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 activity: Recent published work has established that in some cancer patients, somatic mutations result in prematurely terminated Wip1 proteins that are enzymatically active, but lack a portion of the terminal protein-encoding exon. Interestingly, these truncated forms of Wip1 show increased stability;cells expressing the truncated forms of Wip1 accumulate high levels of the phosphatase and exhibit severely compromised activation of p53 (Ruark et al. 1013, Nature 493:06-410). To investigate the relationship between Wip1 mutations and cancer cases, we interrogated public databases to identify missense mutations of Wip1 and identified 41 mutation sites in Wip1 among cancer cases. Two thirds of the cases were located in the C-terminal domain and about one fourth were located in the phosphatase domain. In general, cases with loss of function mutations of Wip1 were likely to harbor mutations of p53, whereas mutations resulting in gain of function for Wip1, often through increased protein stability, were likely to contain no mutations in the p53 gene. These results indicate that the stability of Wip1 is important in its contribution to tumorigenesis. Moreover, the pattern of expression of Wip1 suggests that its activity supports proliferation and preserves proliferative potential in both humans and mice. Under conventional culture conditions, Wip1-/- mouse embryonic fibroblasts (MEFs) undergo premature senescence. We have investigated the mechanism by which Wip1 suppresses premature senescence in MEFs. We found that reduced oxygen pressure only partially rescued the premature senescence resulting from loss of Wip1. Under both 20% and 3% oxygen conditions, early passage Wip1-/- MEFs exhibited increased activation of p53 and increased levels of cyclin-dependent kinase inhibitors, compared with wild type cells. These findings suggest that Wip1 prevents cellular senescence by regulating ATM- and p53-dependent DNA damage response (DDR) signaling resulting from endogenous sources of DNA damage. Notably, increased DDR signaling was associated with S phase cells in the absence of Wip1. The premature aging exhibited by Wip1 knockout mice suggests that the ability of Wip1 to forestall senescence is also important in preserving tissue maintenance capabilities in vivo. Current results characterizing the effects of Wip1 deletion in mice are based on animals in which Wip1 is genetically deleted in all tissues. Despite the utility of this mouse, it does not allow exploration of 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 crossing these conditional knock-out mice with mice bearing an allele for the tissue specific expression of the Cre recombinase to generate conditional knock-out mice. Wip1 phosphatase activity and substrate identification: Wip1 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. To provide an unbiased characterization of the substrate specificity of the Wip1 phosphatase, we have undertaken a quantitative phospho-proteomic analysis of the phosphoproteins present in cells following a stress under conditions of high or low Wip1 activity. Structure of the Wip1 catalytic domain: 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 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 Wip1 phosphatases. We have used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize the millimolar-affinity binding of magnesium ions to PP2C-alpha. Interestingly, mutation of the corresponding active site residues in PP2C-alpha and Wip1 has similar effects on the catalytic activity of each enzyme, as measured in vitro using purified proteins. These results suggest that the binding of a third metal ion to these phosphatases is essential for catalytic activity, involves amino acids conserved in both enzymes and identifies a critical process that could be abrogated by the binding of a specific inhibitor.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011197-06
Application #
8938003
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
Sakai, Hiroyasu; Fujigaki, Hidetsugu; Mazur, Sharlyn J et al. (2014) Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication. Cell Cycle 13:1015-29
Tanoue, Kan; Miller Jenkins, Lisa M; Durell, Stewart R et al. (2013) Binding of a third metal ion by the human phosphatases PP2C? and Wip1 is required for phosphatase activity. Biochemistry 52:5830-43
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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