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. 2010 Dec 1;13(2):184–194. doi: 10.1093/neuonc/noq163

AMPK/TSC2/mTOR-signaling intermediates are not necessary for LKB1-mediated nuclear retention of PTEN tumor suppressor

Juinn-Lin Liu 1, Zhenyu Mao 1, Gary E Gallick 1, W K Alfred Yung 1,✉
PMCID: PMC3064623  PMID: 21123367

Abstract

The regulation of the subcellular localization of phosphatase and tensin homologue (PTEN) is critical to its tumor-suppressing functions. Previously, we found that the activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR/S6 protein kinase (S6K) cascade triggers the nuclear export of PTEN during the G1/S transition. Because mTOR can be alternatively downregulated by tuberous sclerosis complex 2 (TSC2) activation mediated by 5′ adenosine monophosphate-activated protein kinase (AMPK), we proposed that the activation of AMPK α1/2 by LKB1 and/or by calmodulin-dependent protein kinase kinase (CaMKK) would also block the nuclear export of PTEN in a manner similar to that of inhibitors of PI3K, mTOR, and S6K. We found that in LKB1-null A549 lung adenocarcinoma cells, an AMPK activator, metformin, failed to block the nuclear export of PTEN, and the reintroduction of functional LKB1 into these cells restored the metformin-mediated inhibition of the nuclear export of PTEN. In addition, the nuclear export of PTEN was blocked in cells treated with the CaMKK activator ATP, and this inhibition was reversed by the addition of inhibitors of either AMPK (compound C) or CaMKK (STO-609). Although the nuclear export of PTEN is blocked by metformin in MCF-7 breast cancer cells carrying wild-type LKB1, this inhibition could not be reversed by an AMPK inhibitor, suggesting that LKB1 could regulate the nuclear export of PTEN by bypassing AMPK α1/2. Moreover, ATP could not block the nuclear export of PTEN in AMPK α1/2−/− or TSC2−/− mouse embryonic fibroblasts. However, metformin was still able to induce the LKB1-mediated inhibition of the nuclear export of PTEN in these cells. Taken together, these findings strongly suggest that although CaMKK mediates the nuclear retention of PTEN mainly through the activation of AMPK, LKB1 can regulate the nuclear-cytoplasmic trafficking of PTEN, with or without the AMPK/TSC2/mTOR/S6K-signaling intermediates.

Keywords: AMPK, LKB1, mTOR, nuclear export, PTEN

The subcellular localization of the phosphatase and tensin homologue (PTEN) tumor suppressor is cell cycle dependent, and the regulation of PTEN import to and export from the nucleus is integral to its diverse biological functions, particularly its tumor-suppressing function. In addition, the shuttling of PTEN between the nucleus and the cytoplasm is important for cell cycle regulation.1 In particular, PTEN is localized predominantly in the nucleus in differentiated and cell cycle–arrested (resting) cells2–5 but preferentially in the cytoplasm in rapidly cycling cells, including the cells of thyroid, endocrine, and pancreatic tumors and primary cutaneous melanomas.2,4,5 PTEN regulates cell growth and survival differentially in the cytoplasm versus the nucleus; thus, it is important to understand the molecular mechanisms involved in the nuclear-cytoplasmic trafficking of PTEN. Several different mechanisms for the regulation of PTEN nuclear import have been proposed. Liu et al.6 suggested that PTEN enters the nucleus by diffusion, in part because it lacks a canonical functional nuclear localization signal. However, the differential distribution of PTEN in differentiated/resting cells and advanced tumor cells suggests that active transport mechanisms are responsible for PTEN trafficking. This possibility is supported by the findings of Gil et al.,7 who showed that a Ran GTPase-dependent pathway mediates the nuclear import of PTEN through an N-terminal nuclear localization domain. Alternatively, others have proposed that the major vault protein can serve as a Ca2+-dependent surrogate shuttle protein that imports PTEN into the nucleus.8,9 Recent studies have shown that PTEN can be ubiquitinated by NEDD4-1.10 Polyubiquitination leads to the degradation of PTEN in the cytoplasm, whereas monoubiquitination mediates the nuclear import of PTEN.11 It is quite possible, however, that the nuclear localization of PTEN is regulated by diverse mechanisms in different cell types.

