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Cancer Research 67, 11054, November 15, 2007. doi: 10.1158/0008-5472.CAN-07-1263
© 2007 American Association for Cancer Research
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Endocrinology

Cell Cycle–Dependent Nuclear Export of Phosphatase and Tensin Homologue Tumor Suppressor Is Regulated by the Phosphoinositide-3-Kinase Signaling Cascade

Juinn-Lin Liu1, Zhenyu Mao1, Tiffany A. LaFortune1, Marta M. Alonso1, Gary E. Gallick2,3, Juan Fueyo1,3 and W.K. Alfred Yung1,3

1 Brain Tumor Center and Department of Neuro-Oncology, 2 Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, and 3 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas

Requests for reprints: W.K. Alfred Yung, Department of Neuro-Oncology, Unit 431, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1285; Fax: 713-794-4999; E-mail: wyung{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tumor suppressor phosphatase and tensin homologue (PTEN) plays distinct growth-regulatory roles in the cytoplasm and nucleus. It has been shown to be preferentially localized to the nucleus in differentiated or resting cells, and to the cytoplasm in advanced tumor cells. Thus, the regulation of PTEN's subcellular localization seems to be critical to its tumor-suppressing functions. In this study, we showed that activation of the phosphoinositide-3-kinase (PI3K) pathway triggers PTEN's cell cycle–dependent chromosome region maintenance 1–mediated nuclear export, as PTEN was predominantly expressed in the cytoplasm of TSC2–/– mouse embryo fibroblasts or activated Akt mutant-transfected NIH3T3 cells. In contrast, dominant-negative mutants of Akt and pharmacologic inhibitors of PI3K, mTOR, and S6K1, but not of MEK, suppressed the nuclear export of PTEN during the G1-S transition. The nuclear-cytoplasmic trafficking of exogenous PTEN is likewise regulated by the PI3K cascade in PTEN-null U251MG cells. The nuclear export of PTEN could also be blocked by short interfering RNA to S6K1/2. In addition, PTEN interacts with both S6K1 and S6K2. Taken together, our findings strongly indicate that activation of the PI3K/Akt/mTOR/S6K cascade, specifically S6K1/2, is pivotal in regulating the subcellular localization of PTEN. This scenario exemplifies a reciprocal regulation between PI3K and PTEN that defines a novel negative-feedback loop in cell cycle progression. [Cancer Res 2007;67(22):11054–63]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor suppressor phosphatase and tensin homologue (PTEN) is the second most frequently mutated or deleted tumor suppressor gene in human cancers (1, 2). PTEN's primary function is its intrinsic lipid phosphatase activity, which antagonizes phosphoinositide-3-kinase (PI3K; ref. 3), thereby down-regulating the PI3K-dependent signaling pathways involved in cell growth and survival. PTEN's activities are modulated by kinase phosphorylation, membrane recruitment, or oxidation (reviewed in ref. 4). Thus, the subcellular localization of PTEN is critical to the regulation of its diverse biological properties. PTEN is localized predominantly to the nucleus in differentiated and cell cycle–arrested (resting) cells (5–10) but preferentially to the cytoplasm of rapidly cycling cells, including tumor cells of thyroid, endocrine, and pancreatic origin, as well as primary cutaneous melanoma cells (5, 8). PTEN's shuttling between the nucleus and cytoplasm is important in cell cycle regulation (reviewed in ref. 11). Recent studies have shown that, when localized to the nucleus, PTEN mediates growth-regulatory activities. For instance, we showed that nuclear PTEN alone is capable of suppressing anchorage-independent growth and facilitating G1 arrest in U251MG cells without inhibiting Akt activity (12). In addition, Chung and Eng (13) found that nuclear PTEN is required for cell cycle arrest, whereas cytoplasmic PTEN is required for apoptosis in MCF-7 cells. Gil et al. (14) showed that nuclear PTEN could enhance apoptotic processes in U87MG and HEK293 cells. Moreover, nuclear PTEN, but not cytoplasmic PTEN, induced p300-dependent G1 arrest in U2OS cells (15). Collectively, these observations show that PTEN's subcellular localization is cell cycle–dependent and suggests that alterations in the regulation of PTEN import and export to and from the nucleus contribute to its tumor suppressor function.

