Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

<div><p>Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.</p></div

PME-1 knockout suppresses cell proliferation.

<p>(A) Loss of PME-1 suppresses cell proliferation. Cell proliferation of wild type (WT) and PME-1 KO (KO) MEFs were determined by Cell Counting Kit-8. N = 4 *: <i>P</i><0.05 vs. WT. (B-D) Effects of PME-1 KO on ERK1/2 and Akt phosphorylation. WT and KO MEFs were stimulated with EGF (50 ng/ml) for indicated time periods and ERK1/2 and Akt phosphorylation was determined by immunoblotting. Representative images (B) from 3 independent experiments, and quantitative data for phospho-ERK1/2 (C) and phosphoT308 Akt (D) are shown. *: <i>P</i><0.05 vs. WT. (E-F) Loss of PME-1 enhances the association of PP2A AB55αC complex. FLAG-B55α or B56α were transiently expressed in WT and PME-1 KO MEFs, and immunoprecipitated with FLAG-M2 beads. Empty vector was used as mock. PP2Ac and A subunit association was detected by immunoblotting. Representative images from 3 independent experiments were shown.</p

PME-1 protects PP2Ac from protein degradation.

<p>(A-B) Loss of PME-1 does not affect PP2Ac mRNA expression. mRNA levels of PP2Ac α (A) and β (B) isoforms were analyzed by real-time qPCR. Quantitative data from 2 independent experiments performed in duplicate are shown. (C-D) PME-1 protects PP2Ac from protein degradation. PP2Ac degradation was analyzed by cycloheximide chase assay. Representative images (C) and quantitative data for PP2Ac protein level (D) from 5 independent experiments are shown. *: <i>P</i><0.05 vs. WT.</p

Proposed working hypothesis.

<p>Proposed working hypothesis for the regulatory mechanism for PP2A protein level by PME-1. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145226#sec018" target="_blank">Discussion</a> for details.</p

Methyl-esterase activity of PME-1 is required to maintain levels of PP2Ac.

<p>(A) PME-1 inhibitor decreases PP2Ac protein levels. WT MEFs were treated with ABL127 (5 or 10 μM) for 48 hr and levels of proteins were determined by immunoblotting. Representative images (A) and quantitative data (B) from 4 independent experiments are shown. *: <i>P</i><0.05 vs. ABL127 untreated. (C-D) Methyl esterase activity of PME-1 is required to maintain PP2Ac levels. PME-1 KO MEFs were expressed FLAG-PME-1 WT or S156A (methyl esterase dead), and levels of proteins were determined by immunoblotting. Empty vector was used as mock. Representative images (C) and quantitative data (D) from 3 independent experiments are shown. *: <i>P</i><0.05 vs. FLAG-PME-1 WT expressed KO MEFs.</p

Presentation1_Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.pptx

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.</p

DataSheet1_Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.docx

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.</p