Wip1, the product of the PPM1D gene, is a PP2C serine/threonine protein phosphatase that was first identified in my laboratory as a gene whose induction following DNA damage required wild-type p53. The gene PPM1D is amplified and/or overexpressed in several types of human cancers. Our results suggest that Wip1 phosphatase promotes tumorigenesis through inactivation of p53. As an enzyme, Wip1 has the potential to be inactivated by low molecular weight chemical compounds. We have identified specific Wip1 inhibitors through combined use of rational design and screening assays and are pursuing various optimization strategies. To better understand the connection between Wip1 activity and tumorigenesis, we are investigating the regulation of Wip1 expression and activity, identifying targets of Wip1 phosphatase activity, and developing inhibitors of Wip1 phosphatase activity.Regulation of Wip1 expression and activityThe transcriptional induction of Wip1 following exposure to DNA damaging agents requires functional p53 protein. p53 and Wip1 form a negative feedback loop, with Wip1 phosphatase activity promoting the return to homeostasis by removing activating phosphorylations of p53 itself and those of its upstream kinases, ATM, Chk1 and Chk2. Following exposure to IR, the level of Wip1 protein changes more dramatically than its mRNA levels. In our current work, we have established that in Jurkat and U2OS cells, two disparate human tumor cell lines, the level of Wip1 protein increased about 4-fold during S phase without appreciable change in the level of Wip1 mRNA. These studies suggest that Wip1 is regulated on many levels.Under conventional culture conditions, Wip1-/- mouse embryonic fibroblasts (MEFs) undergo premature senescence. We have investigated the mechanism by which Wip1 reduces premature senescence in MEFs. We found that reduced oxygen pressure only partially suppressed premature senescence. Compared with wild type cells, early passage Wip1-/- MEFs under both 20% and 3% oxygen conditions exhibited increased activation of p53 and increased levels of p21, p16Ink4a and p19Arf. The absence of p38 MAPK activation implies that Wip1 may regulate the Ink4a/Arf locus through a novel pathway. These findings suggest that Wip1 prevents cellular senescence by regulating both the DNA Damage Response and p16Ink4a/p19Arf signaling.Current results characterizing the effects of Wip1 deletion in mice are based on a non-conditional knockout mouse. Despite the utility of this mouse, it does not allow us to determine the specific effects of Wip1 deletion in a single tissue. Ubiquitous Wip1 deletion in mice affects the immune system, organismal metabolism and the tumor micro-environment, any of which may affect tumorigenesis in the organ of interest. To overcome these limitations, we have developed a conditional knock-out mouse in which Wip1 deletion can be directed to a single tissue through tissue-specific expression of Cre recombinase or through inducible expression of Cre recombinase to induce deletion at a specified time. We are in the process of generating these conditional knock-out mice, which will be used to study the effects of Wip1 in several mouse tumor models. Wip1 phosphatase activity and substrate identificationWip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs, and we have used biochemical methods to characterize its substrate specificity. Many of the known pTQ/pSQ substrates of Wip1 are phosphorylated by ATM. In addition to its role in the response to DNA damage, ATM regulates the cellular response to insulin through phosphorylation of Ser112 of the inhibitor 4EBP1, resulting in its dissociation from the translation initiation protein, eIF-4E. We are investigating Ser112 of 4EBP1 as a potential Wip1 substrate. Currently, the majority of anticancer therapies use the p53 pathway to induce tumor cells death. We have explored an alternative approach, based on activation of Wip1 phosphatase, toward sensitizing tumors that lack wildtype p53 to anticancer drugs, while at the same time protecting normal tissues. In response to anticancer drugs in tumors with inactive p53, Wip1 overexpression led to the induction of Bax through dephosphorylation-dependent activation of the transcription factor RUNX2. We found that Wip1 interacted with RUNX2 in Saos-2-Wip1-on cells. Mutation of RUNX2 Ser432 to alanine resulted in significantly increased transcriptional activity of the Bax promoter, compared with WT RUNX2. To confirm that phospho-Ser432 of RUNX2 was a target for Wip1, we synthesized a human RUNX2 peptide containing phosphorylated Ser432 and measured the peptide concentration dependence of Wip1 phosphatase activity. The resulting kinetic constants were similar to those obtained with the Chk1 345pS phosphopeptide, a well known substrate for Wip1.Wip1 Inhibitors and structure of the Wip1 catalytic domainMany human tumors in which the PPM1D gene is amplified and/or overexpressed contain wild type p53. Our published results indicate that Wip1 phosphatase promotes tumorigenesis through inactivation of wild-type p53. Phosphatases in general play critical physiological roles as antagonists of kinase activity and, as such, represent important targets in a number of diseases, including cancer. We have continued to develop pyrrole-based small molecule inhibitors of Wip1. The synthesis of these inhibitors has been improved by decreasing the number of synthetic steps and optimization of the synthetic scheme for solution phase synthesis. The new synthesis is scalable, allowing us to prepare 100 mg amounts. We have already synthesized 30 different analogs. The new synthesis has been adapted to the synthesis of a pro-drug, in which the phosphate groups are masked to improve cellular uptake. We have already synthesized a pro-drug form of the pyrrole-based inhibitors and have successfully tested its activity in a cell-based assay. PP2C serine/threonine protein phosphatases are critical regulators of stress responses and are distinguished by divalent metal ion-dependent stimulation of in vitro phosphatase activity. In humans, PP2C-alpha (PPM1A) functions as a tumor suppressor whereas Wip1 (PPM1D) negatively regulates several tumor suppressors. Although a crystal structure of human PP2C-alpha was shown to contain two bound Mn2+ ions, details of the catalytic mechanism and determinants of substrate specificity remain incompletely understood. Recently, structural studies of several prokaryotic PP2C phosphatases demonstrated the presence of three or four bound metal ions. As most of the coordinating residues for the additional metal ions are highly conserved, these results anticipate additional metal binding sites in human PP2C-alpha and PPM1D phosphatases. We have used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize additional metal binding site(s). These results suggest that the binding of additional metal ions to human PP2C phosphatases is essential for catalytic activity and open prospects for the development of specific inhibitors.
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