Given the critical roles of PTEN in both the cytoplasm and nucleus, it is important to elucidate the molecular mechanisms involved in PTEN's nuclear-cytoplasmic trafficking, the overall aim of this study. It has been proposed that PTEN enters the nucleus by diffusion (16), 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's trafficking. This possibility was supported by the findings of Gil et al. (14), who showed that nuclear import of PTEN is mediated through an NH2-terminal nuclear localization domain by a Ran GTPase-dependent pathway. Alternatively, it was suggested that the major vault protein could serve as a Ca2+-dependent surrogate shuttle protein that imports PTEN into the nucleus (17, 18). Recent studies showed that PTEN could be ubiquitinated by NEDD4-1 (19). Polyubiquitination leads to PTEN's degradation in the cytoplasm, whereas monoubiquitination mediates PTEN's nuclear import (20). In this study, we focused on examining the molecular mechanisms involved in PTEN's nuclear export. We confirmed that PTEN is preferentially localized to the nucleus during G0/G1 and is exported to the cytoplasm during the G1-S transition. Furthermore, we systematically showed that the export of endogenous and exogenous PTEN from the nucleus was triggered by the activated PI3K/Akt/mTOR/S6K cascade, providing a negative-feedback loop in cell cycle progression. We also showed that the nuclear export of PTEN is blocked by short interfering RNA (siRNA) to either S6K1 or S6K2. In addition, PTEN interacts with both S6K1 and S6K2, suggesting that S6K1 and S6K2 are directly involved in regulating PTEN's nuclear export.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection. Normal mouse astrocytes were prepared from the brain cortex of postnatal day 4 mice as described previously (21). Mouse cell lines, NIH3T3, 3T3-L1, and mouse embryo fibroblasts (MEF), and rat embryonic fibroblast cell line, Rat-2, as well as human cell lines, U251MG, MCF-7, HaCaT, and A549 were obtained from American Type Culture Collection. All cells were maintained in DMEM/F12 (high glucose) medium supplemented with 10% fetal bovine serum. RAD001 (everolimus) was provided by Novartis. Leptomycin B (LMB), LY294002, wortmannin, sodium salicylate (NaSal), and PD98059 were obtained from Sigma-Aldrich. Plasmids were transfected into NIH3T3 and U251MG cells with FuGENE6 (Roche Applied Science) according to the manufacturer's protocol.

Indirect immunofluorescence and confocal laser scanning microscopy. Immunofluorescence staining was done as described elsewhere (22). Briefly, cells were seeded at a concentration of 1 x 105 cells per well in six-well plates with coverslips inside and synchronized in 0.1% serum for 48 h before treatment with inhibitors for 24 h. The following day, medium was aspirated and the cells were washed once with PBS before being fixed with 3.7% formaldehyde in PBS for 20 min. After another PBS wash, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min followed by blocking with 3% bovine serum albumin/0.1% Tween 20/PBS for 1 h. Cells were then incubated with primary antibodies [mouse anti-PTEN (clone 6H2.1, Cascade Bioscience; or BD Transduction Lab) and rabbit anti–phospho-histone H1 (Upstate USA, Inc.)], anti–Ki-67 (Santa Cruz Biotechnology), or anti-hemagglutinin tag (QED) for 1 h. After two washes with PBS (0.1% Tween 20), cells were incubated with the secondary antibodies conjugated with FITC or Texas red (Invitrogen Molecular Probes) for 1 h and then examined under an Olympus Fluoview (x60 objective) confocal laser scanning microscope (Olympus).

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

Western blotting. Cells were washed with ice-cold PBS and lysed in a buffer containing 50 mmol/L of HEPES (pH 7.5), 1.5 mmol/L of MgCl2, 150 mmol/L of NaCl, 1 mmol/L of EGTA, 20 mmol/L of NaF, 10 mmol/L of Na4P2O7 (sodium PPi), 10% glycerol, 1% Triton X-100, 3 mmol/L of benzamidine, 10 mmol/L of phenylmethylsulfonyl fluoride, 1 µmol/L of pepstatin, 10 µg/mL of aprotinin, 5 mmol/L of iodoacetic acid, and 2 µg/mL of leupeptin to prepare whole-cell lysates. Lysates were clarified by centrifugation at 14,000 x g for 5 min. Proteins equivalent to 5 x 105 cells per lane were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore). The polyvinylidene difluoride membranes were then probed with monoclonal antibodies (mAb) against PTEN (Santa Cruz) and a nucleus-specific protein PARP-1 (EMD Biosciences). Specific proteins were detected by chemiluminescence (Amersham Pharmacia Biotech) following incubation with horseradish peroxidase–conjugated secondary antibodies.

