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Protein and Vesicle Trafficking, Cytoskeleton
ERK1/2 pathway1Department of Physiology, 2Department of Biochemistry and Life Sciences Imaging Facility, McGill University, Montreal H3G 1Y6, Canada
Submitted 26 April 2007 ; accepted in final form 10 August 2007
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ABSTRACT |
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extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEKERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease. 5'-AMP-activated protein kinase; nuclear transport
The heterotrimeric AMPK contains a catalytic 
-subunit, encoded by two genes (1 and 2). The regulatory β- and 
-subunits are encoded by two and three genes, respectively (reviewed in Ref. 7). Activation of AMPK includes the phosphorylation of Thr172 of the -subunit, which can be mediated by one of the two upstream regulatory kinases LKB1 and Ca2+/calmodulin-dependent kinase kinase (CaMKK) (2, 9, and references therein). Previous reports suggest that the 2-subunit of AMPK maybe somewhat enriched in the nucleus (24), and mutations in the β1-subunit can increase its amount in nuclei (34).
The only direct means of communication between the nucleoplasm and cytoplasm is through nuclear pore complexes (NPCs, reviewed in Refs. 8, 26, and 27). There are several mechanisms of macromolecular translocation along NPCs, molecules with a mass of 40 kDa or less may diffuse across the NPC. By contrast, larger macromolecules rely on active transport and, in most cases, on specific carriers that promote transport across the NPC (8, 26, 27). Members of the importin-β family in particular play a crucial role in protein trafficking in and out of the nucleus. For instance, the nuclear exporter Crm1 recognizes hydrophobic leucine-rich signals that target a protein for export to the cytoplasm (16). Crm1-mediated nuclear export is inhibited by the drug leptomycin B (LMB), which covalently modifies the carrier (15).
To date, little is known about the nuclear and cytoplasmic pools of AMPK and how its intracellular distribution is controlled. Such a regulated localization to different cell compartments should be critical for the proper response to extra- and intracellular stimuli, leading to the phosphorylation of distinct AMPK targets. Although AMPK phosphorylates proteins in both the nucleus and the cytoplasm, it has not been determined whether its distribution is sensitive to stress or other changes in cell physiology. This knowledge is important, because it will set the stage to identify specific AMPK functions in either compartment that are dictated by physiological changes. This includes oxidative stress, a key factor that adds to the pathophysiologies in diabetic patients (22, 29, 31). To begin to answer these questions, we analyzed AMPK phosphorylation and localization in human culture cells. Since LKB1 has been associated with the activation and actions of AMPK (reviewed in Ref. 7), we used HeLa cells, which do not synthesize LKB1 (9), as well as HEK293 cells, a human kidney cell line that does express LKB1 (25). Our studies demonstrate that stress, cell density, and signaling through the extracellular signal-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK) module control the subcellular distribution of AMPK. Moreover, we show that AMPK shuttles between the nucleus and the cytoplasm and identified Crm1 as the nuclear carrier that translocates AMPK to the cytoplasm. Together, this multilayered control of intracellular localization provides a unique set of tools to rapidly adjust the distribution of AMPK to changes in cell physiology.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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70% confluency or high density (>100% confluency). This correlates with 4 x 105 and >2 x 106 cells/cm2, respectively. Heat shock was for 1 h at 45.5°C followed by recovery at 37°C for 2, 3, and 5 h. All other treatments were at 37°C. Incubation with 2 mM diethyl maleate was for 4 h. Pharmacological tools. Energy depletion was performed with 50 mM deoxyglucose combined with 10 mM NaN3 for 30 min. For inhibition of the ERK1/2 pathway, cells were incubated with 25 µM PD98059 (Calbiochem) for 5 h or 50 µg/ml ERK peptide inhibitor II (Calbiochem) for 1 h. PD98059 inhibits the MAPK kinase (MEK) thereby preventing the activation of the downstream kinase ERK1/2. The ERK peptide inhibitor II contains 13 residues derived from the NH2-terminal portion of MEK1, binds to ERK, and prevents its activation by inhibiting the binding of MEK1 (11). LMB (LC laboratories, Woburn, MA) has multiple actions, which includes inhibition of the nuclear exporter Crm1, inhibition of cell cycle progression, and antitumor activity. LMB was present at 10 ng/ml for 21 h, and controls were treated with the solvent only under identical conditions. For serum starvation, cultures were grown for 18 h without serum followed by 5 min incubation with fresh medium with or without 8% serum. After stress exposure or drug treatment, cells were immediately fixed for immunofluorescence or stored at –70°C.
