). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine (SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of fulllength Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damageinduced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.