Immunoprecipitation. Immunoprecipitation was done according to the manufacturer's specifications, with slight modifications. Briefly, 5 µL of mAbs against S6K1 (Santa Cruz), and PTEN (Santa Cruz), as well as rabbit anti-S6K2 (Bethyl Laboratories), and anti-PTEN (rabbit mAb; Cell Signaling Technology) were added to 1 mL of NIH3T3, Rat-2, or MCF-7 cell lysate (equivalent to 4 x 106 cells), respectively, overnight at 4°C, followed by 40 µL of 50% protein A/G agarose beads (Pierce) for an additional 3 h at 4°C. The pellet was washed five times with 500 µL of cell lysis buffer and resolved by SDS-PAGE. The blots were probed with mAb against S6K1, or rabbit antibody against PTEN (Upstate).

siRNA and transfection. Mouse S6K1 and S6K2 siGENOME SMARTpool reagents (Dharmacon) were transfected into mouse astrocytes using X-tremeGENE siRNA transfection reagent according the manufacturer's instructions (Roche).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subcellular localization of PTEN is cell cycle–dependent. To confirm that PTEN localization is cell cycle–regulated, NIH3T3 cells were either serum-starved (0.1% serum) for 72 h or released into 10% serum for 24 h after 48 h in 0.1% serum. Nuclear and cytoplasmic fractionations were then done. The immunoblot showed that there was more PTEN in the nucleus of serum-starved NIH3T3 cells; whereas increased levels of PTEN were found in the cytosolic fraction after the cell cycle blockade was released by placing cells in 10% serum (Fig. 1A ). These data suggest that PTEN's subcellular distribution is cell cycle–dependent. However, because not all cells are synchronized by the serum deprivation method (fluorescence-activated cell sorting analysis showed that >22% of cells were not arrested at the G1 phase in serum-starved NIH3T3 cells, and ~34% were in the G1 phase in serum-released NIH3T3 cells), it is imperative to substantiate the immunoblotting result using other methods. We subsequently did double-immunostaining using antibodies against PTEN (Cascade mAb) and a surrogate marker for the G1-S transition, i.e., phospho-histone H1 (histone H1 is phosphorylated by cyclin-dependent kinase 2/cyclin E during S phase entry; ref. 23), on cells prepared under the same conditions as for the subcellular fractionation studies. This method allowed us to measure PTEN's subcellular localization in relation to cell cycle progression at the cellular level. Some limited nonspecific staining was observed in the cytoplasm of PTEN–/– mouse astrocytes regardless of cell cycle progression (Fig. 1B1). However, as Fig. 1B2 shows, PTEN was expressed predominantly in the nucleus of NIH3T3 cells when histone H1 was not phosphorylated (73.3 ± 5.0%). In contrast, the expression of PTEN in the cytoplasm was appreciably increased and its nuclear accumulation was reduced when histone H1 was phosphorylated (26.8 ± 5.0%). Similar localization patterns were also observed in normal mouse astrocytes (Fig. 1B3) and in 3T3-L1 (data not shown), as well as in human A549 (as phospho-histone H1 antibody did not work very well for immunostaining assays in human cells, we resorted to use Ki-67 as an alternative cell cycle marker. It is completely absent during G0 arrest and starts accumulating in the nuclear foci during early G1 phase, and localizes to the nucleoli during late G1, S, and G2 phases; Fig. 1B4), HaCaT and MCF-7 cells (data not shown).


Figure 1
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  		Figure 1. Subcellular localization of PTEN is cell cycle–dependent. A, to substantiate the subcellular localization of PTEN, immunoblotting with a PTEN mAb was done on cytoplasmic (C) and nuclear (N) fractions collected from NIH3T3 cells undergoing serum starvation or released into 10% serum, respectively. mAb against nucleus-specific poly(ADP ribose) polymerase 1 (PARP-1) was used to monitor cross-contamination between the nuclear and cytoplasmic fractions. B, the subcellular localization was corroborated with indirect immunostaining assays. PTEN–/– mouse astrocytes, NIH3T3 cells, and normal mouse astrocytes were double-stained with antibodies against PTEN (1:400; Cascade mAb) and a surrogate cell cycle marker, phospho-histone H1 (1:300). The double-immunostaining was also done on human A549 cells using antibodies against PTEN (1:300; BD Transduction Lab) and Ki-67 (1:400). The subcellular distribution of PTEN was evaluated in 100 cells from each experiment. B5, averages of four experiments.

