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Involvement of 14-3-3 proteins in nuclear localization of telomerase

2000; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês

10.1093/emboj/19.11.2652

1460-2075

Hiroyuki Seimiya, Hiroko Sawada, Yukiko Muramatsu, Mayuko Shimizu, Kumiko Ohko, Kazuhiko Yamane, Takashi Tsuruo,

14-3-3 protein interactions

Article1 June 2000free access Involvement of 14-3-3 proteins in nuclear localization of telomerase Hiroyuki Seimiya Corresponding Author Hiroyuki Seimiya Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Present address: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, 2nd Floor, New York, NY, 10016 USA Search for more papers by this author Hiroko Sawada Hiroko Sawada Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Yukiko Muramatsu Yukiko Muramatsu Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Mayuko Shimizu Mayuko Shimizu Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Kumiko Ohko Kumiko Ohko Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Kazuhiko Yamane Kazuhiko Yamane Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Takashi Tsuruo Takashi Tsuruo Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Hiroyuki Seimiya Corresponding Author Hiroyuki Seimiya Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Present address: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, 2nd Floor, New York, NY, 10016 USA Search for more papers by this author Hiroko Sawada Hiroko Sawada Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Yukiko Muramatsu Yukiko Muramatsu Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Mayuko Shimizu Mayuko Shimizu Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Kumiko Ohko Kumiko Ohko Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Kazuhiko Yamane Kazuhiko Yamane Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Search for more papers by this author Takashi Tsuruo Takashi Tsuruo Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Author Information Hiroyuki Seimiya 1,2, Hiroko Sawada1, Yukiko Muramatsu1, Mayuko Shimizu1, Kumiko Ohko1, Kazuhiko Yamane1 and Takashi Tsuruo1,3 1Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, 170-8455 Japan 2Present address: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, 2nd Floor, New York, NY, 10016 USA 3Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2652-2661https://doi.org/10.1093/emboj/19.11.2652 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post-translational regulation of telomerase activity. Here we identify the 14-3-3 signaling proteins as human TERT (hTERT)-binding partners. A dominant-negative 14-3-3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT-3A, a mutant that could not bind 14-3-3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1-mediated nuclear export, or disruption of a nuclear export signal (NES)-like motif located just upstream of the 14-3-3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild-type hTERT, hTERT-3A increased its association with CRM1. 14-3-3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14-3-3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES-like motif. Introduction The telomere is a unique structure found in the termini of most eukaryotic chromosomes. Tandem repeats of the highly conserved telomeric DNA (TTAGGG in vertebrates) (Moyzis et al., 1988) shorten after each cell cycle due to the end replication problem of chromosomal DNA (Harley et al., 1990; Hastie et al., 1990). Since critically shortened telomeres are often associated with the replicative senescence of normal somatic cells, telomeres have been thought to be a mitotic clock that counts the number of times a cell can divide before eliciting the senescence program (Harley et al., 1990; Hastie et al., 1990; Allsopp et al., 1992; Counter et al., 1992). Based on these situations, maintenance of telomeres is implicated in chromosome stabilization and cell immortalization (Zakian, 1995; Greider, 1996). Most immortal cells, including germ line cells and cancer cells, possess enzymatic activity of telomerase (Kim et al., 1994; Shay and Bacchetti, 1997), which catalyzes de novo synthesis of telomeric repeats at chromosome ends (Greider and Blackburn, 1985; Morin, 1989). Since introduction of the telomerase catalytic subunit gene (TERT) (Meyerson et al., 1997; Nakamura et al., 1997) into normal somatic cells extends the life of the cells (Bodnar et al., 1998), the immortalized phenotype of most