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Vol. 17, Issue 9, 4002-4013, September 2006
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Centro de Investigación Príncipe Felipe, Valencia 46013, Spain
Submitted May 3, 2006;
Revised May 25, 2006;
Accepted June 20, 2006
Monitoring Editor: John Cleveland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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proteins. The lipid- and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mayo and Donner, 2002; Waite and Eng, 2002; Leslie and Downes, 2004; Parsons, 2004; Sansal and Sellers, 2004). The PTEN gene is mutated or lost in a wide variety of human tumors, including malignant glioblastomas, an aggressive tumor of the CNS in humans (Bonneau and Longy, 2000; Eng, 2003). The major tumor suppressor function of PTEN is mediated by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3; Maehama and Dixon, 1998). Through this lipid phosphatase activity, PTEN counteracts the action of the prosurvival proto-oncogenes, the phosphatidylinositol 3-kinases, and protein kinase B/Akt, which control the function of key downstream effectors of this pathway, including cell cycle regulators (such as p27Kip1, cyclin D1, and CHK1) and transcription factors (such as NF-
B and FKHR; Li and Sun, 1998; Nakamura et al., 2000; Gustin et al., 2001; Weng et al., 2001; Radu et al., 2003; Puc et al., 2005). To control the PIP3 levels at the plasma membrane, PTEN possesses, in addition to its N-terminal phosphatase catalytic domain (residues 14–185), a C-terminal phospholipid-binding C2 domain (residues 186–350), which is critical for optimal binding to membranes and PIP3 dephosphorylation (Lee et al., 1999). In addition, the N-terminus of PTEN contains a phosphatidylinositol-4,5-diphosphate (PIP2) binding motif (residues 6–15), which is also essential for PTEN membrane binding and activity (Maehama et al., 2001; Funamoto et al., 2002; Iijima and Devreotes, 2002; Campbell et al., 2003; McConnachie et al., 2003; Iijima et al., 2004; Walker et al., 2004; Vazquez et al., 2006). On the other hand, PTEN possesses a C-terminal tail (residues 350–403) that plays a major role in the stabilization of the molecule and that is the target of posttranslational modifications, including phosphorylation by the protein kinase CK2 and cleavage by caspase-3 (Georgescu et al., 1999; Vazquez et al., 2000; Torres and Pulido, 2001; Torres et al., 2003). The PTEN C-terminal tail also contains a PDZ-domain binding motif that mediates binding to anchoring and/or regulatory proteins and that also controls PTEN binding to membranes (Wu, X. et al., 2000; Wu, Y. et al., 2000; Das et al., 2003; Valiente et al., 2005). Thus, the recruitment of PTEN to sites of the plasma membrane where signaling takes place is dictated by the combination of electrostatic interactions with plasma membrane lipids and protein–protein interactions with membrane-associated proteins.
