PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response Ting Wang, Qiujing Yu, Juan Chen, Bo Deng, Lihua Qian, Yingying Le* Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Graduate School of the Chinese Academy of Sciences, Shanghai, China Abstract Background: Under stress, AMP-activated protein kinase (AMPK) plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP) expression under stress is unclear. Methodology/Principal Findings: We found that heat stress induced dephosphorylation of AMPKa subunit (AMPKa) in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKa under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A) inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKa dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl2, celastrol, MG132)-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKa expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability. Conclusions/Significance: These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress. Citation: Wang T, Yu Q, Chen J, Deng B, Qian L, et al. (2010) PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response. PLoS ONE 5(10): e13096. doi:10.1371/journal.pone.0013096 Editor: Michael Polymenis, Texas A&M University, United States of America Received July 23, 2010; Accepted September 7, 2010; Published October 1, 2010 Copyright: 2010 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Chief Scientist Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SIBS2008006, Y. Le). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yyle@sibs.ac.cn. Introduction Mammalian AMP-activated protein kinase (AMPK) is a heterotrimeric complex consisting of a, b, and c subunits. AMPK is activated allosterically by an increase in the intracellular AMP/ ATP ratios and/or by the phosphorylation of threonine 172 in the a subunit [1]. AMPK is initially viewed as a ‘fuel gauge’ to monitor cellular energy status in response to nutritional environ- mental variations. Physiological or pathological stimuli that deplete cellular energy levels such as prolonged exercise, metabolic poisoning, oxidative stress, hypoxia, ischemia, or nutrient deprivation, result in an increased AMP/ATP ratio that in turn activates AMPK [1]. The activation of AMPK coordinates a cellular program that limits further ATP depletion and promotes compensatory changes that maintain cellular ATP levels. Recent studies revealed that AMPK can also be activated by various stimuli independent of AMP/ATP ratio [2]. AMPK also plays important roles in cell survival and growth [3,4]. Heat shock response is a universal protective mechanism for cell survival under various environmental and physiological stresses. A family of molecular chaperones, named heat shock proteins (HSPs), is dramatically increased in response to these stresses. HSPs are involved in protein folding, assembly, degradation, intracellular localization, etc. They are classified into families, and among them the HSP70 family appears to be the most evolutionarily conserved and distributed in animals [5]. HSP70 transcription in response to heat stress is mainly mediated by binding of heat shock transcription factor 1 (HSF1) to heat shock elements (HSE) in the promoter region of HSP70 gene. Before binding with HSE, HSF1 undergoes phosphorylation, trimerliza- tion and nuclear translocation upon heat stress [6,7]. However, the signaling pathway(s) involved in HSP expression in response to heat stress is unclear. Corton et al. [8] reported that exposed rat primary hepatocytes to heat stress significantly activated AMPK. However, Kodiha et al. [9] reported that heat stress inhibited AMPK in both Hela and 293 cells. Recently, Jung et al. [10] reported that activation of AMPK significantly inhibited HSP70 expression in Hela cells. In the present study, we first examined the effect of heat stress on AMPK activity in various cell types, then investigated the involvement of AMPK in HSP70 expression in PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13096