cancer cells would involve activation of telomerase. Consistently, genetic disruption of the telomerase RNA component (mTR), which abolishes telomerase activity, causes telomere erosion and chromosomal aberrations, resulting in functional defects in highly proliferative organs (Blasco et al., 1997; Lee et al., 1998). Based on these observations, it is likely that cancer cells bypass telomere crisis by activating telomerase. Recent studies have demonstrated that c-Myc directly induces TERT transcription and telomerase activity (J.Wang et al., 1998; Wu et al., 1999). In some immortal cells, differentiation induction downregulates telomerase activity with transcriptional repression of the TERT gene (Meyerson et al., 1997). These observations indicate that telomerase activity is mainly regulated by TERT transcription. On the other hand, telomerase activity could also be regulated by post-transcriptional and/or post-translational modifications of the enzyme. For example, decline in telomerase activity in confluent NIH 3T3 fibroblasts is not associated with downregulation of TERT expression (Greenberg et al., 1998). Similarly, telomerase activity in human lymphocytes is not correlated with the levels of human TERT (hTERT) transcript (Liu et al., 1999). One possible mechanism for the post-translational modification of telomerase is the interaction of hTERT with accessory proteins, including other telomerase subunits, enzymes, adaptors or chaperones. Introduction of hTERT gene into normal cells induces telomerase activity and confers a continuous replicative capacity (Bodnar et al., 1998) whereas alteration of the C-terminus of hTERT disrupts its abilities to maintain telomere length and to immortalize a cell in spite of relevant telomerase activity (Counter et al., 1998). These observations suggest that the C-terminus of hTERT interacts with other factors required for telomere maintenance and cell immortalization. The 14-3-3 proteins, which constitute a highly conserved isoform of homo- and heterodimeric molecules, associate with a number of different signaling proteins, including Raf-1, Cbl, nitrate reductase, Bad, the epithelial keratins K8/18, Cdc25C and the IGF-1 receptor (Liao and Omary, 1996; Moorhead et al., 1996; Muslin et al., 1996; Zha et al., 1996; Craparo et al., 1997; Liu et al., 1997; Peng et al., 1997). Based on their interaction with various ligands, 14-3-3 proteins have been proposed to be important in controlling intracellular signaling pathways (Aitken, 1996). In addition, 14-3-3 proteins work as molecular chaperones or regulate intracellular localization of their binding partners (Liao and Omary, 1996; Kumagai and Dunphy, 1999; Yang et al., 1999). Here, we have identified 14-3-3 family proteins as hTERT-binding partners. We found that hTERT and 14-3-3 specifically bound at their respective C-termini, and this interaction was required for efficient accumulation of hTERT in the nucleus. We also demonstrated that 14-3-3 repressed the interaction of hTERT with CRM1/exportin 1, a receptor for the nuclear export machinery. These observations suggest that 14-3-3 is a post-translational modifier of telomerase that functions by controlling the intracellular localization of hTERT. Results Identification of 14-3-3 proteins as hTERT-interacting partners with a yeast two-hybrid screening Using the C-terminal 129 amino acids of hTERT (non-characterized C-terminus, NCC; Figure 1A) as bait, we performed a yeast two-hybrid screening either on a HeLa or a human testis library. As a result, we obtained positive clones, two from HeLa and the other two from testis (Figure 1B). Both the HeLa clones contained the same cDNA fragment of 14-3-3Θ, an isoform of the 14-3-3 family proteins. These clones lacked the N-terminus (residues 1–69) of the intact protein. The testis clones contained cDNAs of another 14-3-3 isoform, 14-3-3ζ, also lacking the N-terminus (residues 1–56 and 1–53, respectively). These prey clones did not interact with other hTERT domains (Figure 1B) or with other unrelated baits, such as a part of the TEP1 telomerase subunit (data not shown). Figure 1.Interaction of hTERT with 14-3-3 in the yeast two hybrid system. (A) Schematic representation of hTERT bait constructs. Bar indicates the bait domain used in the analysis. Values in parentheses indicate the positions of amino acid residues. SXP, a region containing a conventional 14-3-3 binding motif-like sequence; TRT, telomerase