Indirect immunofluorescence and microscopy.
All steps were carried out at room temperature following published procedures (14). Primary antibodies were used at the following concentrations: AMPK-1/2 (Cell Signaling Techn no. 2532), 1:200; AMPK-β1/2 (Cell Signaling Techn no. 4150), 1:200; and Cy3-coupled secondary antibodies (Jackson ImmunoResearch) were diluted 1:500. No signals were obtained when primary antibodies were omitted (not shown). Images were either acquired for 1-µm slices with a Zeiss LSM510 inverted microscope using a x63 oil-immersion objective with 1.4 numerical aperature or with the Molecular Devices (Sunnyvale, CA) ImageXpress Micro equipped with a 300-W Xenon light source and a CoolSnapHQ (Photometrics, Tucson, AZ). For the ImageXpress, Micro images of 4',6-diamidino-2-phenylindole (DAPI, cube no. 49000) or Cy3 (no. 49005) were collected with cubes from Chroma Technology (Rockingham, VT). Cells were grown on glass coverslips before being labelled and mounted with Vectashield (Vector Laboratories, Burlingame, CA) after labeling was completed. Up to 64 fields of view were imaged at x40 (PlanFluor ELWD 0.6NA) using 2 x 2 binning and ensuring at least 55 cells were sampled for each experimental condition. Image analysis was done with MetaXpress software. DAPI labeling was used to identify the number of cells in each field of view; protein staining with Cy3 was used to identify the entire cell. Care was taken to adjust the threshold level used to ensure that all cells were detected accurately even for lower expressing samples. The total intensity of antibody labeling in the nucleus was measured based on segmentation using DAPI staining, and the intensity of the entire cell was calculated based on segmentation with the Cy3 labeling. The difference between these two intensities was taken as the total intensity of the cytosolic fraction of the protein. All images were corrected for contributions due to background intensity based on intensity measurements off of the cells for each image field. Finally, the ratio of the total intensity in the nucleus versus the total intensity of the cytosol was measured for each sample. Images were processed in Adobe Photoshop 8.0 for publication.
Western blot analysis.
Cells grown on 10-cm culture dishes to 70% confluency or at high density were exposed to stress or pharmacological inhibitors as described above. After treatment, plates were rinsed with phosphate-buffered saline and stored at –70°C until use. Crude extracts were prepared by solubilizing proteins in gel sample buffer, pH 8.0, containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride; aprotinin, leupeptin, and pepstatin, each at 1 µg/ml), 20 mM β-glycerophosphate, 1 mM NaN3, and 2.5 mM NaF. Samples were incubated for 15 min at 95°C and vortexed with glass beads to shear DNA. After centrifugation (5 min, 13,000 rpm, microfuge), equal amounts of protein were separated in SDS-PA gels. Proteins were blotted to nitrocellulose and filters were processed as described (3). Antibodies against AMPK and dually phospho-ERK1/2 (Cell Signaling, no. 9106) were diluted 1:1,000; anti-ERK1/2 (StressGen, KAP-MA001C) was used at 1:2,000, anti-LDH (100–1173, Rockland, Gilbertsville, PA) at 1:2,000, and anti-lamin B (SC-6217, Santa Cruz Biotechnology, CA) at 1:2,000. ECL signals (Amersham Biosciences) were quantified by densitometry (21). Results are shown as means ± SD of at least three independent experiments.
Cell fractionation.
To isolate cytoplasmic and nuclear fractions, cells were incubated in lysis buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, and a mixture of protease inhibitors (Roche)] for 15 min on ice. Cells were drawn through a 26-gauge needle and centrifuged 10 min at 2,000 g. Sediments were resuspended in lysis buffer, centrifuged, and washed once in lysis buffer containing 0.005% Nonidet-40. After 1 min centrifugation at 20,000 g, nuclear proteins were obtained in the sediments. Combined supernatants contained the cytoplasmic marker protein LDH, whereas lamin B was restricted to nuclear fractions. For comparison, one equivalent of cytoplasmic proteins and two equivalents of nuclear proteins were analyzed side-by-side by Western blot analysis with different antibodies. ECL signals were quantified for AMPK-1/2 phosphorylated on Thr172 (p-AMPK-1/2), total AMPK-1/2 (t-AMPK-1/2), and AMPK-β1/2, and nuclear-to-cytoplasmic ratios (nuc/cyt) were calculated. Based on AMPK-1/2 Thr172 phosphorylation and the nuc/cyt distribution of p-AMPK-1/2, we determined the net nuclear levels of p-AMPK-1/2. Note that the amount of t-AMPK-1/2 was not drastically altered by the different stressors. For all conditions the net nuclear levels of controls were defined as 1.