 
We further studied the kinetics of PTEN's nuclear-cytoplasmic trafficking from 0 to 24 h after serum release. As shown in Fig. 2A , in NIH3T3 cells, the cytoplasmic localization of PTEN started to increase 4 h after serum release and peaked between 12 and 16 h. Similar results were observed for phospho-histone H1, a finding consistent with the course of cell cycle progression.


Figure 2
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  		Figure 2. PTEN's CRM1-dependent nuclear export is abrogated by inhibition of the PI3K/Akt/mTOR/S6K signaling cascade. A, NIH3T3 cells were serum-starved (0.1% serum) for 48 h and then released in 10% serum to study the kinetics of PTEN's nuclear-cytoplasmic trafficking. B, CRM1-specific inhibitor, LMB, was added at a concentration of 5 ng/mL in conjunction with 10% serum for 16 h in the medium of previously synchronized NIH3T3 cells. C, pharmacologic inhibitors for PI3K (10 µmol/L, LY294002), mTOR (100 nmol/L, RAD001), p70S6K (10 mmol/L, NaSal), or MEK1 (10 µmol/L, PD98059) were used to identify the pivotal signaling component(s) involved in PTEN nuclear export. All inhibitors were added for 16 h except for NIH3T3 cells, which were treated with RAD001 for 4 h only and then released in 10% serum for 12 h. D1, the subcellular localization of PTEN in NIH3T3 cells was assessed by immunoblotting and indirect immunostaining assays as described in the legend to Fig. 1. The localization of PTEN was examined in 100 cells from each experiment except in LMB-, LY294002-, or RAD001-treated wells, in which only 50 cells were scored because fewer cells were present due to the cytostatic effect of the inhibitors. D2, averages of four experiments.

 
Inhibition of PI3K/Akt/mTOR/S6K signaling cascade abrogates PTEN's nuclear export. To determine whether the retention of PTEN in the cytoplasm during the G1-S transition is due to accelerated nuclear export or inhibited nuclear import, the effect of LMB, a selective inhibitor of the exportin, chromosome region maintenance 1 (CRM1), on PTEN localization was evaluated. As shown in Fig. 2B, when LMB was added to NIH3T3 cells that had been released into 10% serum, PTEN accumulated in the nucleus in most (91.5 ± 3.4%) cells positive for phospho-histone H1, i.e., cells that had entered the S phase. These results strongly suggest that the cytoplasmic translocation of PTEN depends on CRM1-mediated nuclear export.

We next determined which signaling pathways must be activated for PTEN to be exported to the cytoplasm. Because both PI3K/Akt/mTOR/S6K and Ras/Raf/MEK1/Erk cascades are activated in response to growth factor stimulations, we first examined the effect of selective inhibitors of signaling components within these two pathways on PTEN localization in conjunction with cell cycle synchronization. However, due to the fact that many signaling inhibitors also impose cell cycle arrest, we used the minimum amount of inhibitors that would still be effective in blocking the targets without significant hindrance on cell cycle progression. The immunoblots in Fig. 2C showed that there was less PTEN in the cytosolic fraction of NIH3T3 cells treated with the PI3K-selective inhibitor LY294002, the mTOR-selective inhibitor RAD001, or NaSal (to inhibit S6K) than in cells treated with the MEK-inhibitor PD98059. These results implicate the PI3K cascade as being critical to cell cycle–dependent PTEN localization.

In congruence with the subcellular fractionation data, the immunostaining study (Fig. 2D) showed that the cytoplasmic translocation of PTEN during the G1-S transition (cells positive for phospho-histone H1) was blocked in NIH3T3 cells treated with LY294002 (61.5 ± 5.5%), RAD001 (61.8 ± 5.0%), or NaSal (81.3 ± 4.3%), but not with PD98059 (20.5 ± 4.2%), compared with untreated NIH3T3 cells (21.5 ± 5.4%). These observations further support a requirement for the PI3K/Akt/mTOR/S6K pathway but not the Ras/Raf/MEK/Erk pathways in the nuclear export of PTEN.