Our group has focused on elucidating the molecular mechanisms involved in the nuclear export of PTEN. We found that PTEN was expressed predominantly in the cytoplasm of TSC2−/− mouse embryonic fibroblasts (MEFs) and in NIH3T3 cells transfected with constitutively activated mutant Akt, which demonstrated that the activation of the PI3K pathway triggers the cell cycle–dependent CRM1-mediated nuclear export of PTEN.12 In contrast, dominant-negative mutants of Akt and pharmacologic inhibitors of PI3K, mTOR, and S6K1, but not of mitogen extracellular kinase, suppressed the nuclear export of PTEN during the G1/S transition. We further observed that the nuclear-cytoplasmic trafficking of exogenous PTEN is also regulated by the PI3K cascade in PTEN-null U251MG cells.12 The nuclear export of PTEN can also be blocked by siRNA silencing of S6K1/2. In addition, PTEN interacts with both S6K1 and S6K2. Taken together, our findings strongly indicated that the activation of the PI3K/Akt/mTOR/S6K cascade, specifically the activation of S6K1/2, is essential for regulating the subcellular localization of PTEN.12 However, in advanced tumor cells, wild-type PTEN is preferentially expressed in the cytoplasm because of the constitutive activation of the PI3K/Akt/mTOR/S6K cascade and/or deletion/mutation of the LKB1/5′ adenosine monophosphate-activated protein kinase (AMPK)/TSC2 tumor suppressors. Loss of nuclear PTEN expression correlates with heightened tumorigenicity.2,4,5,13,14 Conversely, nuclear expression of PTEN has been positively linked to better prognosis in many tumor types.13,15,16 Thus, there may be a potential clinical benefit to sequestering PTEN in the nucleus.

It has been well documented that TSC2 can be activated by AMPK-mediated phosphorylation, which leads to the downregulation of mTOR.17 We reasoned that the activation of the AMPK pathway would block the nuclear export of PTEN during the G1/S transition in a manner similar to inhibition of the PI3K/Akt/mTOR/S6K cascade. In this report, we corroborated our previous findings by showing that the inhibition of the nuclear export of PTEN mediated by inactivation of mTOR/S6K can be alternatively achieved through the LKB1/Ca++/calmodulin-dependent protein kinase kinase (CaMKK)–dependent activation of AMPK α1/2. Interestingly, we also discovered that LKB1 can induce nuclear retention of PTEN independently of the AMPK α1/2/TSC2/mTOR cascade. Further identification of LKB1's novel substrates involved in blocking the nuclear export of PTEN will lead to the discovery of pharmaceutical agents that facilitate nuclear retention of PTEN as a novel therapeutic strategy for advanced tumors expressing PTEN in the cytoplasm.

Materials and Methods

Cell Culture and Transfection

The human breast cancer cell line MCF-7 and the human lung adenocarcinoma cell line A549 were obtained from American Type Culture Collection. All cells were maintained in Dulbecco's modified Eagle's medium/F12 (high glucose) supplemented with 10% fetal bovine serum. The compounds 2-deoxy-glucose, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), ATP, compound C, metformin, oligomycin, vitamin D3, and STO-609 were obtained from Sigma-Aldrich. Plasmids were transfected into A549 cells using FuGENE6 (Roche Applied Science) according to the manufacturer's protocol.

