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. Author manuscript; available in PMC: 2017 Apr 28.
Published in final edited form as: Prostate. 2015 Oct 6;76(2):172–183. doi: 10.1002/pros.23106

Gsk-3β Controls Autophagy by Modulating LKB1-AMPK Pathway in Prostate Cancer Cells

Aijing Sun 1,2, Changlin Li 2, Ruibao Chen 2, Yiling Huang 2,3, Qi Chen 4, Xiangjun Cui 5, Huafeng Liu 6, J Brantley Thrasher 2, Benyi Li 2,3,5,6,*
PMCID: PMC5408751  NIHMSID: NIHMS856909  PMID: 26440826

Abstract

BACKGROUND

Glycogen synthase kinase 3β (GSK3B, GSK-3β) is a multi-functional protein kinase involved in various cellular processes and its activity elevates after serum deprivation. We have shown that inhibition of GSK-3β activity triggered a profound autophagic response and subsequent necrotic cell death after serum deprivation in prostate cancer cells. In this study, we dissected the mechanisms involved in GSK-3β inhibition-triggered autophagy.

METHODS

Prostate cancer PC-3 and DU145 cells were used in the study. Multiple GSK-3β specific inhibitors were used including small chemicals TDZD8, Tideglusib, TWS119, and peptide L803-mts. Western blot assay coupled with phospho-specific antibodies were used in detecting signal pathway activation. ATP levels were assessed with ATPLite kit and HPLC methods. Autophagy response was determined by evaluating Microtubule-associated proteins 1A/1B light chain 3B (LC3B) processing and p62 protein stability in Western blot assays. Immunofluorescent microscopy was used to detect LKB1 translocation.

RESULTS

Inhibition of GSK-3β activity resulted in a significant decline of cellular ATP production, leading to a significant increase of AMP/ATP ratio, a strong trigger of AMP-activated protein kinase (AMPK) activation in prostate cancer PC-3 cells. In parallel with increased LC-3B biosynthesis and p62 protein reduction, the classical sign of autophagy induction, AMPK was activated after inhibition of GSK-3β activity. Further analysis revealed that Liver kinase B1 (LKB1) but not Calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) is involved in AMPK activation and autophagy induction triggered by GSK-3β inhibition. Meanwhile, GSK-3β inhibition promoted LKB1 translocation from nuclear to cytoplasmic compartment and enhanced LKB1 interaction with its regulatory partners Mouse protein-25 (MO25) and STE20-related adaptor (STRAD).

CONCLUSIONS

In conclusion, our data suggest that GSK-3β plays an important role in controlling autophagy induction by modulating the activation of LKB1-AMPK pathway after serum deprivation.

Keywords: GSK-3β, autophagy, AMPK, LKB1, serum deprivation

INTRODUCTION

Autophagy is initiated after activation of the Unc-51-like kinase 1/2 (ULK1/2) complex that controls the nucleation step of autophagosome formation in mammalian cells [1,2]. As the major energy sensor, the enzyme AMP-activated protein kinase (AMPK) activity increases once cellular AMP levels elevate or ATP levels decrease under nutrient/metabolic stress such as ischemia [3,4]. AMPK is a heterotrimeric complex with one catalytic α and two regulatory β/γ subunits encoded by distinct genes and AMPKα phosphorylation on threonine 172 (pT172) is required for full activation. Several protein kinases are capable of phosphorylating this site, including the ubiquitously expressed liver kinase B-1 (LKB1) and Ca2+/calm-odulin-dependent protein kinase kinase β (CaMKKβ) [4,5]. The initial evidence of AMPK modulation of autophagy in mammalian cells came from analyzing degradation of long-lived proteins [6]. Currently, it is conceivable that among several cellular signal pathways that regulate autophagy response under physiological or pathological conditions, LKB1-AMPK pathway was demonstrated to directly modulate autophagy activity by suppressing protein translation activator mammalian target of rapamycin (mTOR) pathway [7–9] and by activating autophagy initiator ULK1 [10–13].