Constitutive activation of Akt promotes PTEN's cytoplasmic translocation. As pharmacologic inhibitors lack complete specificity, we next employed molecular strategies to examine PTEN's trafficking. To examine a requirement for Akt activation, NIH3T3 cells were transfected with the hemagglutinin-tagged dominant-negative mutant for Akt, pcDNA3-Akt-AAA (hemagglutinin). Expression of this dominant-negative Akt blocked the nuclear export of PTEN in 65.5 ± 5.5% of transfected cells in the presence of 10% serum (Fig. 3A1 ). Conversely, the transient transfection of a hemagglutinin-tagged constitutively active mutant of Akt, pcDNA3-Akt-DD (hemagglutinin), led to increased nuclear export of PTEN. As shown in Fig. 3A2, this enhanced cytoplasmic expression of PTEN occurred even in 59.5 ± 5.0% of Akt-DD–expressing cells that had undergone serum withdrawal. These findings thus provide further evidence that PTEN's nuclear export requires the activation of the PI3K/Akt/mTOR/S6K pathway.


Figure 3
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  		Figure 3. Constitutive activation of Akt/mTOR pathway promotes PTEN's cytoplasmic translocation. A, NIH3T3 cells were transiently transfected with hemagglutinin-tagged dominant-negative mutants of Akt, pcDNA3-Akt-AAA, or constitutively active mutant, pcDNA3-Akt-DD. The transfected NIH3T3 cells were double-stained with antibodies against hemagglutinin-tag (1:300) and PTEN. The localization of PTEN was evaluated in 50 positively transfected cells from each experiment. The results are the average of four experiments. B, the subcellular localization of PTEN was evaluated by immunostaining assays in TSC2–/–, p53–/– MEFs and TSC2+/+, p53–/– MEFs following the exact same experimental procedures as described in Figs. 1 and 2 except that 1 µmol/L of wortmannin was used to inhibit PI3K. Fixed cells were double-stained with antibodies against PTEN (Cascade) and phospho-histone H1. PTEN's subcellular localization was examined in 100 cells from each experiment except that 50 cells were scored in wortmannin- or RAD001-treated TSC2+/+, p53+/+ MEFs. B3, averages of four experiments.

 
PTEN is preferentially localized to the cytoplasm in TSC2–/– MEFs. TSC2 has been shown to be the negative regulator of mTOR's upstream activator, Rheb. Specifically, once TSC2 is phosphorylated by Akt, Rheb is released from its inhibition of the TSC1/2 complex (reviewed in ref. 24). As a consequence of this, mTOR and its downstream effector S6K are often constitutively activated in cells harboring mutations and/or deletions of the tuberous sclerosis complex (TSC) genes TSC1 or TSC2. As shown in Fig. 3B1, very few TSC2–/–, p53–/– MEFs were synchronized at the G0/G1 phase by mere serum starvation. PTEN was preferentially expressed in the cytoplasm (only 2.3 ± 1.0% of phospho-histone–positive cells expressed PTEN in the nucleus only), even in some G1-arrested cells due to constitutive basal activation of mTOR/S6K. Although LMB could block PTEN's nuclear export in only 29.5 ± 3.4% of TSC2–/–, p53–/– MEFs, this is still a considerable fraction because very little PTEN was present in the nucleus before the LMB treatment. Furthermore, the nuclear export of PTEN in TSC2–/–, p53–/– MEFs was substantially suppressed by inhibitors of mTOR (RAD001; 29.8 ± 4.1%) and S6K1 (NaSal; 39.8 ± 4.4%), but not by the PI3K inhibitor wortmannin (3.5 ± 1.9%). These findings strongly indicate that the PI3K cascade downstream effectors, mTOR and/or S6K, but not PI3K or Akt, are intimately involved in PTEN's nuclear export. In contrast, p53 does not seem to participate in this process because there is virtually no difference in the localization patterns of PTEN between TSC2+/+, p53–/– MEFs (Fig. 3B2) and wild-type p53-containing mouse astrocytes and NIH3T3 cells (Fig. 1B2 and B3).

