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. Resulting from genomic amplification, transcriptional dysregulation, or impaired protein degradation, the Wip1 protein is frequently overexpressed in several human cancers, and this increased expression is generally associated with a worse prognosis. Cellular studies have shown that overexpression of Wip1 compromises the functioning of several tumor suppressors, 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 restricted 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 are proving to 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 identify sites of phosphorylation that are affected by Wip1 activity. 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 provide critical insights into Wip1 substrates and function. PP2C serine/threonine protein phosphatases are critical regulators of stress responses and are distinguished by a requirement for supplementation with millimolar concentrations of divalent metal ions to exhibit 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 the catalytic activity of Wip1 and that of a related PP2C phosphatase, PP2Calpha, and therefore identified a critical process that could be abrogated by the binding of a specific inhibitor. The binding of the third Mg2+ to PP2Calpha phosphatase has millimolar affinity and is entropically driven, suggesting it may have a structural as well as a catalytical role. We have used hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and molecular dynamics to investigate conformational changes in the PP2Calpha phosphatase between the active and inactive states. HDX-MS was used to study PP2Calpha and a mutant with an aspartic acid to alanine mutation at residue 146. The D146A mutant lacks the third metal binding site and is not catalytically active, although it is still able to bind phosphorylated substrates. 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 wildtype. These observations suggest that the binding of Mg2+ to the D146 binding site stabilizes the conformation of the active site and the Flap subdomain and results in a more rigid active site structure. This research has been published in Biochemistry. 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 are collaborating with the National Center for Advancing Translational Sciences (NCATS) to screen for novel activators and inhibitors. We have designed and optimized two different assays to measure Wip1 phosphatase activity that are compatible with the small-volume robotics used at NCATS. 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011197-09
Application #
9556488
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Alam, Muhammad S; Gaida, Matthias M; Debnath, Subrata et al. (2018) Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation. PLoS Biol 16:e2004111
Tagad, Harichandra D; Debnath, Subrata; Clausse, Victor et al. (2018) Chemical Features Important for Activity in a Class of Inhibitors Targeting the Wip1 Flap Subdomain. ChemMedChem 13:894-901
Giardino Torchia, Maria Letizia; Dutta, Debjani; Mittelstadt, Paul R et al. (2018) Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome. Proc Natl Acad Sci U S A 115:2174-2179
Debnath, Subrata; Kosek, Dalibor; Tagad, Harichandra D et al. (2018) A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity. J Biol Chem 293:7993-8008
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Dudgeon, Crissy; Shreeram, Sathyavageeswaran; Tanoue, Kan et al. (2013) Genetic variants and mutations of PPM1D control the response to DNA damage. Cell Cycle 12:2656-64
Goloudina, Anastasia R; Tanoue, Kan; Hammann, Arlette et al. (2012) Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents. Proc Natl Acad Sci U S A 109:E68-75
Lowe, Julie; Cha, Hyukjin; Lee, Mi-Ok et al. (2012) Regulation of the Wip1 phosphatase and its effects on the stress response. Front Biosci (Landmark Ed) 17:1480-98
Goloudina, Anastasia R; Mazur, Sharlyn J; Appella, Ettore et al. (2012) Wip1 sensitizes p53-negative tumors to apoptosis by regulating the Bax/Bcl-xL ratio. Cell Cycle 11:1883-7

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