Statistics. For Western blot analysis, ECL signals were quantified as described in Ref. 21. Data were acquired for at least three independent experiments. Results are shown as means ± SD. Bonferroni tests for multiple statistical comparisons (Figs. 1 and 2 and online supplment Fig. 1) and Student's t-test (two-tailed) for unpaired samples were carried out to identify significant differences. For each experiment, all test results were compared with the control.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1/2 and -β1/2-subunits in cultured human cells. The results are shown here for HeLa (Figs. 1–10, online supplemental Figs. 2 and 3) and HEK293 cells (online supplemental Fig. 1). When analyzed by indirect immunofluorescence, - and β-subunits of AMPK were nuclear and cytoplasmic in HeLa cells under nonstress conditions, with -subunits somewhat concentrated in nuclei. When cells were exposed to heat, energy depletion, or oxidative stress, the levels of - and β-subunits in nuclei increased (Figs. 1, A and B, and 3, A and B). In control experiments, the cytoplasmic enzyme lactate dehydrogenase (LDH) did not relocate to the nucleus for any of the stresses analyzed. Furthermore, the nuclear marker protein lamin B was confined to the nucleus under all conditions tested (online supplemental Figs. 2 and 3).
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1/2 and -β1/2 was particularly prominent after heat stress and when cells were allowed to recover at 37°C for 2 to 3 h; after 5 h recovery nuclear levels of AMPK began to decrease again. The distribution of AMPK subunits under different conditions was quantified by determining nuclear and cytoplasmic fluorescence (see MATERIALS AND METHODS) and calculating the nuc/cyt ratio of fluorescence (Fig. 1B). Since the nucleus occupies less than 10% of the mammalian cell volume, a small increase in the nuc/cyt ratio of AMPK represents a drastic change in the nuclear concentration of the protein.
The results obtained for immunolocalization were further substantiated by cell fractionation (Figs. 2A and 4A). Cytoplasmic and nuclear fractions were analyzed by quantitative Western blot analysis and quantification of ECL signals (Figs. 2B and 4B). (Note that for all cell fractionations one equivalent of cytoplasmic and two equivalents of nuclear proteins were separated side-by-side.) As observed for immunolocalization, the nuc/cyt ratio of total AMPK-1/2 and -β1/2 increased upon heat shock and started to decline upon 5 h of recovery.
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-subunits (Figs. 2, 4, and 6; p-AMPK-1/2). In crude extracts, heat stress significantly reduced the modification of Thr172 (Fig. 1, C and D). By contrast, the nuc/cyt ratio of p-AMPK-1/2 did not drastically change and slightly increased at 3 and 5 h of recovery (Fig. 2B). Thus the heat-induced changes in the nuc/cyt distribution of t-AMPK-1/2 and p-AMPK-1/2 were clearly different (Fig. 2B).
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1/2 does not necessarily lead to a change in the net amount of p-AMPK-1/2 in the nucleus. To address this point, we calculated how the net amount of p-AMPK-1/2 in nuclei changes upon exposure to heat and during recovery (see MATERIALS AND METHODS). After heat shock and after 2 or 3 h of recovery, net nuclear levels of p-AMPK-1/2 were reduced compared with unstressed cells (Fig. 2B, net nuclear).
Like heat, energy depletion with deoxyglucose-NaN3 (NDG) or oxidative stress triggered by diethyl maleate (DEM) induced t-AMPK nuclear accumulation as determined by quantitative immunolocalization and Western blot analysis (Figs. 3B and 4). Energy depletion increased the phosphorylation of AMPK-1/2 on Thr172 as well as the nuc/cyt ratio of p-AMPK-1/2. By contrast, DEM treatment reduced Thr172 phosphorylation and the nuc/cyt ratio of p-AMPK-1/2 (Figs. 3, C and D, and 4B). Taken together, changes in the distribution of AMPK-1/2 phosphorylated on Thr172 did not correlate with the redistribution of t-AMPK-1/2. Table 1 summarizes the results obtained for AMPK phosphorylation and distribution and ERK1/2 activation under different experimental conditions.
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1/2 Thr172 correlates with the activation of ERK1/2.
