.The phosphorylation of Chk2 at Thr68 does not correlate with the ATM-dependent mobility shift of Chk2 following ionizing radiation. While Chk2T68 phosphorylation is rapid and transient (see Fig. 1 C), the mobility shift-associated hyperphosphorylation of Chk2 appears to be gradual and persists at least 24–48 h following DNA damage (17Buscemi G. Savio C. Zannini L. Micciche F. Masnada D. Nakanishi M. Tauchi H. Komatsu K. Mizutani S. Khanna K. Chen P. Concannon P. Chessa L. Delia D. Mol. Cell. Biol. 2001; 21: 5214-5222Crossref PubMed Scopus (179) Google Scholar). Indeed, a Chk2T68A mutant, which cannot be phosphorylated at Thr68 and is defective in activation following DNA damage, still shows the same mobility shift on SDS-PAGE as wild-type Chk2 (5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar). Thus, the mobility shift-associated hyperphosphorylation of Chk2 depends largely on the phosphorylation of sites distinct from Thr68. While Thr68phosphorylation of Chk2 correlates with the activation of Chk2 (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the function of this mobility shift-associated hyperphosphorylation of Chk2 remains to be solved. One possibility is that this mobility shift-associated hyperphosphorylation of Chk2 represents a certain negative feedback mechanism. Such complex regulation of kinase activity by various phosphorylation events has been documented for Raf-1 protein kinase. While Raf-1 is initially activated by specific phosphorylation events, the subsequent mobility shift-associated hyperphosphorylation of Raf-1 down-regulates Raf-1 activity and represents a negative feedback mechanism contributing to the desensitization of the signaling pathway (for example, see Ref. 18Wartmann M. Hofer P. Turowski P. Saltiel A.R. Hynes N.E. J. Biol. Chem. 1997; 272: 3915-3923Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Similar feedback mechanism may exist for Chk2 in the DNA damage-signaling pathway. Chk2 (Cds1), an evolutionary conserved protein kinase, is an important component of the DNA damage response pathway. Chk2−/− ES cells are defective in maintaining ionizing radiation (IR)1-induced G2arrest, and Chk2 null thymocytes fail to stabilize p53 and to induce G1 arrest and apoptosis (1Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1042) Google Scholar). Chk2 has also been reported to directly phosphorylate BRCA1 and to regulate its response to DNA damage (2Lee J.S. Collins K.M. Brown A.L. Lee C.H. Chung J.H. Nature. 2000; 404: 201-204Crossref PubMed Scopus (459) Google Scholar). Moreover, heterozygous mutations in the Chk2 gene have been identified in a subset of patients with Li-Fraumeni syndrome (3Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M. Li F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (749) Google Scholar), suggesting that Chk2 acts as a tumor suppressor. The activation of Chk2 in response to DNA damage requires phosphorylation at threonine 68 (Thr68) (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Chk2T68 mutants show reduced Chk2 kinase activation and a diminished induction of the p53-dependent G1 arrest in response to ionizing radiation (5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar). The phosphorylation of Chk2 at Thr68 in response to IR is ATM (ataxia-telangiectasia-mutated)-dependent, although an ATM-independent pathway exists in response to ultraviolet radiation (UV) and hydroxyurea (HU) treatment (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, little is known how Chk2 activation is initiated or how the activity of Chk2 is down-regulated following DNA repair. Given the physiological impact of the DNA damage pathway in genome stability and cancer prevention, it is necessary to gain a better understanding of the mechanisms underlying DNA damage signal transduction. Here we show that the Thr68-phosphorylated form of Chk2 (Chk2T68P) forms distinct nuclear foci in response to ionizing radiation. Only this activated form localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decrease with time. Together, our findings suggest that Chk2 activity is initiated and regulated at the sites of DNA strand breaks