The serine-threonine kinase LKB1 (STK11) was originally found defective in Peitz–Jeghers syndrome that exerts multiple malignancies in human [14]. Later on LKB1 was also found to be mutated or lost in multiple sporadic cancers [15]. Genetically modified mouse models with Lkb1 deficiency developed tumors in various organ/tissue systems including prostate gland [16,17], demonstrating the tumor suppressor nature of LKB1 gene. The importance of LKB1 in cancer cell metabolism was unveiled by the discovery of its control over the cellular energy sensor APMK [18,19].

Glycogen synthase kinase 3 (GSK-3) is a constantly active kinase with multiple functions involving in numerous aspects of cellular fate determination and its activity increases after serum deprivation. There are two GSK3 isoforms, α and β, in mammals [20,21]. We recently showed that inhibition of GSK-3β activity triggered a profound autophagic response in cells under serum-free condition [22]. This phenomenon was also observed in vivo from ischemic mouse models [23,24]. However, the mechanism underlying GSK-3β inhibition-triggered autophagy is not fully clear. In this study, we demonstrated that activation of LKB1-AMPK pathway is responsible for GSK-3β inhibition-triggered autophagy induction under serum-free condition.

MATERIALS AND METHODS

Antibodies and Chemicals

Antibodies for LKB1, GSK-3β, LC3B, mTOR, p70S6K1, and AMPK were obtained from Cell Signaling Inc., (Danver, MA). Antibodies for MO25 and STRAD were from Abgent Inc., (San Diego, CA). Antibodies for Flag and HA tags, ULK1, TIP60, Actin and p62, CaMKKβ, secondary antibodies, as well as TDZD8, 7AIPM, and all pre-verified siRNAs were purchased from Santa Cruz Biotech (Santa Cruz, CA). Anti-HMGB1 antibody was from GeneTex Inc., (Irvine, CA). L803-mts was described previously [22]. The small chemical Wnt agonist was purchased from EMD Biosciences (Catalog #681665, Billerica, MA).

Cell Culture, Drug Treatment, and Transfection

Human prostate cancer cell lines PC-3 and DU145 were obtained from ATCC (Manassas, VA) and cultured in RPMI 1,640 medium supplied with 10% fetal bovine serum (FBS) plus antibiotics as described [22]. The solvent DMSO was added at the same volume in separate well as control.

The plasmid constructs for wild-type or mutant of LKB1 (Flag-tag, #8592/#8593) [19] were obtained from Addgene (Cambridge, MA). The constructs of GSK-3 mutants (HA tag) were gifts friendly provided by Dr Woodgett [25]. Cells were transfected with Plasmid DNAs in Lipofectamine® (Invitrogen, Carlsbad, CA) overnight. Transfection of the siRNAs was conducted with Oligofectamine® (Invitrogen).

ATP-Dependent Luciferase Assay, Glycolysis Assay, and AMP/ATP Measurement by HPLC

After treatment, cells were harvested and the ATP levels were determined using the ATPLite™ assay kit obtained from PerkinElmer (Boston, MA) following the protocol provided by the manufacturer. Cell culture media were collected for the determination of glycolysis activity with a cell-based assay kit, which was designed to detect extracellular levels of L-lactate, the end-product of cellular glycolysis (Catalog #600450, Cayman Chemical, Ann Arbor, MI).

For HPLC-based measurement, cells were harvested, washed, and re-suspended in PBS. Nucleotides (ATP and AMP) were extracted by fast lysing the cells in 0.05 M KOH solution, and then immediately neutralized to pH 6 with 0.1 M KH2PO4. After centrifuge, the supernatant was analyzed by a gradient HPLC method on a Waters e2695 HPLC with UV detection at 254 nm and 340 nm (Waters e2,489 diode array UV detector, Waters, MA). The reversed-phase chromatography was performed with an XBridge™ C18 column 3.5 μm (Waters, Milford, MA). Mobile phase (pH 6) contained acetonitrile (2% for Solvent A and 30% for Solvent B), 0.1 M KH2PO4, and 0.008 M Tetrabutylammonium hydrogen sulfate. The Empower II software (Waters, MA) was used for instrument control and data analysis. All values were normalized to the protein content of whole homogenaties using the bicinchoninic acid method (Pierce Biotechnology, IL).