Subcellular localization of exogenous PTEN is similarly regulated by the PI3K cascade. Because all PTEN antibodies have exhibited certain cross-reactivity in immunoblotting and immunostaining assays, it is a legitimate concern that whether our observations on endogenous PTEN could have been tainted with artifacts. To adequately address this issue, we also examined the localizing pattern of exogenous PTEN. We first transiently transfected wild-type PTEN into PTEN-null U251MG cells, cells were then treated with RAD001 or salicylate or serum-deprived, 2 days after transfection. Fixed cells were double-stained with another mAb against PTEN (BD Transduction Lab) and Ki-67. As shown in Fig. 4 , even though only a small portion of serum-starved U251MG cells were arrested in the G0 phase, most of the exogenous PTEN was preferentially localized to the nucleus in the arrested (Ki-67 negative) cells (66.5 ± 6.6%). Conversely, the cytoplasmic expression of exogenous PTEN was substantially elevated in cells that were committed to cell cycle progression (84.5 ± 4.4% of Ki-67–positive, PTEN-expressing cells). Furthermore, the nuclear export of exogenous PTEN was suppressed by RAD001 (33.0 ± 2.6%) and salicylate (21.5 ± 4.4%), a behavior similar to that of endogenous PTEN in mouse cells. These above experiments involving the use of two different PTEN antibodies show that the cell cycle–dependent nuclear-cytoplasmic trafficking of both endogenous and exogenous PTEN is regulated by the PI3K cascade.


Figure 4
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  		Figure 4. Subcellular localization of exogenous PTEN is similarly regulated by PI3K cascade. A, U251MG cells were transiently transfected with hemagglutinin-tagged pcDNA3.1-PTEN constructs. Forty-eight hours after transfection, cells underwent serum starvation (0.1%), or were treated with RAD001 (100 nmol/L) or salicylate (10 mmol/L) overnight. Fixed cells were double-stained with antibodies against PTEN and Ki-67 as described in Fig. 1. B, the localization of exogenous PTEN was evaluated in 50 transfected cells from each experiment (averages of four experiments).

 
PTEN interacts with both S6K1 and S6K2. As both S6K1 and its homologue, S6K2, are key downstream effectors of mTOR, we further examined if S6K1 and/or S6K2 were directly involved in regulating PTEN's localization. To this end, we transiently transfected siRNA to S6K1 or S6K2, or both, into normal mouse astrocytes for 3 days. As shown in Fig. 5 , more PTEN accumulated in the nucleus as the concentration of siRNA to either S6K1 or S6K2 increased; however, the combination of siRNAs to both S6K1 and S6K2 did not have additive/synergistic effects on blocking PTEN's nuclear export. These observations suggest that both S6K1 and S6K2 are required for PTEN's nuclear export, potentially through separate mechanisms.


Figure 5
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  		Figure 5. PTEN's nuclear export is blocked by siRNA to S6K1 or S6K2. A, normal mouse astrocytes were transiently transfected with siRNA to S6K1 or S6K2 for 3 d followed by immunostaining assays using PTEN mAb to study the effect of siRNA on PTEN's nuclear-cytoplasmic trafficking. B, 100 cells were examined from each experiment (averages of two experiments). C, immunoblotting with antibodies against S6K1, S6K2, and phospho-S6 (Ser240/244; Cell Signaling Technology) was also done to verify the efficiency of siRNA.

 
We next determined if PTEN associated with S6K1 or S6K2. For this analysis, coimmunoprecipitation assays were done using antibodies against PTEN, S6K1, or S6K2. As shown in Fig. 6A , S6K1 was coprecipitated by PTEN antibodies from Rat-2, MCF-7, and to a lesser extent, NIH3T3 cell lysates. Reciprocal immunoprecipitation showed that S6K1 antibodies could also bring down PTEN protein, but it is much less efficient. It is possible that the epitope to S6K1 antibodies is located very close to the interacting domain to PTEN, thus the interaction between S6K1 and PTEN is disrupted by S6K1 antibodies. Interestingly, S6K2 antibodies also pulled down S6K1 and PTEN, suggesting that S6K1, S6K2, and PTEN all exist in the same complex. However, there is no evidence to support that S6K1 and S6K2 interact directly. The size of S6K2 is too close to that of heavy chains for it to be detected effectively by reciprocal immunoprecipitation (data not shown).