PTEN has also been found in the nucleus of some cell lines and tissue cells (Gimm et al., 2000; Lachyankar et al., 2000; Perren et al., 2000; Torres et al., 2001; Ginn-Pease and Eng, 2003). Remarkably, PTEN exerts part of its function in coordination with the nuclear tumor suppressor transcription factor p53. Thus, p53 induces the transcription of the PTEN gene, whereas PTEN stimulates the transcriptional activity of p53 by both catalytically dependent and independent mechanisms that include direct binding of the two tumor suppressor proteins (Stambolic et al., 2001; Mayo et al., 2002; Freeman et al., 2003; Zhou et al., 2003; Tang and Eng, 2006). These findings underscore the complexity of PTEN functions at distinct subcellular compartments and imply that the tumor suppressor and/or proapoptotic functions of PTEN rely, at least in part, in actions executed by PTEN in the cell nucleus. Very recently, Chung and coworkers have reported the existence of several nuclear localization signal (NLS)-like sequences within the PTP and the C2 domains of PTEN, which could be important in regulating the cell cycle and apoptosis (Chung et al., 2005; Chung and Eng, 2005). On the other hand, Liu, F. et al. (2005) have reported that PTEN enters the nucleus by diffusion. Thus, the molecular basis of PTEN nuclear localization remains elusive. In this study, we investigated the molecular basis of the accumulation of PTEN in the cell nucleus, as well as the nuclear PTEN function upon apoptotic stimulation. We provide evidence for a Ran-dependent mechanism that controls PTEN nuclear/ cytoplasmic accumulation based on the existence of multiple nuclear exclusion motifs at distinct PTEN regions and a nuclear localization domain at the PTEN N-terminus. Furthermore, we demonstrate a proapoptotic role for catalytically active nuclear PTEN.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Sigma, St. Louis, MO) plus 10 µg/ml cycloheximide (Sigma) during the indicated times, or in the presence of 1 µM of doxorubicin (Alexis Biochemicals, San Diego, CA) for 24 h. To induce apoptosis in 3T3, Rat1, and HeLa cell lines, cells were incubated in the presence of 0.5 M sorbitol for 30 min. To induce apoptosis in HEK293 cells, cell cultures were incubated in the presence of 50 µM etoposide (Sigma) for 24 h. PTEN was detected using the anti-PTEN 425A monoclonal antibody (mAb), which recognizes an epitope within the PTEN C2 domain (Andrés-Pons et al., 2005). In Figures 2C, 8C, and 10B, the anti-PTEN 421B mAb (which recognizes an epitope at the C-terminus of PTEN; Andrés-Pons et al., 2005) or a rabbit polyclonal anti-PTEN N-terminal antiserum (obtained by immunization with a peptide containing the PTEN residues 1–15) were used. In Figures 9C and 10B, a rabbit polyclonal anti-PTEN antibody (Upstate Biotechnology, Lake Placid, NY) was used that recognizes the PTEN C-terminal region (residues 239–403). The anti-GAPDH mAb has been previously described (Aniento et al., 1993). The anti-RhoA and anti-PCNA mAbs were from Santa Cruz Biotechnology (Santa Cruz, CA), the polyclonal anti-cleaved-PARP was from Cell Signaling (Beverly, MA), and the anti-Flag mouse mAb was from Sigma. Horseradish peroxidase–conjugated goat anti-rabbit and anti-mouse secondary antibodies were from Oncogene Research Products (Boston, MA) and Promega (Madison, WI), respectively. The fluorescein-conjugated goat anti-mouse and the rhodamine-conjugated goat anti-rabbit antibodies were from Sigma. pRK5 PTEN constructs have been previously described (Torres and Pulido, 2001; Torres et al., 2003). pIRES PTEN and pIRES PTEN QMA were made by subcloning of PCR-obtained PTEN cDNAs from the corresponding pRK5 constructs into pIRES. The PTEN truncation and deletion mutants and the PTEN N-terminal amino acid substitution mutants, were made by PCR oligonucleotide site-directed mutagenesis. pRK5 GST-GFP was made by fusing in frame the coding sequences of GST (from pGEX-5X2) and GFP (from pEGFP-N2) after PCR amplification and subcloning into pRK5. The 1–16 and 1–32 N-terminal residues of PTEN were fused to GST-GFP by PCR and subcloned into pRK5 (pRK5 PTEN 1–16/GST-GFP and pRK5 PTEN 1–32/GST-GFP). pRK5 GST-importin 1 and pRK5 GST-importin 5 (wild-type and 
1–70 mutations) were made by subcloning from the appropriate pGEX constructs, which were made by subcloning into pGEX of PCR-obtained cDNA fragments from pDNR-LIB importin 1 and pOTB7 importin 5 (Mammalian Gene Collection, IMAGE ID 4105058 and 2822859, respectively). pCDNA3-Flag-Ran and pCDNA3-Flag-RanQ69L were provided by M.-C. Hung (Giri et al., 2005). All mutations and chimeras were confirmed by DNA sequencing.