Western Blot, Co-Immunoprecipitation, and Immunofluorescent Microscopy

Cells were harvested, rinsed in cold PBS, and lysed in RIPA buffer (Cell Signaling Inc., Danvers, MA). Equal amount of total cellular proteins was subjected to SDS–PAGE followed by immunoblotting with antibodies as indicated. HMGB1 nuclear release detection was conducted as described [22]. For detection of LKB1 interaction with MO25/STRAD, total cellular proteins were prepared in Chaps-containing lysis buffer (Cell Signaling Inc.,) and used for anti-LKB1 immunoprecipitation overnight. Precipitation was carried out with protein A/G agarose plus beads (Santa Cruz Biotech, Dallas, TX) and the immunoprecipitates were eluted in Laemmli sample buffer (Bio-Rad, Hercules, CA). The blot was lastly re-probed with anti-LKB1 antibody as protein loading control.

For examining LKB1 cellular localization in PC-3 cells, immunocytofluorescent assay was conducted with anti-LKB1 and FITC-conjugated secondary antibodies. Cell nuclear were stained with DAPI-containing mounting media. Fluorescent signal was evaluated under an Olympus inverted microscope and microscopic images were taken as described [22].

For LC3B puncta analysis, GFP-LC3 expression constructs (Addgene, plasmid #22405) were transfected in PC-3 cells. GFP puncta were counted from more than 10 microscopic fields. Microscopic images were taken under fluorescent microscope as described previously [22].

Statistical Analysis

Western blot data and fluorescent images presented were from a representative experiment. ATP luciferase and glycolysis data, HPLC measurement of ATP/AMP levels, as well as quantitative data of LC3B puncta and LKB1 cellular localization were presented as mean ± SEM and analyzed using SPSS software (SPSS, Chicago, IL). The differences between drug treatment and the solvent control were considered statistically significant when the P is less than 0.05.

RESULTS

GSK-3β Inhibition Reduces Cellular ATP Production

To elucidate the mechanisms underlying GSK-3β inhibition-trigged autophagic response as reported in our recent publication [22], we first determined if GSK-3β inhibition attenuated cellular ATP levels since energy crisis is a key factor for autophagy induction. PC-3 cells were serum-starved for 24 hr, followed by treatment with GSK-3β specific inhibitors [26,27]. As shown in Figure 1A, cellular ATP levels moderately declined along with time after serum starvation as measured by an ATP-dependent luciferase assay [28]. Treatment with three small molecule inhibitors TDZD8, Tideglusib [29], and TWS119 [30] resulted a significant decline of cellular ATP levels (Fig. 1A and B). Another peptide GSK-3β specific inhibitor L803-mts also caused a similar effect on ATP levels (Fig. 1C). This decline of cellular ATP levels was confirmed by a secondary measurement, high-performance liquid chromatography (HPLC), on cellular AMP/ATP contents. Meanwhile, cellular AMP/ATP ratio increased significantly after TDZD8 treatment (Fig. 1D). These data suggest that GSK-3β inhibition attenuated cellular ATP production after serum deprivation.

Fig. 1.

Fig. 1

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GSK-3 inhibition reduces cellular ATP production. (A–C) PC-3 cells were seeded in 12-well plates overnight and were then serum-starved for 24 hr before treatment. At the end of experiments, cells were harvested, rinsed in cold PBS, and then subjected to ATP-dependent luciferase assay using the ATPLite™ kit. Asterisks indicate a significant difference compared to the control (ANOVA, P <0.05). (A) Cells were treated with the solvent (DMSO) or TDZD-8 (10 μM) for indicated time points. (B) Cells were treated with the solvent, Tideglusib or TWS119 for 16 hr at the indicated drug doses in serum-free media. Time point 0 indicates the condition before any treatment. (C) Cells were treated with the solvent in 10% FBS (FBS+) and in serum-free condition (FBS−), or with L803-mts (100 μM) for 16 hr. (D) PC-3 cells were seeded in 12 plates and then serum-starved for 24 hr. After treatment with the solvent or TDZD8 as indicated, cells were harvested for HPLC-based measurement of nucleotides ATP and AMP levels. Data presented (Mean ± SEM) were from three independent experiments and the error bars indicate SEM. Asterisks indicate a significant difference compared to the control at each time point (ANOVA, P <0.05). (E and F) PC-3 cells were seeded in 12-well plates overnight and were then serum-starved for 24 hr. Cells were treated with the solvent, TDZD8 or Tideglusib in 10% FBS-supplied (E) or FBS-free media (F) for 16 hr. The L-lactate levels in the media were normalized to cellular protein levels in the corresponding sampling well. Data presented (Mean ± SEM) were from three independent experiments and the asterisks indicate a significant difference compared to the DMSO control (ANOVA, P <0.05).

