Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

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.

The Wip1 phosphatase (PPM1D) antagonizes activation of the Chk2 tumour suppressor kinase

“…While this manuscript was in preparation, Fujimoto et al (2005) published results similar to ours regarding the binding of Wip1 to Chk2 and the dephosphorylation of phospho-Thr68 . However, there are key differences in some of our results and interpretations.…”

“…However, there are key differences in some of our results and interpretations. Fujimoto et al (2005) did not determine whether or not IR stimulates the interaction of Wip1 and Chk2 within cells as we did. They also did not report dose-response analyses that allowed us to distinguish the increased catalytic efficiency and specificity of Wip1 towards phospho-Thr68 compared with the other Chk2 phosphorylation sites.…”

“…Fujimoto et al (2005) also found that the addition of a large excess of GST-Wip1 (1 mg) to preactivated Chk2 led to phospho-Thr68 dephosphorylation and a 50% inhibition of Chk2 kinase activity towards a GSTCdc25C substrate. They concluded that Wip1 treatment resulted in a 'drastic inhibition of Chk2 kinase activity [y] in vitro' and they suggested that this effect might be important for Chk2 inhibition by Wip1 in vivo (Fujimoto et al, 2005). We also observed a partial inhibition of preactivated Chk2 kinase activity in vitro upon the addition of large quantities of Wip1 (100-500 ng).…”

“…Finally, they did not find phosphoSer516 to be a substrate for Wip1, whereas we found that it is readily dephosphorylated by Wip1, PP2Ca and CIP in vitro. We do not know the basis for these discrepancies, but it has some import in that Fujimoto et al (2005). showed that Wip1 antagonizes Chk2-dependent apoptosis of g-irradiated MCF7 breast cancer cells, and Chen and colleagues previously established, in the HCT15 cell line, a role for Chk2 Ser516 phosphorylation in stimulating Chk2-dependent apoptosis (Wu and Chen, 2003).…”

The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis

“…Previous analysis showed that Wip1 could modulate apoptotic response and proliferation (Bulavin et al, 2004;Belova et al, 2005), which could explain the early onset of tumors observed in MMTV-ErbB2/ PPM1D mice. While there was no significant difference in the rate of apoptosis between analyzed samples Several potential downstream targets of Wip1 have been identified in vitro: p38 MAPK, p53, Chk1, Chk2 and ataxia-telangiectasia mutated (ATM) (Takekawa et al, 2000;Lu et al, 2005;Fujimoto et al, 2006;Shreeram et al, 2006). The role of p38 MAPK is particularly interesting, as we previously found that Wip1-deficient mice show a p38 MAPK-dependent delay in the onset of breast cancer when crossed with MMTV-ErbB2 transgenic mice (Bulavin et al, 2004).…”