Subcellular Fractionation and Immunoblot
For nucleus/cytoplasm fractionation, cells were harvested with EDTA-trypsin, centrifuged (1500 rpm, 6 min at 4°C), and washed twice with ice-cold phosphate-buffered saline (PBS). The cell pellet was lysed with buffer A (10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) and vortexed for 10 s. The cells lysates were incubated 5 min on ice and centrifuged 6 min, 1500 rpm, at 4°C. The supernatant was considered as the cytoplasmic fraction. The cellular pellets were resuspended with buffer A and treated again as above to eliminate the cytoplasmic remainders. Finally, the cellular pellet was extracted with SDS-PAGE sample buffer during 5 min at 95°C, followed by centrifugation (14,000 rpm, 5 min at 20°C), and the supernatant was considered as the nuclear fraction. For detection of cleaved-PARP, HEK293 cells were lysed in buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 2 mM Na3VO4, 20 mM Na4P2O7). The cell lysates were centrifuged (14,000 rpm, 10 min at 4°C), and the supernatants were processed for immunoblot. The proteins were resolved by SDS-PAGE and analyzed by immunoblot as described (Torres et al., 2003).
Immunofluorescence
Cells were cultured on poly-D-lysine–coated glass coverslips, transfected, and subjected to treatments as indicated. The cells were washed twice with PBS and then fixed and permeabilized in ice-cold methanol for 5 min at –20°C. The coverslips were washed three times with PBS containing 3% bovine serum albumin (PBS-BSA) and incubated 1 h at 37°C with ascites fluid of mouse monoclonal anti-PTEN 425A, or with mouse monoclonal anti-PTEN 421B (for Figure 2C) or rabbit polyclonal anti-PTEN N-terminal (for Figures 2C and 8C; all diluted 1:200 in PBS-BSA). The coverslips were washed three times with PBS and incubated for 1 h at 20°C with fluorescein-conjugated anti-mouse antibody and/or with rhodamine-conjugated anti-rabbit antibody, diluted 1:200 in PBS-BSA, followed by three washes with PBS. The coverslips were mounted onto glass slides using Fa Mounting-fluid (Becton Dickinson, Lincoln Park, NJ) and examined using a Zeiss fluorescence microscope (Thornwood, NY). For quantitation of PTEN subcellular distribution, at least 100 positive cells were scored for each experiment. Cells were rated as showing nuclear staining (N), cytoplasmic staining (C), or staining within both the nucleus and the cytoplasm (N/C). Nuclei were identified by Hoescht 33258 (Molecular Probes, Eugene, OR) staining. All pictures were taken under a 400x magnification.
Apoptosis Assays
Quantitation of apoptosis in transiently transfected U87MG cells was made by nuclear condensation visualization. Cells were processed for immunofluorescence and PTEN- or GST-expressing cells (at least 100 positive cells) were monitored for nuclear condensation by Hoechst 33258 staining. Apoptosis assays in HEK293 cells stably expressing ectopic PTEN were made by immunoblot detection of cleaved-PARP, a proteolytic product of the effector caspase-3, using anti-cleaved-PARP antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was observed (Figure 1, A and B). Similar results were obtained using HEK293 or COS-7 cells (Figure 1A; and unpublished data). On the other hand, the C-terminal truncation PTEN 1–400, which lacks the PDZ-domain binding motif, mostly displayed cytoplasmic localization (Figure 1, C and D). Remarkably, the C-terminal deletion PTEN 370–385 (see scheme in Figure 1C) accumulated in the nucleus, whereas the deletion PTEN 350–368 was mostly found in the cytoplasm (Figure 1D). These results indicate the existence of regulatory determinants of PTEN nuclear distribution in the region spanning the residues 370–385. Interestingly, this region contains both the sites of phosphorylation by CK2 (Ser370, Ser380, Thr382, Thr383, and Ser385) and of cleavage by caspase-3 (Asp371, Asp375, and Asp384; Torres and