It has been shown that under nutritional stress as such as ischemia cellular glycolysis increases to boost ATP production [31]. Therefore, we determined if cellular glycolytic activity was affected after GSK-3β inhibition. PC-3 cells were serum-starved for 24 hr and then treated with TDZD8 or Tideglusib. Glycolytic activity was assessed by measuring the levels of L-lactate, the end product of glycolysis, in culture media. As shown in Figure 1E, GSK-3β inhibitors had only a mild effect on cellular glycolysis activity under FBS-supplied condition. Consistent with a recent report [32], cellular glycolytic activity (in other word, L-lactate release) elevated about two-fold under FBS-free condition, however, GSK-3β inhibitors significantly reduced glycolysis activity in a dose-dependent manner (Fig. 1F). These data indicate that GSK-3β activity is essential for cellular glycolysis and ATP production under serum starvation condition.

GSK-3β Inhibition Leads to AMPK Activation

As a major energy sensor, AMPK is activated once cells suffer energy crisis [4]. Because GSK-3β inhibition attenuated cellular ATP production, we assessed if AMPK was activated in parallel to autophagy induction triggered by GSK-3β inhibition after serum deprivation. As shown in Figure 2A, TDZD8 treatment triggered a drastic autophagy response, as evidenced by increased LC3B biosynthesis and processing (increased level of both LC3BI and II) as well as p62 degradation, two major parameters of autophagy induction [33]. In later time points, TDZD8 treatment induced cellular necrosis at 16–24 hr as evidenced by the release of nuclear protein HMGB1, which is consistent with our previous report [22].

Fig. 2.

Fig. 2

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GSK-3β inhibition leads to AMPK activation and autophagy induction. (A and B) PC-3 cells were seeded in six-well plates overnight and then serum-starved for 24 hr. Cells were treated with the solvent (DMSO) or TDZD8 (10 μM) for the indicated time before harvesting. Equal amount of cellular proteins were subjected to Western blot with the antibodies indicated in the figure. (C and D) PC-3 cells seeded in six-well plates were transfected with the control siRNA or gene-specific siRNAs for GSK-3β (C) or GSK-3α (D, 100 nM) for 2 days in serum-supplied media (10% FBS). After serum starvation for 24 hr, cells were harvested for Western blot analysis with the antibodies as indicated. Actin blot served as protein loading control.

Then, we determined if TDZD8 treatment led to AMPK activation by evaluating its phosphorylation at residue T172 [34]. As expected, AMPK pT172 Levels dramatically increased at 4 hr after TDZD8 treatment compared to the solvent treatment (Fig. 2B). Similar effect was observed when GSK-3β but not GSK-3α gene expression was knocked down with gene-specific small interfering RNAs (siRNAs) (Fig. 2C and D). In parallel, LC3B processing (LC3B-II level increased) was drastically enhanced after TDZD8 treatment (Fig. 2B) while only LC3B biosynthesis was induced (LC3B-I level increase) under the solvent treatment. These data clearly indicated that LC3B biosynthesis was induced by serum starvation but its processing was enhanced after GSK-3β inhibition in parallel to AMPK activation. These data are supported by a recent report showing AMPK activation and autophagy induction in renal cancer cells after treatment with a structurally different GSK-3 inhibitor 9-ING-41 [35].

