Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/07/18/mol.116.105494.DC1 1521-0111/90/4/460–468$25.00 http://dx.doi.org/10.1124/mol.116.105494 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:460–468, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics ACCELERATED COMMUNICATION AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization s Hicham Mahboubi, Antonis E. Koromilas, and Ursula Stochaj Downloaded from Departments of Physiology (H.M., U.S.) and Oncology (A.E.K.), Faculty of Medicine, McGill University, Montreal, Quebec, Canada Received June 3, 2016; accepted July 14, 2016 ABSTRACT molpharm.aspetjournals.org Eukaryotic cells assemble stress granules (SGs) when translation signaling, cytoskeletal organization, and the abundance of trans- initiation is inhibited. Different cell signaling pathways regulate lation initiation factors. Specifically, AMPK activation increased SG production. Particularly relevant to this process is 59-AMP– stress-induced eukaryotic initiation factor (eIF) 2a phosphorylation activated protein kinase (AMPK),whichfunctionsasastress and reduced the concentration of eIF4F complex subunits eIF4G sensor and is transiently activated by adverse physiologic condi- and eIF4E. At the same time, the abundance of histone deacetylase tions. Here, we dissected the role of AMPK for oxidant-induced SG 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied formation. Our studies identified multiple steps of de novo SG by increased acetylation of a-tubulin on Lys40. Pharmacological assembly that are controlled by the kinase. Single-cell analyses studies further confirmed this novel AMPK–HDAC6 interplay and demonstrated that pharmacological AMPK activation prior to its importance for SG biology. Taken together, we provide mech- stress exposure changed SG properties, because the granules anistic insights into the regulation of SG formation. We propose at ASPET Journals on September 27, 2021 became more abundant and smaller in size. These altered SG that AMPK activation stimulates oxidant-induced SG formation but characteristics correlated with specific changes in cell survival, cell limits their fusion into larger granules. Introduction assembly. As such, phosphorylation of translation eukaryotic initiation factor (eIF) 2a on Ser51 destabilizes the 43S In eukaryotic cells, stressful conditions cause a reduction and preinitiation complex and produces canonical SGs (Kedersha reprogramming of protein synthesis; this is accompanied by et al., 2002). Furthermore, a disruption of the eIF4F complex stress granule (SG) assembly in the cytoplasm (Thedieck et al., destabilizes the 48S preinitiation complex and generates 2013). Aside from environmental and disease-related stress, noncanonical SGs (Kedersha et al., 2013). Both routes produce pharmacological compounds can also induce or modulate de novo granules that contain common factors. SG production (Fournier et al., 2010; Fujimura et al., 2012; SG assembly is a two-step process that includes: 1) the local Mahboubi et al., 2015). aggregation of SG-nucleating proteins into cytoplasmic foci SGs are composed of poly(A)-RNAs, microRNAs, RNA bind- and 2) the recruitment of additional factors and the increase of ing proteins, translation factors, signaling components, and granule size (Fujimura et al., 2009). In addition to SG-nucleating proteins of the small ribosomal subunit (Kedersha et al., 2013; proteins, other components crucial to SG production have been Mahboubi and Stochaj, 2014). Several pathways promote SG identified. For example, microtubules play a key role in the production and maturation of SGs (Nadezhdina et al., 2010). This research was supported by the Natural Sciences and Engineering Research Council of Canada [Grant 155509-2009], Fonds de recherche du SGs are critical for signaling in stressed cells. Notably, their Québec–Nature et technologies, Heart and Stroke Foundation of Canada, role goes beyond the passive recruitment of signaling mole- Canadian Cancer Society Research Institute [Grant 700886], and Canadian cules, because they constitute intracellular signaling hubs Institutes of Health Research [Grant MOP-38160]. This work was also supported by a doctoral fellowship from the Natural Sciences and Engineering that control cell fate (Kedersha et al., 2013; Mahboubi and Research Council of Canada. Stochaj, 2014). SGs not only modulate signaling, but their dx.doi.org/10.1124/mol.116.105494. s This article has supplemental material available at molpharm. assembly is also regulated by specific kinase activities. aspetjournals.org. 59-AMP–activated protein kinase (AMPK) is a key regulator of ABBREVIATIONS: AMPK, 59-AMP–activated protein kinase; A769662, 6,7-dihydro-4-hydroxy-3-(29-hydroxy[1,19-biphenyl]-4-yl)-6-oxo-thieno[2,3- b]pyridine-5-carbonitrile; DEM, diethyl maleate; eIF, eukaryotic initiation factor; G3BP1, ras GTPase-activating protein SH3-domain-binding protein 1; HDAC6, histone deacetylase 6; hnRNP K, heterogeneous nuclear ribonucleoprotein K; HuR, human antigen R; MEF, mouse embryonic fibroblast; Rock1, Rho-associated protein kinase 1; SG, stress granule; TIA-1, T-cell intracellular antigen-1. 