Pulido, 2001; Torres et al., 2003), suggesting the possibility that phosphorylation and/or caspase-3 cleavage of PTEN might determine PTEN nuclear accumulation. To test this hypothesis, immunofluorescence experiments were done using PTEN point mutations defective in phosphorylation and/or caspase-3 cleavage. PTEN mutations that affected combinations of the PTEN CK2-phosphorylated residues, including PTEN DMA (S370A/S385A), TMA (S380A/T382A/T383A), and QMA (S370A/S385A/S380A/T382A/T383A) mutations, equally favored PTEN nuclear accumulation (Figure 2, A and B). On the other hand, single amino acid substitutions targeting the individual phosphorylation sites, differentially affected PTEN nuclear localization: mutations S380A, T383A, and S385A, showed a strong nuclear accumulation; distribution of mutation T382A was shifted toward the nucleus, although few cells showed intense PTEN nuclear accumulation and mutation S370A was localized mostly in the cytoplasm (Figure 2B). Thus, mutations that affect PTEN phosphorylation also affect the nuclear/cytoplasmic distribution of PTEN. Next, the PTEN nuclear localization of mutations that abrogate the caspase-3 cleavage at the PTEN C-terminal tail were investigated. As shown in Figure 2, A and B, the localization of the PTEN mutations D384N, CM2 (D371N/D375N), and CM3 (D371N/D375N/D384N) was also nuclear; therefore, cleavage by caspase-3 is not necessary for PTEN nuclear accumulation. The observed PTEN nuclear accumulation was not due to a particular staining obtained with the anti-PTEN mAb used, because identical results were obtained using two other monoclonal and polyclonal anti-PTEN antibodies (Figure 2C). Therefore, distinct mutations in the 370–385 C-terminal region favor the nuclear accumulation of PTEN.
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2 region] at the C2 domain; see Figure 3A) regulate PTEN activities, including PTEN subcellular localization and function (Lee et al., 1999; Das et al., 2003; Orchiston et al., 2004; Chung et al., 2005; Chung and Eng, 2005). To test the role of these motifs in nuclear localization of PTEN, compound mutations were generated (R161A/K163A/K164A [RKK-PTP]; R233A/R234A/K237A [RRK-C2]; K263A/M264A/L265G/K266A/K267A/K269A [CBR3]; and K327A/N329G/K330A/K332A/ R335A [C2]), and assessed for effects on subcellular localization in U87MG cells (Figure 3, B and C). None of the mutations prevented the nuclear accumulation of PTEN 1–375, excluding their roles as putative nuclear localization signals. Furthermore, when present alone, these mutations had distinct effect on the nuclear accumulation of PTEN. The highest degree of nuclear localization was achieved by the mutation C2, followed by the mutations RKK-PTP and CBR3, whereas the mutation RRK-C2 displayed a moderate level of nuclear accumulation (Figure 3, B and C). Together, these results indicate the existence of multiple nuclear exclusion motifs on PTEN, including regions of the C-terminal tail, the PTP and the C2 domains.
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) was assessed in U87MG cells. The nuclear/cytoplasmic distribution of the PTEN C124S mutation was similar to wild-type PTEN; however, on a C-terminal mutation background that caused the accumulation of PTEN in the nucleus, the nuclear accumulation of the C124S mutation was less pronounced (Figure 4, A and B). Interestingly, a different distribution pattern was observed with the G129E mutation. The nuclear localization of PTEN G129E was more pronounced than wild-type PTEN, and there where no effects of this mutation on a C-terminal mutation background (Figure 4, A and B). Therefore, the C124S mutation inhibits, whereas the G129E mutation augments, nuclear accumulation. Together, these results indicate that subcellular distribution of PTEN on U87MG cells is differentially modulated by its protein- and lipid-phosphatase activities.