Next, we examined if GSK-3β inhibition caused inactivation of mTOR pathway, which is negatively regulated by AMPK [36]. As shown in Figure 3A, TDZD8 treatment reduced mTOR phosphorylation levels at the S2448 site. Meanwhile, phosphorylation of mTOR downstream targets p70S6K (pT389) and 4E–BP1 (pT37/T46), the common hallmarks of mTOR activation, were also reduced after TDZD8 treatment. Taken together, these data suggest that GSK-3β inhibition resulted in inactivation of mTOR pathway.

Fig. 3.

Fig. 3

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GSK-3β inhibition leads to inactivation of mTOR pathway. PC-3 cells were seeded in six-well plates and then serum-starved for 24 hr. Cells were treated with the solvent (DMSO) or TDZD8 (10 μM) for 2–4 hr (A) or 4 hr (B), or treated with the Wnt agonist at different doses as indicated (C). Cell lysates were subjected to Western blot assay as described above. Data represent three independent experiments.

It was recently reported that GSK-3β phosphorylates lysine acetyltransferase 5 (KAT5, also called HIV-1 Tat interacting protein 60, TIP60) at S86 site, leading to ULK1 activation and autophagy induction after serum starvation in human colorectal cancer HTC116 cells [37], we went on to determine if TIP60 phosphorylation was altered after GSK-3β inhibition. As shown in Figure 3B, TDZD8 treatment in PC-3 cells increased ULK1 pS317 Level, a sign of ULK1 activation [12], which is consistent with AMPK activation and autophagy induction as shown above. However, TIP60 pS86 Level had no significant alteration after TDZD8 treatment, indicating that GSK-3β-dependent TIP60 activation after serum starvation has a cell-specific role in autophagy regulation.

It has been shown that activation of canonical Wnt pathway negatively regulates autophagy induction, and vice versa [38,39], while GSK-3β inactivation is a common consequence of canonical Wnt activation [40]. Thus, we went on to determine if canonical activation of Wnt pathway was involved in GSK-3β inhibition-triggered autophagy. A small chemical Wnt agonist [41], which has no inhibitory effect against GSK-3β activity, was used to treat PC-3 cells under serum-free condition. As shown in Figure 3C, there was no obvious change in LC3B biosynthesis or processing, as well as p62 protein level after Wnt agonist treatment, although AMPK pT172 Levels were increased by Wnt agonist treatment at a dose-dependent manner. These data suggest that the canonical Wnt pathway is not involved in GSK-3β inhibition-triggered autophagy induction.

LKB1 Is Required for AMPK Activation After GSK-3 Inhibition

It has been shown that LKB1 is the upstream kinase that phosphorylates and activates AMPK in response to nutrient restriction [5,19,42]. We then went on to determine if LKB1 is responsible for AMPK activation after GSK-3β inhibition. LKB1 siRNAs were transfected in PC-3 cells followed by treatment of TDZD8 or L803-mts. As shown in Figure 4A and B, knocking down LKB1 expression abolished GSK-3β inhibitor–induced AMPK (pT172) phosphorylation. In contrast, siRNAs for CaMKKβ, another upstream kinase of AMPK [43], had not obvious effect on GSK-3β inhibition-induced AMPK phosphorylation (Fig. 4A). Consistently, after LKB1 knockdown were largely attenuated GSK-3β inhibition-induced LC3B processing and p62 degradation (Fig. 4B), as well as LC3B puncta formation (Fig. 4C, quantitative data shown in the bar graph) in comparison to the control siRNA. These data indicate that LKB1 but not CaMKKβ is involved in GSK-3β inhibition-induced AMPK phosphorylation and autophagy response.

Fig. 4.

Fig. 4

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LKB1 is responsible for AMPK activation after GSK-3β inhibition. (A–B) PC-3 cells seeded in six-well plates were transfected with the control or gene-specific siRNAs as indicated at 100 nM in media for 2 days in serum-supplied media (10% FBS). Mock transfection omitted the siRNA content. After serum starvation for 24 hr, cells were treated with the solvent (DMSO), TDZD8 (10 μM), or L803-mts (100 μM) for 4 hr before harvesting. Immunoblotting was conducted with the antibodies as indicated. Actin blot served as protein loading control. (C) PC-3 cells were transfected with the siRNAs as indicated for 2 days followed by another transfection with GFP-LC3 construct for 24 hr. Cell were then serum-starved overnight followed by treatment with the solvent or TDZD8 for 4 hr. GFP puncta were counted from more 10 microscopic fields and the representative images were shown from each treatment conditions (magnification: ×200). Quantitative data of GFP-LC3 puncta were summarized in the bar graph panel. The asterisk indicates a significant difference (Student t-test, P <0.05) compared to the LKB1 siRNA transfection (siLKB1).

