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Research ArticleAccelerated Communication

AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization

Hicham Mahboubi, Antonis E. Koromilas and Ursula Stochaj
Molecular Pharmacology October 2016, 90 (4) 460-468; DOI: https://doi.org/10.1124/mol.116.105494

Abstract

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5′-AMP–activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK–HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules.

Introduction

In eukaryotic cells, stressful conditions cause a reduction and reprogramming of protein synthesis; this is accompanied by stress granule (SG) assembly in the cytoplasm (Thedieck et al., 2013). Aside from environmental and disease-related stress, pharmacological compounds can also induce or modulate de novo SG production (Fournier et al., 2010; Fujimura et al., 2012; Mahboubi et al., 2015).

SGs are composed of poly(A)-RNAs, microRNAs, RNA binding proteins, translation factors, signaling components, and proteins of the small ribosomal subunit (Kedersha et al., 2013; Mahboubi and Stochaj, 2014). Several pathways promote SG assembly. As such, phosphorylation of translation eukaryotic initiation factor (eIF) 2α on Ser51 destabilizes the 43S preinitiation complex and produces canonical SGs (Kedersha et al., 2002). Furthermore, a disruption of the eIF4F complex destabilizes the 48S preinitiation complex and generates noncanonical SGs (Kedersha et al., 2013). Both routes produce granules that contain common factors.

SG assembly is a two-step process that includes: 1) the local aggregation of SG-nucleating proteins into cytoplasmic foci and 2) the recruitment of additional factors and the increase of granule size (Fujimura et al., 2009). In addition to SG-nucleating proteins, other components crucial to SG production have been identified. For example, microtubules play a key role in the production and maturation of SGs (Nadezhdina et al., 2010).

SGs are critical for signaling in stressed cells. Notably, their role goes beyond the passive recruitment of signaling molecules, because they constitute intracellular signaling hubs that control cell fate (Kedersha et al., 2013; Mahboubi and Stochaj, 2014). SGs not only modulate signaling, but their assembly is also regulated by specific kinase activities. 5′-AMP–activated protein kinase (AMPK) is a key regulator of cellular homeostasis (Hardie et al., 2012) and is particularly sensitive to stress. We recently showed that AMPK associates with SGs under diverse stress conditions (Mahboubi et al., 2015). However, the effect of AMPK activation on SG biology has not been defined. Our work now reveals the effects of AMPK pharmacological activators on SG formation. Moreover, we identify specific pathways through which AMPK regulates SG biogenesis.

Materials and Methods

Cell Culture, Stress, and Drug Treatments.

The generation of knock-in and control mouse embryonic fibroblasts (MEFs) was described previously (Rajesh et al., 2013). HeLa cells and MEFs were maintained in Dulbecco’s modified Eagle’s medium as described (Mahboubi et al., 2015). MEF medium was supplemented with 2.5 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO). All cells were analyzed between passages 3 and 12. Oxidative stress was generated with 2 mM diethyl maleate (DEM) for 4 hours; controls were incubated with the vehicle ethanol. Alternatively, cells were treated with 0.5 mM sodium arsenite or the vehicle water for 1 hour.

For AMPK activation, cells were preincubated for 1 hour with 50 µM A769662 [6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile] (dissolved in dimethylsulfoxide) or 10 mM sodium salicylate (in water). The histone deacetylase 6 (HDAC6) inhibitor tubastatin A (LC Laboratories, Woburn, MA) was dissolved in dimethylsulfoxide and used at 0.5 µM. AMPK activators, tubastatin A, or vehicle were also present throughout the subsequent stress or recovery period.

Immunocytochemistry.

Protocols for immunocytochemistry were described previously (Mahboubi et al., 2015). Information about primary antibodies is provided in Supplemental Table 1.

Microscopy, Quantitative Image Analysis, and Three-Dimensional Reconstruction.