Indirect Immunofluorescence and Confocal Laser Scanning Microscopy

Immunofluorescence staining was performed as described elsewhere.12,18 Briefly, cells were seeded at a concentration of 1 × 105 cells/well in 6-well plates, with coverslips inside the bottom of the wells. The cells were synchronized in 0.1% serum for 48 hours before being treated with inhibitors for 24 hours. The following day, the medium was aspirated and the cells were washed once with phosphate-buffered saline (PBS) before being fixed with 3.7% formaldehyde in PBS for 20 minutes. After another PBS wash, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, followed by blocking with 3% bovine serum albumin–0.1% Tween 20–PBS for 1 hour. The MEFs were then incubated with either a mouse anti-PTEN primary antibody (clone 6H2.1, Cascade Bioscience) or a rabbit antiphospho-histone H1 primary antibody (Upstate USA). The human cells were double-stained with rabbit monoclonal antibodies against PTEN (Cell Signaling Technology) and mouse anti–Ki-67 antibodies (Santa Cruz Biotechnology) for 1 hour. After 2 washes with 0.1% Tween 20 in PBS, the cells were incubated with the fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies (Invitrogen Molecular Probes) for 1 hour and then examined with an Olympus Fluoview (X60 objective) confocal laser scanning microscope (Olympus).

Subcellular Fractionation

The nuclear and cytoplasmic fractions of cells were separated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce) according to the manufacturer's specifications.

Western Blotting

The cells were washed with ice-cold PBS, and whole cell lysates were prepared by lysing cells in a buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid, 20 mM NaF, 10 mM Na4P2O7 (sodium pyrophosphate), 10% glycerol, 1% Triton X-100, 3 mM benzamidine, 10 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 10 µg/mL of aprotinin, 5 mM iodoacetic acid, and 2 µg/mL of leupeptin. The cell lysates were clarified by centrifugation at 14 000× g for 5 minutes. Samples were loaded (5 × 105 cells/lane), and the proteins were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were then probed with monoclonal antibodies against PTEN (Santa Cruz Technology), and a nucleus-specific protein, PARP-1 (EMD Biosciences), as well as rabbit polyclonal antibodies against LKB1 (Santa Cruz Technology), phospho-AMPK, and phospho-S6 (Cell Signaling Technology, Inc.). Specific proteins were detected by chemiluminescence (Amersham Pharmacia Biotech) following incubation with horseradish peroxidase–conjugated secondary antibodies.

Statistics

Statistical analysis was performed using an unpaired (equal-variance) t-test. Data are presented as the mean ± standard deviation. Each group was compared with untreated controls; a P-value of <.05 is considered significant.

Results

The Nuclear Export of PTEN Is Suppressed by Activation of AMPK

It has been shown that mTOR can be downregulated by AMPK-mediated TSC2 activation.17 We reasoned that the activation of the AMPK pathway would block the nuclear export of PTEN during the G1/S transition in a manner similar to inhibitors of PI3K, mTOR, and S6K. Coincidentally, Eng's group has found that either Ca2+ ionophore9 or ATP depletion19 enhances the nuclear accumulation of PTEN. AMPK α1/2 has been shown to be activated mainly by LKB1 in an AMP-dependent manner, but it can also be activated by CaMKK in an AMP-independent manner. To determine by which mechanism AMPK α1/2 is activated in the nuclear export of PTEN, we tested the effects of a variety of AMPK α1/2 activators on AMPK wild-type MEFs and MCF-7 human breast cancer cells that express wild-type PTEN and have no defects in the LKB1, AMPKs, and CaMKK genes. The cells were serum starved (in 0.1% serum) for 2 days and then released in 10% serum, with or without the AMPK α1/2 activators, for 24 hours. The subcellular localization of PTEN was evaluated by indirect immunofluorescence by double-staining with antibodies against PTEN and the cell cycle progression markers Ki-67 in MCF-7 cells and phospho-histone H1 in MEFs, as described previously.12 As shown in Fig. 1, the nuclear export of PTEN during the G1/S transition was effectively suppressed in Ki-67–positive MCF-7 cells and phospho-histone H1–positive AMPK (+/+) MEFs by all of the AMPK α1/2 activators tested. AMPK α1/2 was activated either through the AMP-dependent LKB1 pathway by AICAR, metformin, and oligomycin,20 through the AMP-independent and Ca2+-dependent CaMKK pathway by vitamin D3 and ATP,21 or through both pathways by 2-deoxy-glucose.22,23 These observations support our hypothesis that the nuclear export of PTEN can be regulated by AMPK α1/2.

