title: Distinct DNA damage determines differential phosphorylation of Chk2.
abstract: Checkpoint kinase 2 (Chk2) has been implicated in DNA damage signaling. By using BJ human fibroblasts, HCT116 colorectal cancer cells and HeLa cervical cancer cells, we further detailed phosphorylation kinetics of Chk2 under treatment with neocarcinostatin (NCS) or doxorubicin (Dox). After NCS treatment, phosphorylation of Chk2 Thr68 occurs in 3 min, followed by phosphorylation of Ser19 and Ser33/35. In ATM deficient fibroblasts, NCS does not induce phosphorylation of NBS1 Ser343 and Chk2 Ser19 and Ser33/35, however Chk2 Thr68 is still phosphorylated, indicating that ATM is essential for phosphorylation of these residues when treated with NCS. By using Chk2-deficient HCT116 cells re-expressing phospho-mutant Chk2 (T68A), we found that inhibition of Thr68 phosphorylation enhances Ser19 phosphorylation in NCS treated cells. Interestingly, in contrast to NCS, Dox does not induce Ser33/35 phosphorylation in HeLa and HCT116 cells. Phosphorylation of Thr68 is sustained until 3 to 4 hours, and phosphorylation of Ser19 occurs 70 to 80 min after Dox treatment. These results demonstrate that Chk2 s involved in the early stages of DNA damage response. Differential phosphorylation kinetics of these residues suggests that DNA damage determines intermolecular and intramolecular interaction of Chk2, which may regulate phosphorylation.
authors.affiliation: a Department of Cancer Genetics; Roswell Park Cancer Center ; Buffalo , NY USA.
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Ouchi
authors.forename: Toru
authors.initials: T
authors.lastname: Ouchi
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2 / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D004317:Doxorubicin; D064447:Checkpoint Kinase 2
keywords: ATM; ATM, ataxia telangiectasia mutated; ATR, ATM related kinase; Chk2; DNA Damage; Dox, doxorubicin; NBS1; NCS: neocarzinostatin; PAGE: poly acryl gel electrophoresis; p53; phosphorylation