Wip1 regulation

Wip1/PPM1D is a type 2C family serine/threonine phosphatase able to dephosphorylate central players in the DNA damage response (DDR). Wip1 removes DNA damage-induced phosphorylation in p53, H2AX, ATM, Chk2 and p38MAPK among others.^1-3 Therefore, Wip1 works like a reset button to inactivate the DNA damage response when the DNA is repaired. Since many of the known targets of Wip1 are tumor suppressors, amplification of the PPM1D gene occurs in some primary cancers, and its deletion in mice causes a tumor resistance phenotype.^3,4

To accomplish these important functions in the cell, Wip1 should be tightly controlled at several levels. In spite the remarkable interest in studying how Wip1 activity/levels are controlled, not much is yet known. For example, Wip1 expression is negatively controlled by miR-16, a microRNA-induced at early time points after DNA damage, to avoid Wip1 activity at the initiation of the DDR.⁵ Also, Wip1 is a transcriptional target for p53, working in a negative feedback loop to inactivate p53.⁶

The elegant work of Macurek and colleagues sheds additional light on the mechanism of regulation of Wip1.⁷ Using state-of-the-art techniques and different rigorous approaches, they demonstrated that the levels of Wip1 are regulated during the cell cycle: high during the S and G₂ phases of the cell cycle and low during mitosis. Wip1 regulation during mitosis is mediated by ubiquitin-dependent proteasome degradation controlled by the anaphase-promoting complex/cyclosome (APC/C) and its activator Cdc20.

Moreover, Macurek and coworkers showed regulation of Wip1 enzymatic activity by phosphorylation of multiple sites in the N-terminal catalytic domain. By mass spectrometry they identified several residues, which, when mutated, did not produce a gel-mobility shift during mitosis that was observed with the wild-type version of the protein. One of these residues (among seven) was shown to be phosphorylated by Cdk1 in vitro. Interestingly, the phosphorylation of these serines and/or threonine inactivates Wip1, as mutations to alanine do not affect the in vitro activity, but a phospho-mimicking mutant is phosphatase inactive.

Consequently, Macurek and colleagues unequivocally showed that Wip1 is degraded and inactivated during mitosis. Why does this occur? The importance of these findings does not seem to be the regulation of mitosis, as the lack of Wip1 did affect mitotic progression, but Wip1 regulation is related to the DDR modulation. Wip1 maintains cells competent for re-entry into the cell cycle after DNA damage in G₂ phase. Therefore, Wip1 high levels and/or activity during G₂ compared with mitosis are likely to be required for this purpose. Does the degradation of Wip1 have a functional role then? Macurek and colleagues suggest that it is, indeed, important for the DDR. During mitosis, DNA damage largely remains unrepaired, but repair can start during the next G₁ phase.^8,9 Therefore, the low levels and/or activity of Wip1 during mitosis might lead to a decrease in the threshold for the DDR (mainly H2AX phosphorylation), which may help in the subsequent repair during the following G₁ phase. Hence, cells may be able to sense low levels of endogenous DNA damage that occurs at underreplicated chromatin regions during normal mitotic progression.⁹ Indeed, Macurek et al. showed that the overexpression of wild type Wip1 during mitosis led to a decrease of γH2AX during mitosis and less 53BP1 focus formation during G₁. In contrast, the expression of a phosphomimicking mutant had a low impact on 53BP1 focus formation during G₁ phase.

The article by Macurek et al. also leaves some open questions that will be interesting to address in the future. From the mechanistic point of view, the work suggests the existence of other post-translational modifications in Wip1 (possible phosphorylations) that might be dependent on cell cycle and/or DNA damage. The molecular details of how Cdc20 recognizes Wip1 to target it for degradation also remain to be elucidated. Finally, the kinase(s) responsible for the mitotic phosphorylation of Wip1 and investigating if these kinases are themselves targets of the phosphatase will be subjects to study. From a pathological point of view, this work opens the possibility to examine the impact of a non-degradable Wip1 or mutants in phosphorylated residues during mitosis on cancer predisposition in mice. Especially interesting to study is the phosphomimicking mutant, as it possibly may be more resistant to tumor formation but might not show some of the secondary effects that the Wip1-knockout mouse has.

Macurek L, Benada J, Müllers E, Halim VA, Krejčíková K, Burdová K, et al. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis. Cell Cycle. 2012;12:251–62. doi: 10.4161/cc.23057.