Abstract 1078: PPM1A acts as a novel RelA phosphatase to negatively regulate NF-κB signaling

Abstract

Abstract Phosphorylation of RelA is required to fully activate NF-κB transcription activity and dephosphorylation of RelA can inhibit its activity. To date, Wip1 (PPM1D) and PP2A have been described as RelA phosphatases. The purpose of this study was to identify novel RelA phosphatase(s) and determine if they play a role in regulation of NF-κB activity related to tumorigenesis. PPM1A belongs to PP2C protein phosphatase family, the same family of Wip1. To begin exploring PPM1A activity toward RelA, serine 536 (S536) phosphorylation of RelA was determined with and without expression or knockdown of PPM1A. Expression of PPM1A in U2OS cells decreased RelA phosphorylation, whereas siRNA-mediated loss of PPM1A increased S536RelA phosphorylation. Luciferase assay using a κB responsive reporter with expression of either PPM1A or phosphatase-dead PPM1A (PPM1A-PD) revealed that wild-type, but not PPM1A-PD, inhibited RelA transcription. Furthermore, in vitro phosphatase assays using bacterially expressed PPM1A with substrate of either full length RelA or phosphorylated RelA peptides, suggest that PPM1A can function as a direct RelA phosphatase at residuesS536 and S276. We could not identify reports of additional phosphatases with activity toward the S276 residue of RelA. Using liquid chromatography and tandem mass spectroscopy (LC-MS/MS) LZAP-associated proteins (LAPs) were sought with PPM1A being identified. LZAP (C53, CDK5RAP3) has tumor suppressor-like activity that is mediated at least partially through inhibition of RelA S536 phosphorylation and activity. PPM1A's ability to decrease RelA phosphorylation at S536 site was largely inhibited in cells with siRNA-mediated loss of LZAP. To determine if PPM1A has tumor suppressor activity, HeLa cells were infected with retrovirus directing shRNA-mediated knockdown of PPM1A and tumorigenic properties of these cells were determined. Preliminary xenograft tumor data suggest PPM1A has a negative effect on tumor initiation and proliferation and studies are underway to determine if PPM1A activity toward NF-κB plays a role. In summary, our data suggest that PPM1A is a novel RelA phosphatase with activity to dephosphorylate RelA at serines 536 and 276. PPM1A inhibits NF-κB transcriptional activity and that PPM1A activity toward RelA is at least partially dependent on LZAP. Our preliminary data also suggest PPM1A might have tumor suppressor-like activity that could be dependent on LZAP or RelA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2011-1078