460 AMPK Activation Alters Stress Granule Assembly 461 cellular homeostasis (Hardie et al., 2012) and is particularly thereby generates oxidative stress (Kodiha et al., 2008). DEM sensitive to stress. We recently showed that AMPK associates efficiently produces cytoplasmic granules that contain core SG with SGs under diverse stress conditions (Mahboubi et al., proteins and poly(A)-RNA (Mahboubi et al., 2013). To date, the 2015). However, the effect of AMPK activation on SG biology signaling events underlying DEM-dependent SG assembly have has not been defined. Our work now reveals the effects of not been elucidated. We addressed this question with MEFs that AMPK pharmacological activators on SG formation. More- carry a knock-in eIF2a (Ser51A) gene. This mutant eIF2a over, we identify specific pathways through which AMPK derivative is nonphosphorylatable (Rajesh et al., 2013) and does regulates SG biogenesis. not promote the formation of canonical SGs. Assessment of several marker proteins revealed that eIF2a- Ser51A did not support the formation of visible DEM SGs Materials and Methods (Supplemental Fig. 1). By contrast, DEM produced obvious Cell Culture, Stress, and Drug Treatments. The generation of SGs in control MEFs with wild-type eIF2a.Theseresults knock-in and control mouse embryonic fibroblasts (MEFs) was described established that eIF2a phosphorylation on Ser51 is required previously (Rajesh et al., 2013). HeLa cells and MEFs were maintained for DEM-dependent SG production. Accordingly, DEM-induced ’ ’ in Dulbecco s modified Eagle s medium as described (Mahboubi et al., granules can be classified as canonical SGs. The compound is 2015) MEF medium was supplemented with 2.5 mg/ml puromycin . thus a valid tool to elicit oxidative stress and examine SG (Sigma-Aldrich, St. Louis, MO). All cells were analyzed between Downloaded from passages 3 and 12. Oxidative stress was generated with 2 mM diethyl biogenesis; it was employed for the experiments described here. maleate (DEM) for 4 hours; controls were incubated with the vehicle Pharmacological Compounds A769662 and Salicylate ethanol. Alternatively, cells were treated with 0.5 mM sodium Efficiently Activate AMPK in HeLa Cells. Using HeLa arsenite or the vehicle water for 1 hour. cells as a model, we previously established AMPK as a SG For AMPK activation, cells were preincubated for 1 hour with 50 mM constituent (Mahboubi et al., 2015). To further define the role of A769662 [6,7-dihydro-4-hydroxy-3-(29-hydroxy[1,19-biphenyl]-4-yl)-6-oxo- AMPK for SG production and SG-related signaling, the enzyme molpharm.aspetjournals.org thieno[2,3-b]pyridine-5-carbonitrile] (dissolved in dimethylsulfoxide) or was activated with pharmacological agents that do not depend 10 mM sodium salicylate (in water). The histone deacetylase 6 (HDAC6) inhibitor tubastatin A (LC Laboratories, Woburn, MA) was dissolved in on liver kinase B1; this upstream kinase is not present in HeLa dimethylsulfoxide and used at 0.5 mM. AMPK activators, tubastatin A, or cells (Jaleel et al., 2005). A769662 (Cool et al., 2006) and vehiclewerealsopresentthroughoutthe subsequent stress or recovery salicylate (Hawley et al., 2012) directly bind AMPK and period. stimulate kinase function independently of AMPKa Thr172 Immunocytochemistry. Protocols for immunocytochemistry were phosphorylation. Because of their direct association with AMPK, described previously (Mahboubi et al., 2015). Information about primary these compounds are believed to have minimal off-target effects. antibodies is provided in Supplemental Table 1. A769662 and salicylate were therefore used for these studies. Microscopy, Quantitative Image Analysis, and Three- Our initial experiments determined the optimal concentra- Dimensional Reconstruction. Images were acquired with a Zeiss at ASPET Journals on September 27, 2021 tions for pharmacological AMPK activation. To this end, LSM510 confocal microscope (Carl Zeiss, Jena, Germany). Appropri- ate filter settings were applied to minimize channel crosstalk. Image the modification of acetyl-CoA carboxylase 1, an established quantification was performed with MetaXpress software (Molecular AMPK substrate, was measured (Supplemental Fig. 2). Based Devices, Sunnyvale,