Next, we used a secondary approach to confirm LKB1 involvement in GSK-3β inhibition-induced AMPK activation. It was reported that DU145 cell line is LKB1-null [44] and we confirmed this LKB1 null pattern (Fig. 5A). As expected, GSK-3β inhibitors did not induced any obvious alteration of AMPK pT172 phosphorylation, LC-3B biosynthesis/processing, and p62 stability in DU145 cells (Fig. 5B). We then reinstalled DU145 cells with either wild-type (LKB1-WT) or kinase-dead LKB1 mutant (LKB1-KD) proteins (Fig. 5C and D). Overexpression of LKB1-WT but not LKB-KD mutant largely increased AMPK pT172 Levels (Fig. 5C lane 5 vs. lane 1), which was further increased by TDZD8 treatment (Fig. 5C lane 6 and Fig. 5D lane 3), while the AMPK activator compound AICAR [45] also increased AMPK phosphorylation even in LKB1-KD transfected DU145 cells (Fig. 5C lane 4), indicating a LKB1-independent activation of AMPK by AICAR treatment as reported by a previous report [46]. Similar to TDZD8 treatment, suppressing GSK-3β by over-expressing a kinase-dead (K85A) mutant together with LKB1-WT but not LKB1-KD also increased AMPK pT172 phosphorylation (Fig. 5D, lane 5 vs. lane 4), while the constitutive active GSK-3 mutant (S9A) had no obvious effect. These data further demonstrated that LKB1 is responsible for AMPK phosphorylation after GSK-3β inhibition.

Fig. 5.

Fig. 5

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Reinstallation of LKB1 activates AMPK in DU145 cells after GSK-3β inhibition. (A) Exponentially grown cells from indicated cell lines were harvested for protein extraction. Equal amount of cellular proteins were subjected to western blot assay with LKB1 or Actin antibodies. (B) DU145 cells were seeded in six-well plates and then serum-starved for 24 hr. Cells were treated with the solvent (DMSO), GSK-3β inhibitors (L803-mts and 7AIPM) at the indicated doses for 4 hr before harvesting. Equal amount of cellular proteins were subjected to Western blot assays. Actin blot was used as protein loading control. (C) DU145 cells in six-well plates were transfected with the plasmid constructs as indicated (1.0 μg plasmid DNA each construct per well) using Lipofectamine® overnight. The mock transfection was conducted by omitting the plasmid DNA. After a 24 hr serum starvation, cells were treated with the solvent DMSO, AICAR (1.0 mM), or TDZD8 (10 μM) for 4 hr in serum-free media. Cells were harvested, rinsed, and lysed for Western blot analysis with the antibodies as indicated. Actin blot served as protein loading control. (D) DU145 cells in six-well plates were transfected with the plasmid constructs as indicated (2.0 μg plasmid DNA each construct per well) using Lipofectamine® overnight. After a 24 hr serum starvation, cells were treated with the solvent (DMSO) or TDZD8 (10 μM) for 4 hr in serum-free media. Cells were harvested, rinsed, and lysed for Western blot analysis with the antibodies as indicated. LKB1 mutants were Flag-tagged and GSK-3β constructs were HA-tagged. Actin blot served as protein loading control. Data represent two separate experiments.

GSK-3β Inhibition Promotes LKB1 Cytoplasmic Translocation

Cytoplasmic translocation from nuclear compartment is a major regulatory mechanism for LKB1 activation [47]. Therefore, we examined if GSK-3β inhibition resulted in LKB1 nuclear-cytoplasm translocation. PC-3 cells were treated with TDZD8 or L803-mts followed by anti-LKB1 immunofluorescent staining. As shown in Figure 6A, LKB1 was mainly resided in the nuclear compartment in solvent-treated control cells. After TDZD8 or L803-mts treatment, LKB1 was mainly localized in cytoplasm. Quantitative analysis revealed a significant difference in terms of LKB1 cellular localization after GSK-3β inhibition (Fig. 6B). These data clearly indicate that GSK-3β inhibition resulted in LKB1 nuclear export to cytoplasmic compartment.