Images were acquired with a Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany). Appropriate filter settings were applied to minimize channel crosstalk. Image quantification was performed with MetaXpress software (Molecular Devices, Sunnyvale, CA); all steps were automated as described (Mahboubi et al., 2013). For single-cell analysis, at least 340 cells were quantified per condition. Methods for three-dimensional reconstruction were previously described (Mahboubi et al., 2013).

Western Blotting.

Whole cell extracts were precipitated with trichloroacetic acid and analyzed by Western blotting as published previously (Mahboubi et al., 2015). Supplemental Table 1 lists the source and dilutions of primary antibodies. Between three and six independent experiments were performed for each antigen.

Evaluation of Cell Viability.

Cells were grown in 96-well plates (Corning, Corning, NY) and cell viability was measured using the CellTiter-Blue kit (Promega, Madison, WI) following the manufacturer’s recommendations. Resorufin fluorescence (560 nm excitation/590 nm emission) was measured with a Tecan Infinite M-1000 plate reader (Tecan, Männedorf, Switzerland). Background fluorescence of the medium was subtracted from all treatment conditions. All treatments were performed at least in triplicate, and three independent experiments were carried out.

Statistical Analyses.

Results represent at least three independent experiments and are shown as means ± S.E.M. Significant differences (P < 0.05) were identified with one-way analysis of variance followed by Bonferroni post hoc analysis or t test, as appropriate. Microsoft Excel (Microsoft Corporation, Redmond, WA) was used for regression analysis and scatter plot generation. Pearson’s correlation coefficient was calculated with the following standard formula: r = cov(A, B)/[σA × σB].

Results and Discussion

DEM-Induced SG Formation Depends on eIF2α Phosphorylation.

DEM depletes cellular glutathione and thereby generates oxidative stress (Kodiha et al., 2008). DEM efficiently produces cytoplasmic granules that contain core SG proteins and poly(A)-RNA (Mahboubi et al., 2013). To date, the signaling events underlying DEM-dependent SG assembly have not been elucidated. We addressed this question with MEFs that carry a knock-in eIF2α (Ser51A) gene. This mutant eIF2α derivative is nonphosphorylatable (Rajesh et al., 2013) and does not promote the formation of canonical SGs.

Assessment of several marker proteins revealed that eIF2α-Ser51A did not support the formation of visible DEM SGs (Supplemental Fig. 1). By contrast, DEM produced obvious SGs in control MEFs with wild-type eIF2α. These results established that eIF2α phosphorylation on Ser51 is required for DEM-dependent SG production. Accordingly, DEM-induced granules can be classified as canonical SGs. The compound is thus a valid tool to elicit oxidative stress and examine SG biogenesis; it was employed for the experiments described here.

Pharmacological Compounds A769662 and Salicylate Efficiently Activate AMPK in HeLa Cells.

Using HeLa cells as a model, we previously established AMPK as a SG constituent (Mahboubi et al., 2015). To further define the role of AMPK for SG production and SG-related signaling, the enzyme was activated with pharmacological agents that do not depend on liver kinase B1; this upstream kinase is not present in HeLa cells (Jaleel et al., 2005). A769662 (Cool et al., 2006) and salicylate (Hawley et al., 2012) directly bind AMPK and stimulate kinase function independently of AMPKα Thr172 phosphorylation. Because of their direct association with AMPK, these compounds are believed to have minimal off-target effects. A769662 and salicylate were therefore used for these studies.

Our initial experiments determined the optimal concentrations for pharmacological AMPK activation. To this end, the modification of acetyl-CoA carboxylase 1, an established AMPK substrate, was measured (Supplemental Fig. 2). Based on results in Supplemental Fig. 2 and the assessment of cytotoxicity (not shown), our subsequent studies were performed with 50 µM A769662 or 10 mM sodium salicylate.

Pharmacological AMPK Activation Stimulates SG Formation during Stress.

