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.  The transcriptional induction of Wip1 following exposure to DNA damaging agents requires functional p53 protein.  Previously, we characterized a functional p53 response element in the Wip1 promoter that is located downstream of the usual start of transcription.  Following the activation of DNA damage response (DDR) signaling, p53-dependent transcriptional activation of the Wip1 promoter results in the production of mRNAs with shorter 5' untranslated regions that may be more efficiently translated.  The increased levels of Wip1 negatively regulate p53 activity through suppression of DDR signaling, enabling the return to cellular homeostasis.  Following exposure to IR, the Wip1 protein levels increase more dramatically than its mRNA levels, resulting from both increased translational efficiency and reduced degradation.  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.  The S phase-specific increase in Wip1 protein levels resulted, at least partially, from increased protein stability.  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 activity, we interrogated public databases to identify missense mutations of Wip1.  We identified four single nucleotide polymorphisms present in the human population that resulted in amino acid changes and characterized the effects of these changes on the activity of Wip1.  For two of the mutations, L120F and P322Q, the altered amino acids are located within the structured portion of the catalytic domain and the mutant proteins exhibited drastically reduced catalytic activity.  The location of a third mutation, S82A, is in an unstructured loop far from the active site and did not affect phosphatase activity.  The fourth mutation, I496V, is located in the poorly characterized C-terminal domain of Wip1 and increased the amount of the protein localized to the cytoplasm.  To investigate the relationship between Wip1 mutations and primary human cancers, we 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.  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 cyclin-dependent kinase inhibitors.  These findings suggest that Wip1 prevents cellular senescence by regulating DDR signaling resulting from endogenous sources of DNA damage.  The premature aging exhibited by Wip1 knockout mice suggests that the ability of Wip1 to forestall senescence is also important in maintaining tissue maintenance capabilities in vivo.  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 crossing these conditional knock-out mice with mice bearing an allele for the tissue specific expression of the Cre recombinase.  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 (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 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.