Fig. 1.

Fig. 1.

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The nuclear export of PTEN is suppressed by activation of AMPK α1/2. Cells were serum starved in 0.1% serum for 48 hours and then released in 10% serum for 24 hours, with or without treatment with the following AMPK activators: metformin (10 mM), 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM), oligomycin (1 μM), ATP (100 μM), vitamin D3 (100 nM), and 2-deoxy-glucose (1 mM). The subcellular localization of PTEN was evaluated by indirect immunofluorescence staining assays. The double-immunostaining assay was performed on human breast cancer (MCF-7) cells using antibodies against PTEN (1:300) and Ki-67 (1:400). AMPK α1/2+/+ MEFs were double-stained with antibodies against PTEN (1:400) and phospho-histone H1 (1:300), a surrogate cell cycle marker. The immunostaining assays were repeated at least 4 times.

AMPK α1/2-Mediated Nuclear Retention of PTEN by Metformin Is Dependent on Functional LKB1

To further characterize the regulation of the nuclear export of PTEN by AMPK α1/2, we used a panel of cell lines carrying different genetic backgrounds, and we studied only the differential effects mediated by metformin (LKB1) or ATP (CaMKK). The LKB1−/− MEFs have a limited life span in culture and are not suitable to study the subcellular localization of PTEN. We resorted to using LKB1-null A549 human lung adenocarcinoma cells (Fig. 2C). Since AMPK α1/2 could not be activated by metformin in the absence of LKB1, metformin failed to block the nuclear export of PTEN in the Ki-67–positive A549 cells during the G1/S transition (Fig. 2A). In contrast, the nuclear export of PTEN was substantially inhibited in Ki-67–positive A549 cells treated with ATP, a CaMKK activator, and this inhibition was reversed by the addition of inhibitors of either AMPK (compound C) or CaMKK (STO-609), as shown in Fig. 2A. Furthermore, the reintroduction of functional LKB1 into A549 cells restored the metformin-mediated inhibition of the nuclear export of PTEN (Fig. 2B). We corroborated the immunostaining results by subcellular fractionation followed by western blotting with PTEN antibodies (Fig. 2D). These findings provide additional evidence to support the notion that the AMPK α1/2 pathway is involved in the regulation of the nuclear export of PTEN, and its activation by metformin is dependent on functional LKB1. Surprisingly, compound C did not reverse the inhibition of the nuclear export of PTEN in the Ki-67–positive A549 cells treated with metformin. These results suggest that LKB1 may also regulate the nuclear-cytoplasmic trafficking of PTEN through alternative pathways.

Fig. 2.

Fig. 2.

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The AMPK α1/2-mediated nuclear retention of PTEN by metformin is dependent on functional LKB1. (A) Serum-starved human LKB1-null A549 cells were released in 10% serum for 24 hours in the presence of 10 mM metformin, 100 μM ATP, 100 μM ATP + 40 μM compound C, or 100 μM ATP + 25 μM STO-609. (B) In comparison, pcDNA3-LKB1–transfected A549 cells were also treated with metformin or metformin + compound C. The cells were fixed and double-stained with PTEN and Ki-67 antibodies. The localization of PTEN was examined in 100 Ki-67–positive cells from each experiment. (C) AMPK is phosphorylated only in LKB1-transfected A549 cells treated with metformin. (D) The subcellular localization of PTEN was corroborated with subcellular fractionation followed by immunoblotting with a PTEN monoclonal antibody. A monoclonal antibody against nucleus-specific poly(ADP ribose) polymerase (PARP-1) was used to monitor cross-contimination between the nuclear and the cytoplasmic fractions. Cy, cytoplasmic fraction; N, nuclear fraction. The average percentages of Ki-67–positive cells with nuclear PTEN from 4 experiments are shown in (E). An unpaired (equal-variance) t-test was performed on all treated groups compared with untreated controls. The P-values of A549 cells treated with ATP, and A549 (LKB1) cells treated with metformin or both metformin and compound C, are statistically significant (<.0001; denoted by asterisk).