To assess the effect of AMPK activation on SG production, HeLa cells were preincubated with A769662 or vehicle. As shown in Supplemental Fig. 2, pretreatment with A769662 resulted in efficient AMPK activation. AMPK activation was followed by DEM exposure; A769662 or vehicle was also present throughout the stress period.

AMPK activation alone was not sufficient to induce SG formation (Fig. 1A), whereas DEM produced large SGs as described previously (Mahboubi et al., 2013, 2015). The combination of A769662 and DEM also generated SGs; these granules contained the marker proteins T-cell intracellular antigen-1 (TIA-1), ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1), and human antigen R (HuR) (Fig. 1A). However, their properties clearly differed from DEM-induced SGs. Thus, SGs produced by AMPK preactivation and subsequent simultaneous treatment with A769662 and DEM were more numerous and smaller in size. The same results were obtained with the AMPK activator salicylate. Accordingly, after kinase activation, the combination of salicylate and DEM generated more and smaller granules compared with DEM alone. This was consistently observed with the SG markers TIA-1, G3BP1, and HuR (Fig. 1A).

Fig. 1.
Fig. 1.

Pharmacological AMPK activation generates oxidant-induced SGs that are more numerous and smaller in size. (A) HeLa cells were treated with the vehicle EtOH or the oxidant DEM. Incubation with the AMPK activator A769662 or sodium salicylate was as described in the Materials and Methods. SGs were visualized by immunostaining of the marker proteins TIA-1, G3BP1, and HuR. Nuclei were stained with DAPI. (B) Western blotting for crude extracts evaluated Acc1 phosphorylation on Ser79; this modification is catalyzed by AMPK. Bar graphs represent the quantification of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Acc1, acetyl-CoA carboxylase 1; DAPI, 4,6′-diamidino-2-phenylindole; EtOH, ethanol; pAcc1, phospho-acetyl-CoA carboxylase 1 (Ser79). Bar, 20 µm.

Control experiments confirmed that in the absence of oxidative stress, A769662 and salicylate significantly increased Acc1 phosphorylation (Fig. 1B). As expected, both agents caused only minor changes in AMPKα modification on Thr172. In line with our earlier work (Kodiha et al., 2007), DEM diminished Thr172 phosphorylation of AMPKα; this was not prevented by A769662 or salicylate. However, as we showed previously, DEM induces a rapid and transient AMPK activation that precludes SG formation (Mahboubi et al., 2015).

Sodium arsenite provided additional evidence for the effect of A769662 on SGs (Supplemental Fig. 3). Arsenite generates oxidative stress and is an established inducer of SGs; the assembly of arsenite SGs relies on eIF2α phosphorylation (McEwen et al., 2005). As for DEM, A769662 modulated the assembly of arsenite SGs; they were more abundant and smaller in size when AMPK was activated before and throughout arsenite exposure. These results further support the role of AMPK activation for eIF2α-dependent SGs.

Since the SG composition is stress specific (Kedersha et al., 2013; Mahboubi et al., 2013; Mahboubi and Stochaj, 2014), we evaluated granules generated by the treatment with A769662 plus DEM. These granules contained AMPKα2, importin-α1, importin-β1, and heterogeneous nuclear ribonucleoprotein K (hnRnP K), proteins that are commonly present in canonical SGs (Fig. 2). In summary, AMPK activation in combination with oxidative stress stimulates the formation of a large number of cytoplasmic granules that contain all of the SG marker proteins we examined.

Fig. 2.
Fig. 2.

SGs produced upon A769662 pretreatment and subsequent incubation with DEM and A769662 contain importin-β1, importin-α1, hnRNP K, HDAC6, and AMPKα2. HeLa cells were treated as described for Fig. 1A; the association of different proteins with SGs was evaluated by immunostaining. DAPI, 4,6′-diamidino-2-phenylindole; EtOH, ethanol. Bar, 20 µm.

AMPK Activation Alters Specific SG Properties.