reverse transcriptase motif; CDE, reverse transcriptase motifs C, D and E (Nakamura et al., 1997); NCC, non-characterized C-terminus. (B) Interaction of hTERT with 14-3-3 in yeast cells. The prey clones used are 14-3-3Θ (residues 70–245) and 14-3-3ζ (residues 57–245). The black (actually blue) signal on the SG-HWU/X plate and the growth on the SG-HWUL plate indicate activation of the reporter genes, lacZ and LEU2, respectively. S, synthetic; G, galactose; X, X-Gal; D, glucose; H, histidine (−); W, tryptophan (−); U, uracil (−); L, leucine (−). Download figure Download PowerPoint hTERT and 14-3-3 specifically bind via their respective C-termini To confirm the direct interaction between hTERT and 14-3-3, we analyzed the binding of recombinant 14-3-3Θ and hTERT (residues 831–1132) fused to glutathione S-transferase (GST). As shown in Figure 2A, these purified proteins bound directly in vitro. Furthermore, in vitro-translated full-length hTERT and hTERT(del109–962) bound to GST–14-3-3Θ(70–245) but not to the control GST (Figure 2B). In the case of full-length hTERT, another band with lower molecular weight also bound to 14-3-3Θ(70–245) (asterisk in Figure 2B). Since this smaller band was similarly detected in hTERT tagged with HA at the C-terminus (hTERT-HA) when analyzed by anti-HA immunoblotting (data not shown), it seemed to be a degraded product of the hTERT C-terminus. Neither hTERT(del220–1108) nor hTERT(del657–1132) bound to 14-3-3Θ(70–245). These observations were consistent with the results of the two-hybrid analysis (Figure 1), because only hTERT constructs with an intact NCC region could bind to 14-3-3Θ. Figure 2.Interaction of hTERT with 14-3-3 via their respective C-termini. Values in parentheses indicate the positions of amino acid residues. (A) Direct interaction between hTERT and 14-3-3. The GST fusion protein was immobilized on glutathione–Sepharose and incubated with recombinant 14-3-3Θ. Bound fractions were analyzed by western blotting with anti-14-3-3Θ. Factor Xa was used to prepare the recombinant 14-3-3Θ and a trace of it (inactivated) remained in the preparation. (B) Binding properties of hTERT deletion mutants with 14-3-3. del, deleted. The GST fusion protein was immobilized on glutathione–Sepharose and incubated with each [35S]hTERT. The radioactivity bound to the beads was detected by phosphoimaging. Control radioactivity (interaction of each hTERT with GST alone) was given a value of 1. The input does not have the Sepharose material. Asterisk indicates an hTERT carboxyl fragment probably due to the degradation of the full-length protein (lane 1). (C) Binding properties of 14-3-3Θ deletion mutants with hTERT. Boxes in the upper scheme indicate α-helices. The GST fusion protein was immobilized on glutathione–Sepharose and incubated with 35S-labeled full-length hTERT or hTERT-HA. The specific signal was detected by phosphoimaging. Control radioactivity (interaction of hTERT with GST alone) was given a value of 1. (D) Association of hTERT-HA and 14-3-3 in intact cells. The lysate of hTERT-HA-transfected 293T cells was incubated with either anti-TEP1 (Santa Cruz Biotechnology), anti-14-3-3 or mouse normal IgG fixed on protein G–Sepharose and the bound fractions were detected by anti-HA western blot analysis. IP, immunoprecipitation: WB, western blot. (E) Association of endogenous hTERT and 14-3-3 in intact cells. The A2780 cell lysate was immunoprecipitated with either anti-14-3-3 or anti-Xpress (control, Invitrogen) and the bound fractions were analyzed by anti-hTERT western blotting (upper panel). The same blot was re-probed with anti-14-3-3Θ (lower panel). Download figure Download PowerPoint Next, we examined the binding of the full-length hTERT to various regions of 14-3-3Θ. 14-3-3 contains nine α-helices, A–I (Liu et al., 1995; Xiao et al., 1995), and all 14-3-3Θ constructs that had the C-terminus (helices H and I) could bind hTERT (Figure 2C). Helix G, required for binding to phosphorylated c-Raf (Gu and Du, 1998), was dispensable for the hTERT binding. The C-terminal fragment of degraded hTERT again bound to these fusion proteins except 14-3-3Θ(1–127) and GST itself. Similarly, hTERT with an HA-epitope tag also interacted with the 14-3-3Θ C-terminus. We also observed co-immunoprecipitation of endogenous 14-3-3 with ectopically expressed hTERT-HA or with endogenous hTERT (Figure 2D and E). These observations indicate that hTERT binds to 14-3-3 via each C-terminus both in vitro and in intact cells. 