Figure 6
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  		Figure 6. PTEN interacts with S6K1 and S6K2. A, NIH3T3, MCF-7, and Rat-2 cell lysates were immunoprecipitated with antibodies against PTEN, S6K1, or S6K2 as well as control mouse and rabbit IgGs, and the immunoblots were then probed with antibodies against S6K1 or PTEN. B, hemagglutinin-tagged pcDNA3.1-PTEN constructs were transiently transfected into U251MG cells. Double-immunostaining with antibodies against hemagglutinin-tag and S6K1 was done 48 h after transfection under serum starvation. C, reciprocal regulation between PI3K and PTEN. It has been shown that activated S6K1 provides a negative feedback loop to the PI3K signaling cascade by inhibiting IRS-1. However, it only down-regulates the PI3K pathway activated by insulin/insulin-like growth factor, but not by other growth factors. Our data showed that at the G0/G1 phase, not only is PTEN sequestered in the nucleus to antagonize PI3K, but it also contributes to maintaining cell cycle arrest, partially through the activation of AMPK. The nuclear export of PTEN is triggered by the fully activated PI3K cascade during G1-S transition. The exported PTEN is then not only released from its nuclear growth–suppressing activity, it could then also dephosphorylate the cytoplasmic PIP3 to prevent the constitutive activation of PI3K cascade. This scenario exemplifies a novel negative feedback loop in cell cycle progression.

 
We then determined if the association between PTEN and S6K1/2 also occurred at the physiologic level. Due to the limitations of the antibodies, we transiently transfected COOH-terminal hemagglutinin-tagged PTEN in the pcDNA3.1 vector into U251MG cells. Double-immunostaining with hemagglutinin and S6K1 (S6K2 antibodies are not suitable for immunostaining assays) antibodies was done 2 days posttransfection. As Fig. 6B shows, a fraction of the PTEN and S6K1 proteins colocalized in the nucleoplasm. Taken together, these data strongly suggest that S6K1/2 is directly involved in regulating PTEN's nuclear export.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phosphorylation plays a pivotal role in regulating the function and activity of proteins. GSK3 and CK2 have been shown to regulate PTEN's stability (25, 26). However, CK2 does not play a role in regulating PTEN's subcellular localization. Chung et al. (17) found no difference in the localization between wild-type PTEN and CK2 dephosphorylation mutants. In this report, we show that the nuclear export of PTEN occurs via a CRM1-dependent mechanism during the G1-S transition and is clearly modulated by an activated PI3K cascade, S6K1/2 in particular, suggesting that PTEN either contains a cryptic nuclear export signal or its nuclear export is mediated through its interaction with other nuclear export signal–harboring nuclear proteins. Our findings are in contrast with those of Liu et al. (16), who suggested that CRM1-dependent nuclear export did not play a significant role in PTEN's trafficking because LMB did not affect the subcellular localization of the exogenous GFP-PTEN protein in HeLa cells. However, the discrepancy between these and our findings is most likely due to the larger size (~83 kDa) of the GFP-PTEN proteins used in that study, which may have prevented it from entering the nucleus as efficiently as endogenous PTEN. In addition, the HeLa cells used by Liu et al. (16) were not synchronized or growth-arrested. As a result, there was little GFP-PTEN present in the nucleus prior to the LMB treatment. This possibility is supported by our observations of PTEN's predominant cytosolic localization in TSC-2–/– MEFs because of constitutive activation mTOR/S6K. It should be noted that, on the other hand, the exogenous PTEN is always observed both in the nucleus and in the cytoplasm in unsynchronized U251MG cells transiently transfected with PTEN and mutant constructs (this study and ref. 12). This is most likely due to the fact that exogenous PTEN could enter the nucleus by diffusion or active transport mechanisms as discussed in the Introduction; however, the normal amount of CRM1 was not enough to fully accommodate all the overexpressed PTEN proteins for their nuclear export efficiently even when mTOR/S6K was activated. Moreover, despite the nuclear export of PTEN being mediated by CRM1, PTEN's interaction with CRM1 is dependent on the activating status of the PI3K cascade and might also rely on the existence of intermediate chaperon molecule(s). These factors may contribute to the preferential localization of PTEN in certain cell types.

The data presented in this study strongly suggest that S6K directly regulates PTEN's nuclear export. Our preliminary results suggested that the S6K1 immune complex phosphorylated PTEN in vitro (data not shown). However, because PTEN could be phosphorylated by several kinases, including CK2 (25), LKB1 (27), CK1, and GSK3ß (28), it is highly plausible that PTEN is phosphorylated by one of these kinases, if they exist in the mix of S6K immune complexes. Further experiments are warranted to verify if PTEN is a physiologic substrate of S6K1/2 and if such phosphorylation plays any direct role in PTEN's nuclear export. Meanwhile, because only multiple nuclear exclusion motifs (14) and an NH2-terminal cytoplasmic localization signal (29) but no bonafide nuclear export signal have been identified in PTEN, it is conceivable that S6K1/2 might regulate PTEN's trafficking indirectly by phosphorylating the protein(s) interacting with PTEN through such motifs.