Crosstalk between AMPK and ERK1/2 may involve positive as well as negative signaling events (29). In our experiments with HeLa cells, heat and DEM significantly activated ERK1/2, as evident from its dual phosphorylation (Figs. 1, C and D, and 3, C and D; p-ERK1/2). At the same time, the phosphorylation of AMPK-1/2 Thr172 (p-AMPK-1/2) was reduced. By contrast, deoxyglucose-NaN3 elevated Thr172 and decreased ERK1/2 phosphorylation. In HEK293 cells upon DEM treatment, we detected neither an increase in the dual phosphorylation of ERK1/2 nor significant changes in the modification of AMPK1/2. Nevertheless, for all stress conditions analyzed there was an inverse relationship between AMPK-1/2 Thr172 phosphorylation and the dual modification of ERK1/2 in both HeLa and HEK293 cells (Figs. 1, 3, and 5 and online supplemental Fig. 1), suggesting that this effect is not restricted to a specific cell line.
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1/2 in nuclei. We further addressed this point using serum-deprived cells. After 18 h incubation in serum-free medium, the addition of serum to HeLa cells rapidly induced ERK1/2 phosphorylation. Concomitantly, there was a small but significant decrease of AMPK-1/2 Thr172 phosphorylation as well as a reduction of the net amount of nuclear p-AMPK1/2. Unlike p-AMPK1/2, t-AMPK-1/2 and -β1/2 subunits accumulated in nuclei (Figs. 5 and 6).
AMPK shuttles between the nucleus and the cytoplasm and is exported from nuclei by the carrier Crm1.
Results for the relocation of AMPK- and β-subunits upon stress and the reversal during recovery may suggest that they shuttle between the nucleus and the cytoplasm using unknown transporters. The carrier Crm1 is involved in the export of a large number of proteins and efficiently inhibited by the drug LMB. As shown for quantitative immunofluorescence (Fig. 7, A and B) and Western blot analysis (Fig. 7, C and D), incubation with LMB resulted in the nuclear accumulation of t-AMPK- and β-subunits in unstressed HeLa cells, in line with the idea that the subunits shuttle and Crm1 serves as their nuclear exporter. No significant changes were detected for the distribution of p-AMPK-1/2 (Fig. 7, C and D).
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- and β-subunits of the kinase were nuclear and cytoplasmic at 70% confluency in unstressed HeLa cells (Figs. 1, 3, 5, and 7) but were restricted to the cytoplasm in high-density cultures when analyzed by indirect immunofluorescence or cell fractionation (Fig. 8, A, C, D). Moreover, in high-density HeLa cultures treated with the inhibitor LMB, AMPK failed to accumulate in nuclei, indicating that shuttling does not take place under these conditions (Fig. 8, A, C, D). This density-dependent redistribution of AMPK is not simply an effect of changes in the medium of high-density cultures, since AMPK was nuclear as well as cytoplasmic and able to shuttle for cells growing in less dense areas of the same coverslip (data not shown). Interestingly, in high-density cells we detected little phosphorylation of AMPK-1/2 Thr172 or activation of ERK1/2 (Fig. 8B). This could be in part attributed to changes in protein levels, as the total amount of AMPK-1/2 and ERK1/2 was somewhat reduced in high-density cultures. By contrast, the levels of AMPK-β1/2 were somewhat increased (Fig. 8B). It is not known why the nuc/cyt ratio of AMPK-β1/2 decreases in the presence of LMB (Fig. 8D).
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1/2 (Table 1). Previous experiments by others indicate a complex crosstalk between AMPK activation and ERK1/2 activation, with positive as well as negative feedback between the two pathways (12). No previous studies have analyzed the possible role of MEKERK1/2 signaling in the intracellular localization of AMPK. In particular, it is not known whether ERK1/2 has any effect on the distribution of AMPK subunits under nonstress conditions. To begin to understand the events that control the nucleocytoplasmic distribution of AMPK in unstressed cells, we tested a potential role of MEKERK1/2 signaling in this process. Two different pharmacological inhibitors, PD98059, which inhibits the upstream kinase MEK, and peptide inhibitor II, which prevents ERK binding to MEK (11), induced the nuclear accumulation of AMPK-1/2 and β1/2 (Figs. 9 and 10). Although the phosphorylation of AMPK-1/2 Thr172 was slightly reduced, the nuc/cyt ratio of p-AMPK-1/2 increased upon treatment with PD98059 (Figs. 9D and 10B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Our studies provide new insights into the complex regulatory processes that determine the modification and subcellular distribution of AMPK. Changes in the phosphorylation of AMPK-1/2 Thr172 were inversely related to changes in the activation of ERK1/2. This is not restricted to HeLa cells, which are lacking the upstream activating kinase LKB1, but was also observed with HEK293 cells, which contain LKB1.