The effect of AMPK activation on SGs was characterized in depth by the assessment of granule properties, single-cell analyses, and statistical inference methods. We focused on A769662, because this compound was optimized for AMPK activation (Cool et al., 2006) and caused moderate and physiologically relevant kinase stimulation (Fig. 1B; Supplemental Fig. 1).

Using G3BP1 as an SG marker, we analyzed 340 DEM-treated cells (4272 SGs) and 407 cells incubated with DEM plus A769662 (5641 SGs). Our established protocols (Mahboubi et al., 2013) evaluated several SG parameters. Quantitative automated measurements (Fig. 3A) revealed that AMPK activation significantly increased the number of SGs per cell. This was accompanied by an overall shift in SG abundance within the cell population (Fig. 3B). In particular, A769662 significantly reduced the subpopulation of cells with few SGs (<5 SGs/cell; Fig. 3B), while increasing the subpopulation with numerous SGs (>20 SGs/cell). At the same time, the compound raised the number of cells with small SGs from 25% to 45%, whereas the percentage for medium and large SGs was diminished.

Fig. 3.
Fig. 3.

A769662 stimulates the formation of smaller and more numerous SGs in stressed cells. Quantitative image analyses assessed different SG parameters. Results are shown for three independent experiments. The total number of cells analyzed was 340 for DEM and 407 for DEM plus A769662. (A) Upon DEM-induced stress, A769662 significantly increased the number of SGs per cell. Note that data were normalized to DEM, our reference for SG production. (B) Binning identifies the subpopulations of SG-producing cells that were affected by A769662. Changes were observed for the number of SGs per cell and SG size. The pie charts show the percentage of SGs that were present in different size groups. (C) Pearson’s correlation coefficients (bars) were calculated for each pair of variables listed. **P < 0.01.

Single-cell analysis and statistical inference defined the relationship between different SG parameters and the changes induced by AMPK activation. To this end, we calculated the slope of linear regression curves and the Pearson’s correlation coefficient (Fig. 3C; Supplemental Fig. 4). Our results support several conclusions for DEM-induced SG production. First, the analysis of more than 340 cells identified a strong (R2 > 0.5) linear relationship between the average SG area and SG number per cell (Supplemental Fig. 4). Interestingly, the slope was < 1.0, suggesting that the doubling of the SG number leads to less than the doubling of the SG area. Second, the total pixel intensity of SGs per cell highly correlated with the number of SGs. Notably, A769662 treatment decreased the slope for both curves.

Together, our single-cell studies provide quantitative evidence that AMPK activation has a significant effect on SG parameters. As such, AMPK activation promotes the formation of a larger number of SGs that are smaller in size. Because SGs have a direct role in stress signaling, our data indicate that AMPK activation reshapes the cellular stress response.

AMPK Activation Modulates eIF2α Phosphorylation.

To gain mechanistic insight into the pathways through which AMPK regulates SG assembly, we measured the phosphorylation of eIF2α on Ser51, a prerequisite for canonical SG biogenesis (Kedersha et al., 2002). As required for DEM-dependent SG production (Supplemental Fig. 1), the oxidant significantly increased the phosphorylation of eIF2α (Fig. 4A). Notably, eIF2α phosphorylation was further enhanced if cells were pretreated with A769662 and then incubated with both DEM and the AMPK activator. This increase was significant compared with DEM treatment alone. In the absence of oxidative stress, A769662 had little effect on eIF2α phosphorylation (Fig. 4A). This is in line with our immunocytochemistry results, because AMPK activation alone was not sufficient to induce SGs (Fig. 1A). Collectively, our data demonstrate that AMPK activation enhances eIF2α phosphorylation in the context of oxidative stress. We propose that this combination of kinase activation and stress stimulates de novo SG assembly.

Fig. 4.
Fig. 4.

