ZIA BC 011197 (ZIA) | |||
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Title | Regulation and Function of WIP1 Phosphatase and its Role in Tumor Cells | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Appella, Ettore | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $583,055 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Breast (50.0%) Ovarian Cancer (50.0%) |
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Research Type | |||
Cancer Initiation: Oncogenes & Tumor Suppressor Genes Systemic Therapies - Discovery and Development |
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Abstract | |||
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 icreased 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. Many of the known pTQ/pSQ substrates are phosphorylated by ATM. We have undertaken a quantitative phosphoproteomic analysis to provide a characterization of the substrate specificity of the Wip1 phosphatase. In our experiments, 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 Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A, the first PPM structure determined, showed two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. We recently determined the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK, a physiological substrate of PPM1A. The structure revealed three metal ions in the active site confirming the presence of a third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational analyses demonstrated that the complex has a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain and that the position of the substrate in the active site allows solvent access to the |