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.

Keywords:

• ATM
• Chk2
• DNA Damage
• NBS1
• phosphorylation
• p53

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank all the members of the Ouchi Laboratory for helpful discussion. We particularly thank Dr Bert Vogelstein at Johns Hopkins University for providing us with HCT116 Chk2(-) cell line.

Funding

This work is supported by grants R01CA79892, R01CA90631, and 5P30CA16056 from National Institutes of Health, Breast Cancer Research Grant from Susan G. Komen Foundation.

Author Contribution

MO and TO planned and generated all the data, discussed the results and wrote the paper.