14-3-3Θ carboxyl fragment inhibits nuclear accumulation of hTERT 14-3-3 forms homo- and heterodimers (Aitken, 1996) that seemed to be required for successful involvement in signaling events, because mutant monomeric forms of 14-3-3, although able to bind Raf-1, do not enable Raf-1 to be activated in vivo, nor do they restore Raf-1 activity after displacement of 14-3-3 in vitro (Tzivion et al., 1998). Consistent with a previous report (Luo et al., 1995), 14-3-3Θ(128–245) (14-3-3Θ-ΔN), which lacked the N-terminal dimerization domain, could not dimerize with endogenous 14-3-3 in vitro or in intact cells (Figure 3A and B), whereas it could still bind efficiently to hTERT (Figures 2C and 3C). As a control, 14-3-3Θ(1–127) (14-3-3Θ-ΔC) could bind to endogenous 14-3-3 but not to hTERT (Figure 3A–C). Interestingly, 14-3-3Θ-ΔN inhibited the nuclear accumulation of hTERT-HA expressed in 293T cells (Figure 3D, lower panel). Among the cells transfected with hTERT-HA in combination with an empty vector or 14-3-3Θ-ΔC, 82% (55 of 67) or 79% (55 of 70) exhibited punctate accumulation of hTERT-HA in their nuclei, respectively (Figure 3D, upper panel). On the other hand, among the cells co-transfected with hTERT-HA and 14-3-3Θ-ΔN, only 41% (15 of 37) did. As shown in Figure 3C, the hTERT–14-3-3Θ-ΔN co-localization was observed in 59.5% (22 of 37) of co-transfected cells (either in the cytoplasm or in the nucleus). On the contrary, the hTERT–14-3-3Θ-ΔC co-localization was observed in only 2.9% (2 of 70) of co-transfected cells, because hTERT-HA and 14-3-3Θ-ΔC existed predominantly in the nucleus and in the cytoplasm, respectively. Furthermore, 14-3-3Θ-ΔNV176D, in which the hydrophobic property of the ligand-binding site was altered (H.Wang et al., 1998), lost 80% of its ability to bind hTERT (data not shown). This mutant did not inhibit the nuclear accumulation of hTERT-HA [91% (31 of 34) of co-transfected cells exhibited punctate localization of hTERT-HA in the nuclei; data not shown], indicating that the inhibition of the nuclear accumulation of hTERT-HA required the hTERT–14-3-3Θ-ΔN interaction. None of the mutations affected the total amount of ectopically expressed hTERT-HA (Figure 3C, lower panel). Since a similar 14-3-3 carboxyl fragment inhibits the Raf-1 activation by the full-length 14-3-3 in vitro and excludes Raf-1 from its activation process in vivo (Luo et al., 1995), it is likely that 14-3-3Θ-ΔN, as a dominant-negative mutant, blocks the association of the wild-type 14-3-3 dimers with hTERT, inhibiting nuclear accumulation of hTERT. In fact, 14-3-3Θ-ΔN but not 14-3-3Θ-ΔC inhibited the association of hTERT-HA with endogenous 14-3-3 in 293T cells, as determined by immunoprecipitation assay (data not shown). Figure 3.Effects of 14-3-3Θ deletion mutants on intracellular localization of hTERT. (A) Failure in dimerization of 14-3-3Θ(128–245) with endogenous 14-3-3 in vitro. 293T cellular lysate was prepared and was incubated with each GST–14-3-3Θ fusion protein immobilized on glutathione–Sepharose. Endogenous 14-3-3 bound to the beads was detected by anti-14-3-3 western blot analysis. (B) Failure in dimerization of 14-3-3Θ-ΔN with endogenous 14-3-3 in intact cells. 293T cells were transfected with pFLAG-CMV-2–14-3-3Θ(1–127) (ΔC) or pFLAG-CMV-2–14-3-3Θ(128–245) (ΔN). Cellular lysates were prepared, and immunoprecipitation followed by western blot analysis was performed. Note that the antibody used for immunoprecipitation directly recognizes the endogenous 14-3-3 but not 14-3-3Θ-ΔC or 14-3-3Θ-ΔN (data not shown). (C) Interaction of hTERT with 14-3-3Θ-ΔN but not with 14-3-3Θ-ΔC in intact cells. 293T cells were transfected with pCR3–hTERT-HA, pCR3–hTERT-HA and pFLAG-CMV-2–14-3-3Θ-ΔC, or pCR3–hTERT-HA and pFLAG-CMV-2–14-3-3Θ-ΔN. Cellular lysates were prepared, and immunoprecipitation followed by western blot analysis was performed. (D) Effects of 14-3-3Θ deletion mutants on intracellular localization of hTERT. 