in response to ionizing radiation. DISCUSSIONOur data show that phosphorylation and activation of Chk2 by ATM correlate with the severity of DNA damage in vivo. The activated form of Chk2 localizes in distinct foci at the sites of DNA strand breaks within minutes following ionizing radiation. The disappearance of these foci coincides with that of γ-H2AX and 53BP1 and correlates with the reported time course of DNA repair (8Kodym R. Horth E. Int. J. Radiat. Biol. 1995; 68: 133-139Crossref PubMed Scopus (19) Google Scholar, 9Nunez M.I. Villalobos M. Olea N. Valenzuela M.T. Pedraza V. McMillan T.J. Ruiz de Almodovar J.M. Br. J. Cancer. 1995; 71: 311-316Crossref PubMed Scopus (72) Google Scholar, 11Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (1948) Google Scholar,12Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (399) Google Scholar). The phosphorylation and activation of Chk2 by ATM occurs most likely at the sites of DNA breaks. First, ATM can be directly activated by DNA breaks in vitro (14Smith G.C. Cary R.B. Lakin N.D. Hann B.C. Teo S.H. Chen D.J. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11134-11139Crossref PubMed Scopus (146) Google Scholar, 15Chan D.W. Son S.C. Block W. Ye R. Khanna K.K. Wold M.S. Douglas P. Goodarzi A.A. Pelley J. Taya Y. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2000; 275: 7803-7810Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Second, following DNA damage, ATM co-localizes with γ-H2AX foci in vivo (16Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). It is not yet known whether ATM directly phosphorylates Chk2 or this phosphorylation event requires a mediator. In budding yeast, phosphorylation and activation of the Chk2 homologue scRad53 by Mec1, an ATM homologue, require scRad9, a protein with C-terminal tandem BRCT motifs (BRCA1 C-terminus). It is intriguing that phosphorylated Chk2 co-localizes with 53BP1, a protein also containing C-terminal tandem BRCT motifs. It remains to be determined whether the phosphorylation and activation of Chk2 requires 53BP1 in mammals.The phosphorylation of Chk2 at Thr68 does not correlate with the ATM-dependent mobility shift of Chk2 following ionizing radiation. While Chk2T68 phosphorylation is rapid and transient (see Fig. 1 C), the mobility shift-associated hyperphosphorylation of Chk2 appears to be gradual and persists at least 24–48 h following DNA damage (17Buscemi G. Savio C. Zannini L. Micciche F. Masnada D. Nakanishi M. Tauchi H. Komatsu K. Mizutani S. Khanna K. Chen P. Concannon P. Chessa L. Delia D. Mol. Cell. Biol. 2001; 21: 5214-5222Crossref PubMed Scopus (179) Google Scholar). Indeed, a Chk2T68A mutant, which cannot be phosphorylated at Thr68 and is defective in activation following DNA damage, still shows the same mobility shift on SDS-PAGE as wild-type Chk2 (5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar). Thus, the mobility shift-associated hyperphosphorylation of Chk2 depends largely on the phosphorylation of sites distinct from Thr68. While Thr68phosphorylation of Chk2 correlates with the activation of Chk2 (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the function of this mobility shift-associated hyperphosphorylation of Chk2 remains to be solved. One possibility is that this mobility shift-associated hyperphosphorylation of Chk2 represents a certain negative feedback mechanism. Such complex regulation of kinase activity by various phosphorylation events has been documented for Raf-1 protein kinase. While Raf-1 is initially activated by specific phosphorylation events, the subsequent mobility shift-associated hyperphosphorylation of Raf-1 down-regulates Raf-1 activity and represents a negative feedback mechanism contributing to the desensitization of the signaling pathway (for example, see Ref. 18Wartmann M. Hofer P. Turowski P. Saltiel A.R. Hynes N.E. J. Biol. Chem. 1997; 272: 3915-3923Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Similar feedback mechanism may exist for Chk2 in the DNA damage-signaling pathway. Our data show that phosphorylation and activation of Chk2 by ATM correlate with the severity of DNA damage in vivo. The activated form of Chk2 localizes in distinct foci at the sites of DNA strand breaks within minutes following ionizing radiation. The disappearance of these foci coincides with that of γ-H2AX and 53BP1 and correlates with the reported time course of DNA repair (8Kodym R. Horth E. Int. J. Radiat. Biol. 1995; 68: 133-139Crossref PubMed Scopus (19) Google Scholar, 9Nunez M.I. Villalobos M. Olea N. Valenzuela M.T. Pedraza V. McMillan T.J. Ruiz de Almodovar J.M. Br. J. Cancer. 