AMPK activation alters key signaling events, with minor effects on the abundance of SG proteins. The effect of A769962 on SG-related signaling and SG components was evaluated by Western blotting of crude extracts generated for HeLa cells. Results are shown for three to six independent experiments. Actin served as the reference. In the absence of oxidative stress, signals for phospho-eIF2α (p-eIF2α) are very low. Therefore, results for p-eIF2α were normalized to the DEM/vehicle sample. (A) A769662 changes the phosphorylation and abundance of translation initiation factors. One-way analysis of variance with simultaneous comparison of all pairs shows that A769662 significantly increases the modification of eIF2α. #P < 0.05; **P < 0.01; ***P < 0.001 (t test or one-way analysis of variance compared with control). (B) Assessment of SG-nucleating proteins and other SG constituents. *P < 0.05; **P < 0.01 (t test compared with control). EtOH, ethanol.

AMPK Activation Reduces eIF4G and eIF4E Concentrations.

Independent of eIF2α phosphorylation, destabilization of the eIF4F complex can also induce SG assembly (Dang et al., 2006). The eIF4F complex consists of the scaffolding protein eIF4G, the helicase eIF4A, and the cap-binding polypeptide eIF4E (Piccirillo et al., 2014); eIF4E is the limiting factor for eIF4F complex formation. Various pharmacological agents target the eIF4F complex and thereby generate noncanonical SGs (Fujimura et al., 2012).

AMPK activation reduced eIF4G and eIF4E abundance (Fig. 4A), mainly under stress conditions. Importantly, this correlated with an enhanced SG production. Our data are consistent with studies by others, which showed that eIF4E depletion compromises eIF4F assembly and thus stimulates SG production (Fujimura et al., 2012). We therefore conclude that AMPK activation diminishes the concentrations of key translation initiation factors. In stressed cells, this reduction may stimulate SG formation.

AMPK Activation Has No Profound Effect on the Abundance of Common SG Proteins.

The concentration of granule-nucleating proteins G3BP1, TIA-1, and TIAR is crucial for SG assembly (Bley et al., 2015). Moreover, an increase in granule-nucleating proteins stimulates SG production (Kedersha et al., 1999; Tourrière et al., 2003). Given that AMPK activation altered the number and size of SGs, we examined core and other common SG proteins. A769662 slightly reduced the concentration of nucleating SG proteins. However, the effect was not significant upon stress (Fig. 4B), which is in line with the cell’s ability to initiate SG assembly (Fig. 1). The same was observed for other established SG constituents, such as the RNA binding proteins HuR and hnRNP K as well as the nuclear transporter importin-β1.

AMPK Activation Reorganizes Microtubules in Stressed Cells through HDAC6.

Microtubules and their associated motors control the production of full-size SGs. For example, pharmacological microtubule stabilization enhances SG formation and increases the number of foci (Nadezhdina et al., 2010). On the other hand, α-tubulin acetylation on Lys40 regulates microtubule–motor interactions (Dompierre et al., 2007). HDAC6 determines Lys40 acetylation, which is particularly high in stable microtubules (Asthana et al., 2013). HDAC6 also interacts with microtubules and SGs (Kwon et al., 2007). Furthermore, HDAC6 deacetylates heat shock protein 90 (Krämer et al., 2014), which in turn affects SG formation (Matsumoto et al., 2011). Thus, HDAC6 affects SG assembly through several pathways.

In our experiments, DEM led to the association of HDAC6 with SGs (Fig. 2) and to profound microtubule disorganization, which was further augmented by A769662 (Fig. 5). Notably, A769662 diminished HDAC6 abundance in DEM-stressed cells, although the decrease did not reach significance; moreover, A769662 increased α-tubulin acetylation on Lys40 (Fig. 5, A and B).

Fig. 5.
Fig. 5.

AMPK activation impinges on the microtubule cytoskeleton. (A) HeLa cells were incubated with DEM and A769662 as in Fig. 1. Ac-K40-tub, α-tubulin, and TIA-1 were detected. (B) Western blotting measured the abundance of HDAC6 and α-tubulin acetylation for Lys40. The bar graphs show the quantification for four independent experiments. ***P < 0.001 (one-way analysis of variance pairwise comparison identified significant differences). (C) Three-dimensional reconstruction of microtubules and SGs. Both panels depict DAPI (blue), TIA-1 (magenta), and tubulin (green). Ac-K40-tub, tubulin acetylated on Lys40; DAPI, 4,6′-diamidino-2-phenylindole; EtOH, ethanol. Bar, 20 µm.