Our results demonstrate that various forms of stress, including heat, energy depletion, and oxidants, not only alter the phosphorylation state of AMPK-1/2, but also concentrate AMPK in nuclei. The simplified model in Fig. 11 summarizes how different physiological conditions regulate the intracellular distribution of AMPK. In unstressed cells (Fig. 11A), AMPK- and -β shuttle between the nucleus and the cytoplasm, using the carrier Crm1 for export from the nucleus. Upon exposure to stress both - and β-subunits accumulate in nuclei (Fig. 11B). This redistribution could be achieved by upregulation of AMPK nuclear import, increase in nuclear retention, reduced export, or a combination of these events. High cell density confines AMPK- and β-subunits to the cytoplasm and prevents them from shuttling (Fig. 11C). It is possible that contacts between neighboring cells regulate the distribution of AMPK; signaling events based on high culture density could prevent nuclear import or induce cytoplasmic anchoring of AMPK subunits. Finally, our results demonstrate a complex role for MEKERK1/2 signaling in the control of AMPK localization. Under nonstress conditions, inhibition of the MEKERK1/2 pathway triggered nuclear accumulation of AMPK - and β-subunits (Fig. 11D).
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1/2 and t-AMPK-1/2 may be controlled differently. Our results indicate that there is no direct link between the phosphorylation of Thr172 of AMPK-1/2 and the distribution of t-AMPK-1/2. However, the changes in net nuclear p-AMPK-1/2 are negatively correlated with ERK1/2 activation; whenever the net nuclear levels of p-AMPK-1/2 were reduced, we observed an increase in ERK1/2 activation, and vice versa (summarized in Table 1).
Taken together, our results support the hypothesis that p-AMPK-1/2 localization can be linked to the activation status of ERK1/2, whereas a more complex regulation directs the distribution of t-AMPK-1/2 and β1/2 in stressed cells. Future experiments will have to determine whether the localization of the MAPK ERK1/2, in addition to its activation, plays a role in the nucleocytoplasmic distribution of AMPK.
At this point, we can only speculate how AMPK redistribution participates in the response to different stressors. One effect of AMPK redistribution could be changing its interactions with kinase substrates. For example, raising the nuc/cyt ratio of activated AMPK in nuclei may increase the phosphorylation of nuclear or reduce the modification of cytoplasmic targets. AMPK controls cell physiology not only by the direct phosphorylation of various metabolic enzymes but also via transcriptional regulation (reviewed in Ref. 28). Many genes change their expression levels when a dominant-negative allele of AMPK-2 is highly overexpressed over the endogenous wild-type -subunit in skeletal muscles of transgenic mice (20). Proteins encoded by these genes participate in various functions, including energy metabolism, cell signaling, transcription, and translation. Furthermore, AMPK activity contributes to the regulation of mRNA stability as the kinase controls the half-lives of p21, cyclin A, B1, and VEGF mRNAs (reviewed in Ref. 6).
A possible connection between the multiple processes that AMPK affects and the results reported here could be the transcriptional regulator p300/CBP and several transcription factors that are acetylated by p300/CBP. These include members of the FOXO family, p53 and NF-
B. AMPK phosphorylates p300/CBP on Ser89 (35) thereby modulating the interactions of p300/CBP with transcriptional regulators (reviewed in Ref. 28). Upon oxidative stress, FOXO proteins move to the nucleus to be bound and acetylated by p300/CBP (reviewed in Refs. 30 and 33). The acetylation state of FOXO proteins seems to control which genes are selected for transcription. A simplified model may propose a link between AMPK activation, p300/CBP phosphorylation, and FOXO protein acetylation; and the cascade AMPKp300/CBPFOXO proteins could contribute to the specific gene expression following oxidative stress. Since we observed a reduction in p-AMPK-1/2 in nuclei upon oxidant exposure, this model would predict that p300/CBP phosphorylation on Ser89 is reduced, which could modulate the subsequent interaction between p300/CBP and FOXO proteins and ultimately gene expression.
Our data show that the nucleocytoplasmic distribution of total and phosphorylated AMPK is regulated differently; AMPK subunits accumulate in nuclei upon stress independent of kinase activation. There are several possible scenarios that could explain this redistribution: 1) the redistribution of AMPK to nuclei controls its accessibility to activating upstream kinases; 2) AMPK nuclear accumulation is caused by inhibition of nuclear export in stressed cells; and 3) alternatively, AMPK subunits have additional functions that are unrelated to the kinase activity but required in nuclei of stressed cells for other processes. Future experiments will have to explore these possibilities.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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