1995; 71: 311-316Crossref PubMed Scopus (72) Google Scholar, 11Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (1948) Google Scholar,12Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (399) Google Scholar). The phosphorylation and activation of Chk2 by ATM occurs most likely at the sites of DNA breaks. First, ATM can be directly activated by DNA breaks in vitro (14Smith G.C. Cary R.B. Lakin N.D. Hann B.C. Teo S.H. Chen D.J. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11134-11139Crossref PubMed Scopus (146) Google Scholar, 15Chan D.W. Son S.C. Block W. Ye R. Khanna K.K. Wold M.S. Douglas P. Goodarzi A.A. Pelley J. Taya Y. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2000; 275: 7803-7810Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Second, following DNA damage, ATM co-localizes with γ-H2AX foci in vivo (16Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). It is not yet known whether ATM directly phosphorylates Chk2 or this phosphorylation event requires a mediator. In budding yeast, phosphorylation and activation of the Chk2 homologue scRad53 by Mec1, an ATM homologue, require scRad9, a protein with C-terminal tandem BRCT motifs (BRCA1 C-terminus). It is intriguing that phosphorylated Chk2 co-localizes with 53BP1, a protein also containing C-terminal tandem BRCT motifs. It remains to be determined whether the phosphorylation and activation of Chk2 requires 53BP1 in mammals. The phosphorylation of Chk2 at Thr68 does not correlate with the ATM-dependent mobility shift of Chk2 following ionizing radiation. While Chk2T68 phosphorylation is rapid and transient (see Fig. 1 C), the mobility shift-associated hyperphosphorylation of Chk2 appears to be gradual and persists at least 24–48 h following DNA damage (17Buscemi G. Savio C. Zannini L. Micciche F. Masnada D. Nakanishi M. Tauchi H. Komatsu K. Mizutani S. Khanna K. Chen P. Concannon P. Chessa L. Delia D. Mol. Cell. Biol. 2001; 21: 5214-5222Crossref PubMed Scopus (179) Google Scholar). Indeed, a Chk2T68A mutant, which cannot be phosphorylated at Thr68 and is defective in activation following DNA damage, still shows the same mobility shift on SDS-PAGE as wild-type Chk2 (5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar). Thus, the mobility shift-associated hyperphosphorylation of Chk2 depends largely on the phosphorylation of sites distinct from Thr68. While Thr68phosphorylation of Chk2 correlates with the activation of Chk2 (4Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (680) Google Scholar, 6Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (260) Google Scholar, 7Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the function of this mobility shift-associated hyperphosphorylation of Chk2 remains to be solved. One possibility is that this mobility shift-associated hyperphosphorylation of Chk2 represents a certain negative feedback mechanism. Such complex regulation of kinase activity by various phosphorylation events has been documented for Raf-1 protein kinase. While Raf-1 is initially activated by specific phosphorylation events, the subsequent mobility shift-associated hyperphosphorylation of Raf-1 down-regulates Raf-1 activity and represents a negative feedback mechanism contributing to the desensitization of the signaling pathway (for example, see Ref. 18Wartmann M. Hofer P. Turowski P. Saltiel A.R. Hynes N.E. J. Biol. Chem. 1997; 272: 3915-3923Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Similar feedback mechanism may exist for Chk2 in the DNA damage-signaling pathway. We thank Drs. Scott Kaufmann, Larry Karnitz, and Jann Sarkaria for stimulating conversations and members of Dr. Junjie Chen's laboratory for helpful discussions.