Activated S6K1 has been shown to trigger a negative-feedback loop to the PI3K signaling cascade by inhibiting IRS-1 (reviewed in ref. 30). However, it would only antagonize PI3K pathways activated by insulin/insulin-like growth factor, but not by other growth factors. Our data show another novel mechanism by which S6K1 regulates cell cycle progression. During the G0/G1 phase, when PTEN is in the nucleus, not only is PTEN sequestered from the plasma membrane, where it antagonizes PI3K, its presence in the nucleus also contributes to maintaining cell cycle arrest, partially through the activation of AMPK (12). PTEN is not exported to the cytoplasm until the PI3K/PDK/Akt/mTOR/S6K signaling cascade is activated upon the stimulation of growth factors during G1-S transition. The exported PTEN is not only released from its nuclear growth–suppressing activity, it can also dephosphorylate the cytoplasmic PIP3 to prevent the constitutive activation of Akt-mediated signaling pathways (Fig. 6C). This scenario thus constitutes a reciprocal regulation between PI3K and PTEN in normal cells.

Such equilibrium between PTEN and PI3K is often disrupted in tumor cells, however, because either PTEN is functionally inactivated or other mechanisms constitutively activate the PI3K/Akt/mTOR/S6K signaling cascade. Thus, it is not surprising that PTEN is preferentially expressed in the cytoplasm in a variety of tumors in which the PI3K cascade is frequently activated. We also observed the constitutive cytoplasmic localization of PTEN in glioblastoma multiforme tumors and cell lines expressing wild-type PTEN, such as LN229, LN428, and LN751 cells (data not shown). Coincidentally, S6K is universally constitutively active in these cells, suggesting that the activation of S6K bypasses PTEN regulation.

The mechanism(s) by which nuclear PTEN controls cell growth are incompletely defined. It has been suggested that nuclear PTEN's growth-suppressing activities may be mediated through p53 (31), which is dependent on p300 interaction (15), the inhibition of MSP58 (32), or maintaining chromosomal integrity (33). Our previous report showed that nuclear PTEN's growth-regulatory function depends on its lipid phosphatase activity (12). Activated PI3K has been shown to translocate into the nucleus (34), and functional PIP3 has also been detected inside the nucleus (35). Ahn et al. (36) suggest that nuclear PI3K signaling mediates the antiapoptotic effects of nerve growth factor through nuclear PIP3 and nuclear Akt. Therefore, nuclear PTEN's growth-suppressing activities could be mediated, at least in part, through a reversal of nuclear PI3K's effects. However, a recent study showed that the nuclear pool of PIP3 is insensitive to PTEN even when it is targeted to the nuclei (37). Alternatively, but not exclusively, nuclear PTEN's biological function might also depend on its production of PIP2, which leads to the activation of growth-suppressing modules. Thereafter, the translocation of PI3K to the nucleus during the G1-S transition could release the growth-suppressing effect imposed by nuclear PTEN by converting nuclear PIP2 into PIP3.

In summary, PTEN has very distinct growth-regulatory roles in the cytoplasm and the nucleus. In the cytoplasm, it has intrinsic lipid phosphatase activity that negatively regulates the cytoplasmic PI3K/Akt pathway. In the nucleus, PTEN displays Akt-independent growth-suppressing activities. In this report, we showed that PTEN's cell cycle–dependent nuclear export is regulated by the PI3K/Akt/mTOR/S6K signaling cascade. Our findings have implications for identifying nuclear proteins in the PTEN pathway that might be novel targets for cancer therapy.


   Acknowledgments
 
Grant support: NCI/NIH R01 grant CA56041 (W.K.A. Yung), the Gilliland Foundation (W.K.A. Yung), the University Cancer Foundation/UTMDACC (J-L. Liu), and Cancer Center Core grant CA16672 (M. D. Anderson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Betty Notzon for her critical reading and editing of this manuscript; Ron DePinho for PTEN–/– mouse astrocytes; David Kwiatkowski for TSC2–/–, p53–/– MEFs and TSC2+/+, p53–/– MEFs; James Woodget for the Akt constructs; and Maria-Magdalena Georgescu for the GST-PTEN–expressing bacteria.

Received 4/ 4/07. Revised 8/22/07. Accepted 9/24/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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