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17–32 mutation (see below), affected the subcellular localization of PTEN (unpublished data). The removal of the first seven residues on a PTEN 1–375 background (PTEN 8–375 mutation) did not affect PTEN nuclear accumulation (Figure 6, B and C). On the other hand, removal of the first 16 residues of PTEN prevented the nuclear accumulation of PTEN 1–375, PTEN RKK-PTP, and PTEN C2 (PTEN 17–375, PTEN 17–403/RKK-PTP, and PTEN 17–403/C2 mutations, respectively; Figure 6, B and C). The deletion of residues 17–32 (PTEN 17–32) also impaired the nuclear accumulation of PTEN 1–375, and both PTEN 17–32 and PTEN 1–375/17–32 showed, in most of the cells, a perinuclear localization with punctate staining in the cytoplasm (Figure 6B and unpublished data). Finally, the deletion of residues 33–48 did not affect the nuclear accumulation of PTEN 1–375 (Figure 6, B and C). These results pointed to residues 8–32 as being important for PTEN nuclear accumulation. In fact, mutation of positively charged residues within this amino acid region (K13A/R14A/R15A mutation; [KRR-NLS]; see Figure 6A), on the appropriate mutant background (1–375/KRR-NLS mutation), prevented PTEN nuclear accumulation (Figure 6, B and C). Alanine mutational scanning of the region 9–17 revealed that residues Lys13, Arg14, Arg15, and Tyr16 (mutations K13A, R14A, R15A, and Y16A) were essential for nuclear accumulation of PTEN 1–375 (Figure 6D). Interestingly, the tumor-associated mutation K13E (Duerr et al., 1998), on the background 1–375, was also excluded from the nucleus (Figure 6D). Additional mutational analysis was performed in the region 17–32. As shown, mutation of residues Thr26, Tyr27, and Tyr29 (mutation T26A/Y27A/Y29A; [TYY]) impaired the nuclear accumulation of PTEN 1–375, whereas mutation of residues Asp22 to Leu25 (D22A/L23A/D24A/L25A; [DLDL]) did not (Figure 6D). Thus, the N-terminus of PTEN is required for PTEN nuclear localization.
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proteins, which mediate classical nuclear transport of NLS-containing proteins in association with importin 
transporters (Görlich and Kutay, 1999; Xu and Massagué, 2004). Wild-type or mutant forms of importin 1 and importin 5 were fused at their N-terminus to GST to facilitate their detection, and their subcellular localization in U87MG cells was tested. Both wild-type importin 1 and importin 5 were accumulated in the cell nucleus, whereas importin 1 1–70 and importin 5 1–70, which lack the binding site for the importin transporters, were present both in the cytoplasm and in the nucleus (Figure 8A, b, d, f, and h). This distribution pattern was independent of the presence of recombinant PTEN (unpublished data). Remarkably, when PTEN nuclear mutants were coexpressed with nuclear, wild-type importin 1 or importin 5, PTEN did not accumulate in the nucleus and remained cytoplasmic (Figure 8A, a and c, and B). On the other hand, the accumulation of PTEN nuclear mutants was not affected when they were coexpressed with cytoplasmic importin 1 1–70 or importin 5 1–70 (Figure 8A, e and g, and B). Finally, the cytoplasmic localization of wild-type PTEN was unaffected by the coexpression with wild-type or the mutated importin 1 or importin 5 (unpublished data). These results indicate that PTEN entry into the nucleus is not mediated by direct binding to importin 1 or importin 5, but rather by an importin-related nuclear transport mechanism.
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proteins is required for importin-mediated nuclear transport (Görlich and Kutay, 1999; Kuersten et al., 2001; Ström and Weis, 2001). To test the involvement of Ran in the nuclear accumulation of PTEN mutations, coexpression experiments were performed using the GTPase-deficient, dominant negative, RanQ69L mutation (Nachury and Weis, 1999). Wild-type Ran was distributed in both the nucleus and the cytoplasm, whereas RanQ69L displayed a diffuse cytoplasmic localization and was also at the nuclear membrane (Figure 8C, b, e, h, and k). Notably, in the presence of RanQ69L, PTEN 1–375 or PTEN QMA did not accumulate in the nucleus, whereas wild-type Ran did not affect the nuclear accumulation of PTEN (Figure 8C, a, d, g, and j). These results demonstrate that PTEN nuclear accumulation is dependent on the activity of Ran GTPase.