Fig. 6.

Fig. 6

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GSK-3β inhibition leads to LKB1 nuclear-cytoplasm translocation. (A and B) PC-3 cells were seeded on coverslips overnight. After serum starvation for 24 hr, cells were treated with the solvent, TDZD8 (10 μM) or L803-mts (100 μM) for 4 hr. Cells were then rinsed, fixed, and permeabilized for immunocytofluorescent assay with anti-LKB1 antibody. FITC-conjugated secondary antibody was used to visualize the signal. Cell nuclear compartment was stained with DNA dye DAPI. Microscopic images were representative from two independent experiments. Magnification: ×200. The percentages of cells with distinct LKB1 Localization (Mean ± SEM) from more 10 microscopic fields were summarized in the graphic panel (B) The asterisk indicates a significant difference of the cytoplasmic localization compared to the nuclear localization (Student t-test, P <0.05). (C) PC-3 cells were seeded in P100 dishes and cultured in different conditions as follow: regular condition (10% FBS) or FBS-free for 24 hr and then treated with the solvent (FBS-free), TDZD8 (10 μM) or L803-mts (100 μM) for 4 hr. Cells were rinsed in cold PBS and harvested for protein extraction. Immunoprecipitation was carried out with anti-LKB1 antibody or IgG control followed by Western blotting with anti-MO25 and anti-STRAD antibodies. Membrane was re-probed with anti-LKB1 for protein loading control. Whole cellular lysate was used as Input control. Data represent two separate experiments.

It has been suggested that LKB1 is activated after interacting with two adaptor proteins MO25 and STRAD [42,47,48]. Therefore, we explored if GSK-3β inhibition promoted the interaction of LKB1 with MO25 or STRAD. An anti-LKB1 co-immunoprecipitation assay was conducted using total protein extract from PC-3 cells cultured in regular condition (10% FBS), FBS-free media plus/minus TDZD8 or L803-mts. As shown in Figure 6C, the adaptor protein MO25 was constantly associated with LKB1. This interaction was largely increased by TDZD8 or L803-mts treatment. Most interestingly, STRAD interaction with LKB1 was observed after GSK-3β inhibitor treatment, indicating a recruitment of STRAD to LKB1-MO25 complex after GSK-3β inhibition. These data suggest that inhibition of GSK-3β activity promotes the formation of LKB1/STRAD/MO25 complex, possibly leading to LKB1 activation (Fig. 7).

Fig. 7.

Fig. 7

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GSK-3β modulation of LKB1-AMPK pathway in autophagy induction. (A) With serum/growth factors supplement, cells are growing normally due to PI3K-AKT pathway activation, which suppresses GSK-3β activity. Meanwhile, sufficient nutrient supply also leads to low activity of LKB1-AMPK pathway. (B) Under serum starvation, cellular AMP accumulates and LKB1-AMPK pathway is activated, resulting in autophagy induction. Meanwhile, inactivation of PI3K-AKT pathway also leads to increase of GSK-3β activity, which serves as a checkpoint of LKB1-AMPK pathway to control the extent of cellular autophagy activity. (C) Under serum starvation, GSK-3β inhibition removes its restrictive action on LKB1-AMPK pathway, leading to profound autophagy and cell death. Bif-1 accumulation is also involved in this process as reported (22).

DISCUSSION

In this study, we demonstrated that inhibition of GSK-3β activity resulted in a rapid decline of cellular ATP production and subsequently AMPK activation through a LKB1-dependent mechanism. These data provide a strong mechanistic insight to our recent report that GSK-3β inhibition induced a profound autophagic response and subsequent necrotic cell death under serum-free condition [22]. As the authors’ aware, this is the first report regarding GSK-3β modulation of AMPK activation via LKB1-dependent mechanism.