293T cells were transfected as in (C) and indirect immunofluorescence staining was performed. Expression of hTERT-HA (colored in red by rhodamine or Texas Red) and FLAG-14-3-3Θ-ΔC or -ΔN (colored green by fluorescein) were detected. Note the cytoplasmic localization of hTERT in the hTERT–14-3-3Θ-ΔN-coexpressed cell. Also shown are nuclei as colored in blue by DAPI. Download figure Download PowerPoint hTERT mutant that cannot bind to 14-3-3 fails to localize into the nucleus At present, conventional 14-3-3 binding motifs, such as RSXpSXP (pS indicates a critical phosphoserine), have been identified in most 14-3-3 binding proteins (Yaffe et al., 1997). However, there is no such consensus motif in the hTERT NCC region. Consistently, alkaline phosphatase treatment of hTERT did not diminish its interaction with 14-3-3Θ (data not shown), suggesting that this interaction was independent of phosphorylation, which is usually required for the association of 14-3-3 with its partners. Also, the SXP bait, which contained RQHSSP, resembling the consensus RSXSXP, did not interact with 14-3-3Θ or 14-3-3ζ (Figure 1). In addition, two-hybrid screenings using the SXP region as a bait either on a HeLa prey cDNA library or on a normal human testis prey cDNA library did not isolate any 14-3-3-related gene fragments as positive clones (H.Seimiya, M.Shimizu and T.Tsuruo, unpublished observations). These situations suggest that the interaction of hTERT with 14-3-3 is mediated by an alternative mode that is independent of serine phosphorylation. Crystal structure analysis predicts that 14-3-3 forms an amphipathic groove and its binding partners would possess an amphipathic helix to fit into the groove (Liu et al., 1995; Xiao et al., 1995). Since both hTERT and mTERT (mouse homolog) NCC regions have a putative amphipathic helix with a characteristic serine/threonine cluster (Du et al., 1996; Figure 4A), we constructed a mutant hTERT, in which Thr1030, Ser1037 and Ser1041 in the serine/threonine cluster were all mutated to alanine (hTERT-3A; Figure 4B). As expected, this mutant lost the ability to interact with 14-3-3Θ both in yeast (Figure 4C) and in 293T cells (data not shown). Finally, we found that hTERT-3A-HA diminished its ability to localize into the nucleus and was accumulated in the cytoplasm both in 293T cells and in normal foreskin fibroblasts (Figure 4D). The percentage of cells with punctate accumulation of hTERT in the nuclei were 87% (87 of 100, 293T/wt), 46% (46 of 100, 293T/3A), 72% (31 of 43, HFF/wt) and 31% (12 of 39, HFF/3A), respectively. In the fibroblasts, the efficiency of ectopic expression of hTERT-HA was much lower than that in 293T cells (as described below; Figure 7B) and it is unlikely that the failure of the mutant in nuclear accumulation is due to artificial overexpression of the protein. These observations suggest that 14-3-3 is involved in nuclear localization of hTERT. Figure 4.Intracellular localization of an hTERT mutant that can not bind to 14-3-3. (A) Putative amphipathic helices in the NCC domains of hTERT and mTERT. Wheel structure analysis was performed by DNASIS (Hitachi Software). Serine/threonine cluster (Du et al., 1996) and hydrophobic residues are indicated by red and blue letters, respectively. (B) Partial amino acid sequence of hTERT and hTERT-3A. Underline indicates a putative amphipathic helix. Residues with asterisks form a serine/threonine cluster as in (A). (C) Two hybrid analysis between NCC-3A and 14-3-3Θ(70–245). LacZ activation by the baits was monitored by measurement of β-galactosidase levels in liquid cultures grown on galactose. (D) Indirect immunofluorescence staining. Expression of hTERT-HA or hTERT-3A-HA was detected (colored in red by rhodamine). Also shown are nuclei as colored in blue by DAPI. wt, wild-type hTERT; 3A, hTERT-3A; HFF, human foreskin fibroblasts. Download figure Download PowerPoint Figure 5.14-3-3 binding is not required for telomerase activity. (A) In vitro reconstitution (IVR) of telomerase activity. IVR telomerase was prepared by using the plasmids for each hTERT construct and hTR RNA subunit as described previously (Weinrich et al., 1997). Telomerase activity in each preparation was detected by the TRAP assay (Kim et al., 1994). Also shown are the results of western blot analysis on each IVR preparation with anti-HA or anti-14-3-3. Note that the reticulocyte lysate used for in vitro translation contained its own 14-3-3 proteins. (B) Telomerase activity in the hTERT-transfected fibroblasts. Human foreskin fibroblasts at population doubling 37 were transfected with indicated expression vectors. Portions of A2780 cell lysate (300 cells) or the fibroblast lysate (2 × 104 cells; much larger cell numbers than in case of A2780 were due to low efficiency of transfection and expression of exogenous hTERT) were analyzed by the TRAP assay. Also shown is the result of anti-HA western blot