Nuclear PTEN Enhances Apoptosis
Treatment of U87MG cells with the proapoptotic cytokine TNF- increases PTEN cleavage by caspase-3, that renders PTEN products lacking the PTEN C-terminal region (Torres et al., 2003). Thus, we tested the effects of TNF- on localization of PTEN in U87MG cells. As predicted, PTEN accumulated in the nucleus following TNF- treatment (Figure 9, A and B). The nuclear accumulation of PTEN upon TNF- stimulation was not affected on the PTEN 8–403 N-terminal truncation, but it was prevented on the PTEN 17–403 truncation (Figure 9B), suggesting that the PTEN N-terminus controls PTEN nuclear/cytoplasmic transport during apoptosis. We also tested the nuclear accumulation of endogenous PTEN from 3T3, Rat1, and HeLa cell lines treated with the apoptotic stimulus sorbitol. As shown, the levels of nuclear PTEN augmented in all these cell lines after sorbitol treatment (Figure 9C). Next, we assessed the effect of PTEN nuclear accumulation on the response of cells to apoptotic stimuli. U87MG cells were transiently transfected with cytoplasmic or nuclear PTEN and treated with TNF- or doxorubicin, followed by immunofluorescence and quantitation of nuclear condensation (as a measurement of apoptosis; Figure 10A). Remarkably, the percentage of cells with condensed nuclei under these conditions was higher when nuclear PTEN (PTEN QMA and PTEN 1–375) was overexpressed, compared with cytoplasmic PTEN (PTEN wt). Furthermore, these effects were dependent on the PTEN catalytic activity, because they were not observed after overexpression of the PTEN C124S or G129E mutants (Figure 10A). Also, overexpression of the PTEN mutant K13E, which prevents nuclear accumulation of PTEN 1–375 or QMA (Figure 6D, and unpublished data), did not augment apoptosis (Figure 10A). The apoptotic response of HEK293 cells stably expressing cytoplasmic or nuclear PTEN was also analyzed by immunoblot analyses of cleaved-PARP, a hydrolysis product of caspase-3. Stable expression of cytoplasmic wild-type PTEN slightly increased the amount of cleaved-PARP in HEK293 cells after etoposide treatment (Figure 10B). Noticeably, cleaved-PARP was more prominent in etoposide-treated cell clones expressing the nuclear PTEN QMA mutant than those expressing cytoplasmic wild-type PTEN (Figure 10B, lanes 5–6 and 9–10 vs. lanes 3–4 and 7–8). Together, our results indicate that PTEN nuclear accumulation is favored upon apoptotic stimulation and that catalytically active nuclear PTEN enhances apoptosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Iijima and Devreotes, 2002; Campbell et al., 2003; McConnachie et al., 2003; Iijima et al., 2004; Walker et al., 2004). Our findings demonstrate that this region is also necessary for PTEN nuclear localization. First, N-terminal tagging of PTEN with an HA epitope, or with the GST-protein, blocked its nuclear accumulation in U87MG cells, suggesting they mask a functional N-terminal sequence required for PTEN nuclear localization. Second, a stretch of positively charged residues, which could be reminiscent of a NLS-like sequence, is present at the N-terminus of PTEN (see Figure 6A). Third, PTEN N-terminal truncations and deletions, as well as point mutations at specific N-terminal residues, abrogate PTEN nuclear accumulation in nuclear mutants of PTEN, or after TNF- treatment. Fourth, the first N-terminal 32 residues of PTEN are sufficient to target a GST-GFP cytoplasmic protein to the nucleus. We hypothesize that both a PIP2-binding motif and a nuclear localization motif overlap within the N-terminal region of PTEN. An exciting possibility is that the precise functionality of one or the other of these targeting motifs may target the protein to a particular subcellular compartment and exclude it from the other. Using chicken PK fused to the N-terminal portion of PTEN, Liu, F. et al. (2005) have found that the N-terminus of PTEN lacks intrinsic NLS activity in HeLa