LKB1 Mediates Nuclear Retention of PTEN Bypassing AMPK in MCF-7 Cells

To corroborate the findings in the LKB1-null A549 cells, we repeated the same experiments in wild-type LKB1-expressing MCF-7 cells. As demonstrated by immunostaining assays and cell fractionation experiments, the inhibitory effect of ATP on the nuclear export of PTEN was substantially reversed by the AMPK inhibitor compound C and by the CaMKK inhibitor STO-609 (Fig. 3), similar to what was observed in A549 cells. In contrast, like experiments with LKB1-transfected A549 cells (Fig. 2B), the metformin-mediated nuclear retention of PTEN in MCF-7 cells could not be reversed by the addition of the AMPK inhibitor compound C (Fig. 3). These data indicate that LKB1 can suppress the nuclear export of PTEN in an AMPK α1/2-independent manner.

Fig. 3.

Fig. 3.

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LKB1 mediates nuclear retention of PTEN, bypassing AMPK in MCF-7 cells. (A ) MCF-7 cells were treated with 100 μM ATP, 100 μM ATP + 40 μM compound C, 100 μM ATP + 25 μM STO-609, 10 mM metformin, or 10 mM metformin + 40 μM compound C after serum release. The cells were fixed and double-stained with PTEN and Ki-67 antibodies. The localization of PTEN was examined in 100 Ki-67–positive cells from each experiment. The average percentages of Ki-67–positive cells with nuclear PTEN from 4 experiments are shown in (B). The P-values of MCF-7 cells treated with ATP, metformin, or both metformin and compound C are statistically significant (<.0001, denoted by an asterisk). (C) The subcellular localization of PTEN was also assessed by subcellular fractionation followed by immunoblotting with a PTEN monoclonal antibody. Cy, cytoplasmic fraction; N, nuclear fraction.

CaMKK-Induced Inhibition of the Nuclear Export of PTEN Is Mediated by AMPK α1/2 Activation

Activation of AMPK α1/2 by CaMKK blocked the nuclear export of PTEN in MCF-7 cells expressing wild-type LKB1 and in LKB1-null A549 cells. This inhibition could be reversed by either an AMPK inhibitor or a CaMKK inhibitor (Figs 2 and 3). These observations suggest that the activated CaMKK-mediated nuclear retention of PTEN is dependent mostly on functional AMPK α1/2. To definitively validate this hypothesis, we studied the effect of ATP on the subcellular localization of PTEN in AMPK α1/2-null MEFs. The inhibition of the nuclear export of PTEN in AMPK α1/2 wild-type MEFs by the CaMKK activator ATP (Fig. 1) was also reversed by either the AMPK inhibitor compound C or the CaMKK inhibitor STO-609, as shown in Fig. 4A. In contrast, ATP could not block the nuclear export of PTEN in phospho-histone H1–positive AMPK α1/2−/− MEFs. These findings confirm our hypothesis that the CaMKK-induced inhibition of the nuclear export of PTEN is mediated mainly through AMPK α1/2 activation.

Fig. 4.

Fig. 4.