GSK-3β modulation of autophagy is an understudied area, and it has been claimed as a potential therapeutic target for several human diseases such as type II diabetes, neuronal degenerative diseases, and cancers [40,49] that are mostly involving metabolic reprograming and autophagic events. GSK-3β inhibitors were shown to enhance autophagy activity in neuronal cells to promote Amyloid-β degradation [50] and in ischemia rat brain to suppress inflammation [24]. Conversely in ischemic mouse heart, GSK-3β inhibition exacerbates ischemic injury by stimulating mTOR pathway and by suppressing autophagy [23]. GSK-3β was also found to phosphorylate the acetyltransferase HIV-1 Tat interactive protein (TIP60) that subsequently acetylates and activates ULK1 to trigger autophagy after serum starvation in human colorectal carcinoma HTC116 cells [51]. These data indicated that GSK-3β exerts distinct effect on autophagy depending on stress conditions and cell types.

Initially, LKB1 was shown as a negative regulator of GSK-3β activation under the canonical Wnt signaling in a study of Xenopus embryo development [52]. Similarly, GSK-3β was found to inhibit mTOR pathway by phosphorylating tuberous sclerosis complex two (TCS2) in concert with AMPK after Wnt stimulation [53]. But in human cervical cancer HeLa cells that are null of LKB1 gene, reinstallation of wild-type but not kinase-dead mutant LKB1 induced de-phosphorylation of GSK-3β at serine nine position, suggesting a positive regulatory effect of LKB1 on GSK-3β activation [54]. In our study, inhibition of GSK-3β activity in prostate cancer cells under serum-free condition resulted in activation of LKB1-AMPK pathway, indicating that GSK-3β can also act as a negative modulator of LKB1-AMPK pathway. This notion is supported by a recent report that GSK-3β negatively regulates AMPK activity to potentiate neutrophil activation in an inflammatory mouse model [55].

It has been shown that LKB1 is predominantly in the nucleus when it is not associated with its regulatory proteins STRAD and MO25 but it is translocated to the cytoplasm after activation [42,47,48]. In our study, we found that inhibition of GSK-3β activity promoted LKB1 nucleo-cytoplasmic translocation and interaction with its adaptor protein STRAD. It might be postulated that GSK-3β negatively regulates LKB1-AMPK pathway by keeping LKB1 inside the nuclear compartment away from its partners. It is also plausible that LKB1 and GSK-3β might be reciprocally modulating each other as a negative feed-back mechanism, although further mechanistic study is needed to elucidate the details.

In summary, this study shed new lights on GSK-3β inhibition-triggered autophagy after serum deprivation. We demonstrated that inhibition of GSK-3β activity caused a rapid decline of cellular ATP production, and subsequently LKB1-depedent AMPK activation and inactivation of mTOR pathway in association with autophagy induction. After serum deprivation, GSK-3β plays an important role in modulating LKB1 activity by restricting its nucleo-cytoplasmic translocation and the formation of LKB1/STRAD/MO25 protein complex. Our studies also demonstrated that LKB1 but not CaMKKβ is responsible for AMPK activation after GSK-3β inhibition. It is reasonable to hypothesize that combining GSK-3β inhibitor (like Tideglusib) with vascular disrupting agents (VDAs) that reducing blood flow in tumor tissue might improve VDA’s efficacy [56,57] although further studies are desirable.

Acknowledgments

Grant sponsor: Zhejiang Provincial Natural Science Foundation of China; Grant number: #LY13H160032; Grant sponsor: NIH/NCI R21; Grant number: 1R21CA175279-01A1; Grant sponsor: Chinese NSF; Grant number: #81172427; Grant sponsor: KU William L. Valk Endowment.

We are grateful for the technical assistance from Stanton Fernald at the KUMC Imaging Core facility. This work was partially supported by a grant from Zhejiang Provincial Natural Science Foundation of China (#LY13H160032) to Dr Aijing Sun, and by grants from NIH/NCI R21 Grant (1R21CA175279-01A1) and Chinese NSF Grant (#81172427), as well as by the KU William L. Valk Endowment to Dr Benyi Li.

Footnotes

Conflict of interest: None.

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