Overall, these changes support the idea that AMPK activation through the reduction of HDAC6 alters several activities relevant to SG biology. To further test this hypothesis, we took advantage of tubastatin A, a recently developed HDAC6 inhibitor (Butler et al., 2010). Tubastatin A is characterized by high potency and specificity for HDAC6. To our knowledge, this compound has not been used previously to investigate SG assembly. Initial studies determined the tubastatin A concentration that reliably increased tubulin acetylation in our experiments (data not shown). Subsequent experiments tested the effects of tubastatin A on SG formation, either alone or in combination with A769662 (Supplemental Fig. 5). Similar to A769662, pretreatment with tubastatin A generated SGs that were more numerous and smaller compared with SGs induced with DEM only. Importantly, when combined, A769662 did not potentiate the effect of tubastatin A. This suggests that HDAC6 is a major effector through which AMPK controls SG formation.

AMPK Activation Alters the Survival of Stressed Cells.

To determine the physiological relevance of AMPK activation during stress, we monitored cell fate after stress withdrawal (Fig. 6A). When cells recovered from the DEM insult in the presence of A769662, SG disassembly was delayed, and SGs were present up to 21 hours poststress. By contrast, SG disassembly began at around 3 hours in vehicle controls. This led to significant differences in the percentage of cells containing SGs, and these differences were most prominent at the later stages of stress recovery (Fig. 6B).

Fig. 6.
Fig. 6.

AMPK activation prolongs the persistence of SGs and improves the survival of stressed cells. HeLa cells were preincubated with the vehicle DMSO or A769662, stressed, and allowed to recover for different time periods. Stress and poststress incubations were in the presence of vehicle or A769662 as indicated. (A) A769662 prolongs the presence of SGs and the sequestration of Rock1. (B) Quantification of SG-containing cells after stress and during recovery. At least 100 cells were scored per time point for each experiment. The error bars represent the S.E.M., determined for three independent sets. (C) Cell viability was assessed after 21 hours of recovery in three independent experiments. Compared with ethanol controls, the survival of DEM-treated cells was significantly lower. ***P < 0.001 (one-way analysis of variance). A769662 significantly increased the survival of cells recovering from stress. #P < 0.05 (t test). DAPI, 4,6′-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; EtOH, ethanol.

In parallel with the effects on SGs, we observed marked changes to the cytoskeleton. Oxidative stress led to a substantial loss of microtubule filaments, both in the absence and presence of A769662. During the subsequent recovery phase, AMPK activation caused striking differences in cytoskeleton organization. In A769662-treated cells, disorganized microtubules began to emerge 4 to 5 hours poststress; they persisted for up to 21 hours. Without A769662, microtubule bundles appeared only transiently 1–3 hours after stress withdrawal. Notably, the SG perseverance induced by A769662 was accompanied by improved cell viability (Fig. 6C). These data are consistent with a prosurvival role of SGs (reviewed in Kedersha et al., 2013; Mahboubi and Stochaj, 2014).

AMPK Activation Prolongs the SG Sequestration of Rho-Associated Protein Kinase 1 in Stressed Cells.

Rho-associated protein kinase 1 (Rock1) links SG formation to cell survival (Tsai and Wei, 2010). Thus, sequestration of Rock1 is one of the mechanisms that contribute to the antiapoptotic function of SGs. Since SG composition is stress dependent and dynamic, it was important to evaluate the subcellular distribution of Rock1 during and after stress. Figure 6A demonstrates that DEM SGs contained Rock1. Rock1 remained bound to SGs throughout the recovery period. Importantly, A769662 not only prolonged SG perseverance but also maintained the confinement of Rock1. These data suggest a mechanistic link between AMPK and cell survival. We propose that AMPK serves as an upstream regulator that alters SG dynamics throughout stress exposure and promotes Rock1 sequestration. Ultimately, these events will stimulate the recovery from oxidative insults and improve cell survival.