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CaMKK-induced inhibition of the nuclear export of PTEN is mediated through AMPK α1/2-dependent TSC2 activation. (A) AMPK α1/2+/+ MEFs and AMPK α1/2−/− MEFs, (B) TSC2+/+ and p53−/− MEFs, and (D) TSC2−/− and p53−/− MEFs were serum starved for 48 hours followed by a serum release for 24 hours and treated with either 100 μM ATP, 100 μM ATP + 40 μM compound C, or 100 μM ATP + 25 μM STO-609. The subcellular localization of PTEN was evaluated by double-immunostaining with antibodies against PTEN and phospho-histone H1. The localization of PTEN was examined in 100 phospho-histone H1–positive cells from each experiment. The average percentages of phospho-histone H1–positive cells from 4 experiments are shown in (C) and (E). The P-values are statistically significant (<.0001, denoted by asterisk) only in ATP-treated MEF (AMPK+/+) and MEF (TSC2+/+) cells.

AMPK α1/2-Mediated Inhibition of the Nuclear Export of PTEN Is Dependent on TSC2 Activation

AMPK α1/2 has been shown to phosphorylate/regulate many target proteins in addition to TSC2. To prove that the AMPK α1/2-mediated inhibition of the nuclear export of PTEN is dependent on TSC2 activity, possibly through its inactivation of mTOR, we used TSC2−/− MEFs to study the effect of ATP on the nuclear-cytoplasmic trafficking of PTEN. As shown in Fig. 4B, the activation of AMPK α1/2 by ATP blocked the nuclear export of PTEN in phospho-histone H1–positive TSC2+/+ MEFs but not in phospho-histone H1–positive TSC2−/− MEFs, suggesting that TSC2 activity is essential for the AMPK α1/2-mediated nuclear retention of PTEN.

LKB1 Regulates the Nuclear-Cytoplasmic Trafficking of PTEN by Bypassing the AMPK/TSC2-Signaling Intermediates

To elucidate whether AMPK α1/2 is necessary for the metformin-mediated nuclear retention of PTEN, we performed experiments using AMPK α1/2-null MEFs. As shown in Fig. 5A, metformin blocked the nuclear export of PTEN in phospho-histone H1–positive AMPK α1/2-null MEFs in a manner comparable with that seen in AMPK wild-type MEFs (Fig. 1). In addition, the metformin-mediated nuclear retention of PTEN could not be rescued by compound C in either the AMPK α1/2-null or AMPK wild-type MEFs (Fig. 5A). These results substantiated our earlier findings that AMPK α1/2 is not required for the metformin/LKB1-mediated inhibition of the nuclear export of PTEN. To investigate the role TSC2 played in the LKB1-mediated regulation of the nuclear-cytoplasmic trafficking of PTEN, TSC2-null and TSC2 wild-type MEFs were treated with metformin, and its effect on the subcellular localization of PTEN was studied by using immunofluorescence staining assays. As shown in Fig. 5B, metformin was still able to induce the LKB1-mediated inhibition of the nuclear export of PTEN in both phospho-histone H1–positive TSC2-null and TSC2 wild-type MEFs, and this inhibition could not be reversed by the addition of the AMPK inhibitor compound C. In summary, our observations presented in this report strongly suggest that the nuclear export of PTEN is additionally regulated by other unidentified downstream targets of LKB1, and the activation of TSC2 is not necessary for the LKB1-mediated inhibition of the nuclear export of PTEN. This hypothesis was further substantiated by the results of western blotting for phospho-AMPK (T172) and phospho-S6 (S240/244), which are shown in Fig. 6A. S6 phosphorylation was effectively inhibited by metformin and ATP and reversed by AMPK inhibitor compound C in AMPK+/+ and TSC2+/+ MEFs. In contrast, neither metformin nor ATP could suppress the S6 phosphorylation in AMPK−/− or TSC2−/− MEFs. These results suggest that LKB1-mediated inhibition of mTOR/S6K is dependent on functional AMPK and TSC2; however, it is dispensable for the LKB1-mediated nuclear retention of PTEN. Taken together, our findings strongly indicate that an alternative pathway exists, whereby LKB1 regulates the nuclear-cytoplasmic trafficking of PTEN in an AMPK/TSC2/mTOR/S6K-independent manner.