cells. However, our findings demonstrate that PTEN residues 1–32 target GST-GFP to the nucleus in U87MG cells. These observations suggest that nuclear localization activity of PTEN N-terminus differs from classical NLS motifs and may be dependent on the molecular and cellular context. In fact, residues 1–16, which contain key residues for PTEN nuclear accumulation, were not sufficient to target GST-GFP to the nucleus. On the other hand, Chung et al. (2005) have reported the existence of several NLS-like sequences within the PTP and C2 domains of PTEN that overlap with our RKK-PTP, RRK-C2, and CBR3 mutations (Figure 3). The results obtained with our mutants show an increase in PTEN nuclear accumulation, indicating that these sequences may behave as nuclear exclusion motifs in the wild-type protein. The discrepancy between these studies could be explained by the mutations analyzed or by the distinct cell systems and conditions of PTEN nuclear accumulation tested, yet they all underscore the importance of these motifs in the control of PTEN nuclear localization.
The PTEN C-terminal tail (residues 350–403) is required for PTEN binding to regulatory proteins, including plasma membrane proteins and PDZ-domain scaffolding proteins (Wu, X. et al., 2000; Wu, Y. et al., 2000; Tolkacheva et al., 2001; Vazquez et al., 2001; Mahimainathan and Choudhury, 2004; Sumitomo et al., 2004; Valiente et al., 2005), and this region has also been shown to interfere with membrane binding and with PTEN-mediated regulation of cell migration (Das et al., 2003; Raftopoulou et al., 2004). We and others have reported the phosphorylation of the region 370–385 of the PTEN C-terminal tail (Torres and Pulido, 2001; Birle et al., 2002; Miller et al., 2002). The results here described, showing PTEN nuclear accumulation in mutants targeting the PTEN 370–385 region, outline the critical regulatory importance of this region on distinct PTEN functions, including those exerted in the nucleus (see below). Moreover, PTEN is cleaved by caspase-3 at residues in this region, rendering PTEN products that lack some phosphorylation sites and the PDZ-domain binding motif (Torres et al., 2003). Our findings indicate that the cleavage of PTEN by caspase-3 is sufficient, but not necessary, for PTEN nuclear accumulation, because PTEN mutations that either mimic or impair caspase-3 cleavage displayed nuclear localization. Our results also suggest that alterations in PTEN phosphorylation may be sufficient to trigger PTEN nuclear accumulation. However, we have not found a clear correlation between PTEN phosphorylation by CK2 and PTEN nuclear accumulation (this report; and unpublished data), suggesting that other factors may also regulate PTEN nuclear localization in U87MG cells. Phosphorylation of the C-terminal tail of PTEN by other protein kinases, such as MAST kinases, LKB1, or GSK3 (Al-Khouri et al., 2005; Mehenni et al., 2005; Valiente et al., 2005), could modulate PTEN subcellular localization. The possibility also exists that cytoplasmic or nuclear anchoring or transport proteins may regulate PTEN localization (Yu et al., 2002; Freeman et al., 2003; Zhou et al., 2003; Chung et al., 2005; Okumura et al., 2005; see also below). Treatment of cells with the nuclear export inhibitor leptomycin B did not affect the subcellular localization of PTEN in our experiments, suggesting that PTEN is not subjected to active CRM1-dependent nuclear export (unpublished data). Our overexpression experiments using wild-type and mutant importin proteins suggest that PTEN entry into the nucleus could be mediated by proteins that can be sequestered upon binding to the N-terminal region of importin 1 and importin 5. Also, the Ran dependence of the PTEN nuclear accumulation suggests an active role for importin transporters in this process. Whether an importin and/or a PTEN-specific importin are directly involved in PTEN nuclear transport deserves further study.
