1. ¹Department of Genome Sciences, Faculty of Medical Sciences, Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
2. ²Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3. ³PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan
4. ⁴School of Allied Health Science, Faculty of Medicine, Osaka University, 1-7, Yamada-oka, Suita 565-0871, Japan
5. ⁵Departments of Gastroenterology and Hematology, Hokkaido University, Graduate School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan
6. ⁶Departments of Hematology and Oncology, Hokkaido University, Graduate School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan

Correspondence: Y Minami, Department of Genome Sciences, Faculty of Medical Sciences, Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: +81-78-382-5560; Fax: +81-78-382-5579; E-mail: minami@kobe-u.ac.jp

⁷These authors contributed equally to this work

Received 1 August 2005; Revised 26 September 2005; Accepted 27 September 2005; Published online 25 November 2005.

Abstract

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.