Fig. 5.

Fig. 5.

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LKB1 regulates the nuclear-cytoplasmic trafficking of PTEN by bypassing the AMPK/TSC2-signaling intermediates. (A) AMPK α1/2+/+ MEFs and AMPK α1/2−/− MEFs, (B) TSC2+/+, p53−/− MEFs and TSC2−/−, p53−/− MEFs were serum starved for 48 hours followed by a serum release for 24 hours and treated with either 10 mM metformin or 10 mM metformin + 40 μM compound C. The subcellular localization of PTEN was evaluated by double-immunostaining with antibodies against PTEN and phospho-histone H1. The localization of PTEN was examined in 100 phospho-histone H1–positive cells from each experiment. The average percentages of phospho-histone H1–positive cells with nuclear PTEN from 4 experiments are shown in (C) and (D). The P-values are all statistically significant (either <.0001[*] or =0.0002 [#]).

Fig. 6.

Fig. 6.

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LKB1-mediated nuclear retention of PTEN is independent of S6K inactivation. (A) Immunoblotting with antibodies against phospho-AMPK (T172) and phospho-S6 (S240/244) was performed on cell lysates extracted from AMPK α1/2+/+ MEFs, AMPK α1/2−/− MEFs, TSC2+/+, p53−/− MEFs, and TSC2−/−, p53−/− MEFs treated with 10 mM metformin, 10 mM metformin + 40 μM compound C, 100 μM ATP, or ATP + 40 μM compound C. (B) The proposed model for the AMPK/TSC2/mTOR/S6K-independent pathway by which LKB1 mediates nuclear retention of PTEN. There may be some unidentified substrates of LKB1, either related or unrelated to AMPK but also dependent on the AICAR/metformin activation, that may contribute to this pathway.

Discussion

We discovered previously that the activation of the PI3K/Akt/mTOR/S6K-signaling cascade is essential for the nuclear export of PTEN during the G1/S transition in normal cells. In this report, we further substantiated that the inhibition of the nuclear export of PTEN mediated by an inactivation of mTOR/S6K can be alternatively achieved through the LKB1/CaMKK-dependent activation of AMPK α1/2. In addition, we also discovered that LKB1 can induce nuclear retention of PTEN independently of the AMPK α1/2/TSC2/mTOR/S6K cascade. Since it has been shown that nuclear sequestration of PTEN in tumors is correlated with better prognosis, identification of the signaling component(s) responsible for this process could offer us an alternative therapeutic strategy for treating advanced cancers. Interestingly, a recent study by Guo et al.24 showed that AICAR inhibits the growth of epidemal growth factor receptor VIII-expressing glioblastomas by inhibiting lipogenesis instead of mTORC1 signaling. However, the lipogenic-signaling pathway is unlikely to be involved in LKB1-dependent nuclear retention of PTEN, since AICAR-induced inhibition of lipogenesis is mediated mainly through the activation of AMPK.

It has been reported that LKB1 phosphorylates PTEN at residues S380, T382, T383, and S385, which are also phosphorylated by CK2.25 Phosphorylation of these residues may affect the  stability and activity of PTEN,26–28 but it does not seem to have a direct impact on the subcellular localization of PTEN.8 In addition, a bonafide nuclear export signal has not been identified in PTEN; only multiple nuclear exclusion motifs7 and an N-terminal cytoplasmic localization signal29 have been identified. Thus, LKB1 might regulate the nuclear-cytoplasmic trafficking of PTEN indirectly through inhibition of proteins that interact with PTEN and are responsible for the nuclear export of PTEN.

