The wild-type p53-induced phosphatase Wip1 (PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. The Wip1 protein is overexpressed and the PPM1D gene is amplified in several human cancers, and this increased expression is generally associated with a worse prognosis. Cellular studies have shown that overexpression of Wip1 compromises tumor suppressor functions, while other studies have demonstrated that mice lacking Wip1 expression are resistant to tumorigenesis. Our current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens to identify specific inhibitors of Wip1 phosphatase activity. Ubiquitous Wip1 deletion in mice affects the immune system, organismal metabolism and the tumor micro-environment, all of which may affect tumorigenesis in the organ of interest. To overcome these limitations, we have generated a conditional knock-out mouse in which the inactivating deletion of Ppm1d exon 3 can be directed to a single tissue through tissue-specific expression of Cre recombinase or implemented at a specified time through inducible expression of Cre recombinase. These conditional Wip1 knock-out mice will be useful in a variety of models of tumorigenesis. Recently, in collaboration with Dr. Oleg Demidov, University of Burgundy, France, we have been developing model systems to investigate the effects of deletion of Wip1 at various times. These model systems will allow us to investigate the roles of Wip1 in tumor initiation, tumor progression and metastasis. Wip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs when the surrounding amino acids are acidic, hydrophobic, or aromatic, whereas adjacent basic amino acids are inhibitory. Many of the known pTQ/pSQ substrates of Wip1 are phosphorylated by ATM. We have undertaken a quantitative phosphoproteomic analysis to provide an unbiased characterization of the substrate specificity of the Wip1 phosphatase. In this experiment, we have used the stable isotope labeling with amino acids in cell culture (SILAC) approach to label cells in culture for quantitation of the relative change in phosphorylation sites following cellular stress under conditions of high or low Wip1 activity. These studies, which are currently ongoing, will identify the sites of phosphorylation that are affected by Wip1 activity. We have optimized the sample preparation workflow for these experiments and are currently analyzing the samples using mass spectrometry. Preliminary experiments identified more than 800 phosphorylation sites of which nearly 10% are affected by modulation of Wip1 activity. Among the proteins containing phosphorylation sites affected by Wip1 activity are several transcription factors and kinases. These studies will provide critical insights into Wip1 substrates and function. 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. Previously, we used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize the binding of a third metal ion that is essential for Wip1 catalytic activity, and therefore identified a critical process that could be abrogated by the binding of a specific inhibitor. Recently, using hydrogen deuterium exchange - mass spectrometry (HDX-MS), we have probed PP2CA and a mutant lacking the third metal binding site to examine how the absence of the third metal modifies the conformational mobility of the enzyme structure. HDX-MS was used to study PP2CA and a mutant with an aspartic acid to alanine mutation at residue 146. The D146Amutant lacks the third metal binding site and is not catalytically active, although it is still able to bind phosphorylated substrates. Proteolytic digestion resulted in 94% and 83% sequence coverage for the wildtype and D146A mutant, respectively, with 71 identical peptides detected for both proteins. Deuterium uptake plots for both proteins showed that the mutant had a more rapid rate of deuterium uptake for peptides containing aspartic acid residues involved in forming the first and second metal binding sites. Additionally, peptides present in the Flap domain and the neighboring region had increased deuterium uptake in the mutant when compared to the wild-type. These data indicate that divalent metal ion binding to the third binding site results in a more rigid active site structure. Inhibition of Wip1 activity has been established as a possible way to limit the growth of tumors that retain wild-type p53. In the past, we have used our understanding of the Wip1 substrate motif to develop small molecule competitive inhibitors of Wip1 phosphatase activity with low micromolar inhibitory activity. To develop potent and highly specific activators and inhibitors of Wip1, we have initiated collaboration with Dr. Nathan P. Coussens at the National Center for Advancing Translational Sciences (NCATS). We have designed and optimized a plate-based assay to screen for compounds that inhibit or activate purified Wip1 activity. This sensitive assay has been miniaturized to a 1536-well format with 4 microL volumes to allow high throughput screening of NCATS compound libraries for inhibitors and activators of the Wip1 phosphatase. Also, we are pursuing several structural approaches to provide additional information about the Wip1 catalytic site, the flap domain, the catalytic mechanism, and determinants of substrate specificity.
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