Remarkably, both the PTEN N-terminus and the PTEN C-terminal tail are unstructured regions within the PTEN molecule (Lee et al., 1999). Such regions might be solvent exposed and are likely to play a dynamic role in the PTEN intramolecular or intermolecular interactions that control PTEN subcellular distribution and access to substrates. In this regard, it has been found that phosphoisoforms of PTEN exist in different conformations (Vazquez et al., 2001). In addition, our findings that mutations of charged residues at surface-exposed regions within the PTP and C2 domains favor nuclear accumulation of PTEN, suggest that a large surface on the PTEN protein dictates its subcellular localization. Our results demonstrate a negative contribution of surface-exposed PTEN nuclear exclusion motifs and a positive contribution of the PTEN N-terminus, for PTEN nuclear localization. We thus hypothesize a nuclear localization domain-masked conformation (located in the cytoplasm) and a nuclear localization domain-unmasked conformation (located in the nucleus) for PTEN. The balance between the action of the PTEN nuclear exclusion motifs and the action of the PTEN nuclear localization domain (see scheme in Figure 11), could determine, in a Ran-dependent manner, the nuclear/cytoplasmic distribution of PTEN.
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; Perren et al., 2000; Whiteman et al., 2002). Also, in some cell lines expressing endogenous PTEN, its nuclear expression has been associated with G0-G1 cell cycle phase arrest (Ginn-Pease and Eng, 2003) or with cell differentiation (Lachyankar et al., 2000), and it has been found that nuclear PTEN is required for cell cycle arrest in MCF-7 cells (Chung and Eng, 2005). Also, it has been reported that nuclear targeted-PTEN leads to growth suppression of U251MG cells (Liu, J. L. et al., 2005). Our results show an increase in PTEN nuclear localization upon apoptotic stimulation, suggesting a relation between nuclear PTEN and apoptosis. Furthermore, we have found that catalytically active nuclear PTEN enhances the apoptotic responses of U87MG and HEK293 cells to distinct stimuli. The scheme shown in Figure 11 summarizes the proposed cellular functions of PTEN in the cell nucleus.
A nuclear PTEN/PI3K/Akt pathway that controls apoptosis has been described in some cell lines, and the existence of PIP3 phosphatase activity on nuclear PTEN has been reported (Deleris et al., 2003; Ahn et al., 2004; Ye and Snyder, 2004; Deleris et al., 2006). In addition, putative nuclear protein substrates of PTEN could account for the proapoptotic or growth suppressive effect of PTEN in the nucleus, and the possibility exists of a differential regulation of PTEN catalysis in the nucleus and in the cytoplasm. Interestingly, we have found that the protein- and lipid-phosphatase activities of PTEN affect its localization, suggesting a putative regulatory mechanism of PTEN nuclear/cytoplasmic distribution dictated by its dual enzymatic activity. On the other hand, recent reports have demonstrated a tumor-suppressor function for PTEN in the nucleus that is independent of its catalytic activity, but is dependent on the physical interaction of PTEN with nuclear effectors, including the tumor suppressor p53 and the oncogene MSP58/MCRS1; such binding cooperates with the transcriptional activity of p53 and inhibits the oncogenic potential of MSP58/MCRS1 (Freeman et al., 2003; Zhou et al., 2003; Okumura et al., 2005; Tang and Eng, 2006). Thus, it is conceivable that PTEN behaves as a multifunctional and versatile molecule that executes its biological functions via multiple mechanisms and at distinct subcellular localizations, including the cell nucleus. The identification of the molecular mechanisms that are important for PTEN nuclear accumulation will help to unravel the specific functions of PTEN in the nucleus.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WCA 3PX, United Kingdom. 
Address correspondence to: Rafael Pulido ( rpulido{at}cipf.es)
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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