We demonstrated previously that the predominantly cytoplasmic expression of PTEN in cycling cells is caused primarily by increased CRM1-dependent nuclear export rather than inhibition of nuclear import.12 However, we cannot definitively exclude the possibility that metformin/AICAR may enhance the nuclear import of PTEN in addition to inhibiting its nuclear export through different mechanisms, such as facilitating ubiquitination of PTEN and/or its interaction with nuclear shuttle proteins such as Ran GTPase or major vault protein.

AICAR and metformin do not activate LKB1 or AMPK directly; instead, they either inhibit ATP synthesis, thereby affecting the ratio of AMP to ATP (eg, metformin), or simulate the function of AMP (eg, AICAR, an AMP analog). When AMPK is bound to AMP/AICAR, it becomes an excellent substrate for the presumably constitutively active LKB1. Although AMPK α1/2 are the only known kinases to be phosphorylated by LKB1 upon activation by AICAR and metformin, it is highly plausible that some unidentified substrates of LKB1, either related or unrelated to AMPK but also dependent on AICAR/metformin activation, may contribute to the AMPK/TSC2/mTOR/S6K-independent pathway by which LKB1 mediates nuclear retention of PTEN (Fig. 6B).

It was shown recently that activation of AMPK α1/2 by AICAR could downregulate mTOR through Raptor by bypassing TSC2.30 On the basis of the authors' hypothesis, the AICAR-induced inhibition of the nuclear export of PTEN in TSC2-null MEFs could be attributed to activated AMPK α1/2-mediated Raptor/mTOR inhibition. However, we and Dowling et al.31 did not observe the same level of mTOR/S6K inhibition in TSC2-null MEFs when a different AMPK activator, metformin, was used instead of AICAR (Fig. 6A). In addition, another study by Inoki et al.17 also failed to show the same level of mTOR/S6K inhibition in TSC2 knockdown HEK293 cells using 2-deoxy-glucose. Neither did we see the inhibition of the nuclear export of PTEN in TSC2-null MEFs when AMPK α1/2 was alternatively activated through CaMKK by ATP. Thus, it is possible that AICAR may modulate kinases other than LKB1 and/or AMPK α1/2, which are responsible for the inactivation of Raptor/mTOR in the absence of TSC2.

Most recently, Song et al.32 discovered that the cytoplasmic retention of PTEN is enhanced by deubiquitination mediated by herpesvirus-associated ubiquitin-specific protease and is inhibited by the promyelocytic leukemia (PML) protein through the inactivation of death domain-associated protein in the PML nuclear body. These results agreed with their earlier findings that NEDD4-1–mediated monoubiqitination of PTEN promotes its nuclear import.11 However, Fouladkou et al.33 demonstrated that the stability and subcellular localization of PTEN are clearly independent of NEDD4-1. In addition, we have observed very limited localization of PTEN in the PML nuclear bodies (data not shown). Nevertheless, ubiquitination/deubiquitination may affect the interaction of PTEN with nuclear-cytoplasmic shuttle proteins in a cell type–specific manner, and these effects may be modulated by the PI3K and/or LKB1/AMPK cascades. Further identification of LKB1's novel substrates involved in blocking the nuclear export of PTEN will lead to the discovery of pharmaceutical agents that facilitate nuclear retention of PTEN as a novel therapeutic strategy for advanced tumors expressing PTEN in the cytoplasm.

Funding

NCI/NIH RO1 (CA56041) to W.K.A.Y., Accelerate Brain Cancer Cure (ABC2) to W.K.A.Y., and Cancer Center Core (CA16672) MD Anderson.

Acknowledgments

We thank Kate Newberry for her critical reading and editing of this manuscript; Jun-Ichi Nezu for LKB1 constructs; Keith Laderoute for AMPK+/+MEFs and AMPK−/− MEFs; and David Kwiatkowski for TSC2+/+, p53−/− MEFs and TSC2−/−, p53−/− MEFs.

Conflict of interest statement. None declared.

References

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