AMPK Activation Stimulates SG Formation through a Multistep Mechanism.

Our studies demonstrate that AMPK activation prior to stress exposure modulates SG formation. We identified several SG-relevant processes that were strongly affected by AMPK stimulation; they provide the basis for our simplified model (Fig. 7). We propose that the kinase impinges on critical steps of SG assembly. Specifically, AMPK activation: 1) increases eIF2α phosphorylation, 2) reduces the abundance of essential eIF4F subunits, and 3) depletes HDAC6 and thereby affects microtubule organization, granule movement, and possibly other activities involved in SG formation. As a consequence of AMPK activation, steps 1 and 2 stimulate SG biogenesis through signaling events that alter translation initiation. Although SGs become more numerous under these conditions, they fail to grow to a larger size. This could be explained by step 3, which impairs the fusion of SG foci, a process required to produce larger granules (Nadezhdina et al., 2010). Together, these AMPK-mediated effects promote recovery and improve survival after oxidative stress.

Fig. 7.
Fig. 7.

Simplified model for the AMPK-mediated control of SG production and its physiological effects. See the text for details.

Conclusions

Our results demonstrate that AMPK activation affects key aspects of SG biogenesis and thereby alters granule assembly and fusion. The combination of these events leads to significant changes in SG parameters. The new insights presented here are important because they are relevant to different human diseases. For example, AMPK activation can interfere with tumor cell proliferation, notably by affecting translation (Grzmil and Hemmings, 2012; Chen et al., 2014). On the other hand, SGs are formed by some types of cancer, where they promote tumor survival and progression (Thedieck et al., 2013; Somasekharan et al., 2015). Furthermore, SG-like granules are also produced in other diseases that are linked to altered AMPK activities, including type 2 diabetes and neurodegeneration (reviewed in Mahboubi and Stochaj, 2014). Because SGs are cytoprotective (Kedersha et al., 2013; Thedieck et al., 2013; Mahboubi and Stochaj, 2014), AMPK-dependent modulation of SG properties could explain some of the beneficial effects of AMPK activator–based therapy. Our results emphasize that therapeutic treatments should consider the effect of AMPK on SG biogenesis as a new avenue to modulate cell survival in various human diseases.

Acknowledgments

The authors thank Drs. R. Jones, J. Presley, N. Sonenberg, J. White, and X. Yang (McGill University) for generous gifts of reagents. The authors also thank R. Avramesc (McGill University) for technical advice.

Authorship Contributions

Participated in research design: Mahboubi, Stochaj.

Conducted experiments: Mahboubi, Stochaj.

Contributed new reagents or analytic tools: Koromilas.

Performed data analysis: Mahboubi, Stochaj.

Wrote or contributed to the writing of the manuscript: Mahboubi, Stochaj.

Footnotes

    • Received June 3, 2016.
    • Accepted July 14, 2016.

  • This research was supported by the Natural Sciences and Engineering Research Council of Canada [Grant 155509-2009], Fonds de recherche du Québec–Nature et technologies, Heart and Stroke Foundation of Canada, Canadian Cancer Society Research Institute [Grant 700886], and Canadian Institutes of Health Research [Grant MOP-38160]. This work was also supported by a doctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

  • dx.doi.org/10.1124/mol.116.105494.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

AMPK
5′-AMP–activated protein kinase
A769662
6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-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

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AMPK Activation Alters Stress Granule Assembly

Hicham Mahboubi, Antonis E. Koromilas and Ursula Stochaj
Molecular Pharmacology October 1, 2016, 90 (4) 460-468; DOI: https://doi.org/10.1124/mol.116.105494
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