analysis on each total lysate. Download figure Download PowerPoint Identification of a nuclear export signal-like motif in hTERT Since hTERT protein is too large to diffuse through a nuclear pore complex channel (Nigg, 1997), the cytoplasmic localization of hTERT observed in Figures 3 and 4 might involve regulated machinery for nuclear export. We examined the hTERT primary amino acid sequence to determine whether it contains a leucine-rich sequence of conserved spacing and hydrophobicity that fits the criteria established for nuclear export signal (NES) (Fischer et al., 1995; Wen et al., 1995). As shown in Figure 5A, we found that residues between amino acids 970 and 981 conform to this motif, as indicated by the similarity to other known NESs such as p53 (Stommel et al., 1999). This putative NES was located just upstream of the 14-3-3 binding amphipathic helix (residues 1030–1047) and was conserved in mTERT. To confirm the interaction between CRM1 and the NES-like motif in hTERT, we performed an in vitro pull-down assay of CRM1 with GST–hTERT(831–1132). As shown in Figure 5B, CRM1 specifically associated with GST–hTERT(831–1132) but not with GST alone. We generated a mutant construct, GST–hTERTnes(831–1132), in which three conserved leucines, Leu974, Leu978 and Leu980, were replaced by alanines. This nes mutant significantly diminished its association with CRM1, indicating that this motif was actually recognized by CRM1. Figure 6.Identification of an NES-like motif in hTERT. (A) Comparative alignment of the NES-like motifs identified in TERT C-termini with other known NESs. Bold letters indicate the conserved Leu/Met/Ile residues. hTERTnes, a mutant in which three conserved leucines were replaced by alanines (asterisks). (B) Interaction between hTERT and CRM1 in vitro. The GST fusion protein was immobilized on glutathione–Sepharose and incubated with the 293T cell lysate (3 × 106 cells/sample). After the extensive washing, bound fractions were analyzed by western blotting with anti-human CRM1 antibody. Equal loading of each GST fusion protein was confirmed by Coomassie Blue staining of the SDS–PAGE gel (data not shown). Download figure Download PowerPoint 14-3-3 enhances nuclear localization of hTERT by inhibiting the CRM1 binding to the hTERT NES-like motif To determine whether the NES/CRM1-mediated nuclear export (Fornerod et al., 1997; Fukuda et al., 1997; Ossareh-Nazari et al., 1997) is involved in cytoplasmic localization of hTERT by 14-3-3Θ-ΔN, we first examined the effect of Leptomycin B (LMB), a potent inhibitor of NES/CRM1-mediated nuclear export (Nishi et al., 1994; Kudo et al., 1998, 1999). As shown in Figure 3, 14-3-3Θ-ΔN inhibited the nuclear accumulation of hTERT (Figure 6A, top). When the cells were treated with 20 nM LMB for 2 h, this effect of 14-3-3Θ-ΔN was completely abolished (Figure 6A, middle). Similarly, cytoplasmic localization of hTERT-3A-HA in foreskin fibroblasts was reversed by treatment with LMB (Figure 6B). LMB did not affect the intracellular localization of the wild-type hTERT-HA (data not shown). Figure 7.Involvement of the CRM1-mediated nuclear export in the cytoplasmic localization of hTERT. (A) Effects of LMB and the nes mutation on 14-3-3Θ-ΔN-mediated inhibition of nuclear localization of hTERT. 293T cells were transfected with pCR3–hTERT-HA and pFLAG-CMV-2–14-3-3Θ-ΔN or pCR3–hTERTnes-HA and pFLAG-CMV-2–14-3-3Θ-ΔN. After 24 h of transfection, cells were fixed and indirect immunofluorescence staining was performed. Middle panel, cells were treated with 20 nM LMB for 2 h before the methanol fixation. Expression of hTERT-HA (colored red by rhodamine) and FLAG-14-3-3Θ-ΔN (colored green by fluorescein) were detected. (B) Effects of LMB and the nes mutation on the 3A mutation-derived cytoplasmic localization of hTERT. Human foreskin fibroblasts were transfected with pCR3–hTERT-HA (wt), pCR3–hTERT-3A-HA (3A) or pCR3–hTERTnes-3A-HA (nes-3A). After 24 h of transfection, cells were fixed and indirect immunofluorescence staining was performed. Expression of each hTERT-HA construct (colored red by rhodamine) was detected. The percentage of cells with punctate accumulation of hTERT in the nuclei were 72% (31 of 43, wt), 33% (43 of 131, 3A), 60% (62 of 103, 3A + LMB), and 55% (78 of 143, nes-3A), respectively. (C) hTERT–CRM1 interaction in intact cells. 293T Cells were transfected with pCR3–hTERT-HA (wt) or pCR3–hTERT-3A-HA (3A). Cellular lysates were prepared, and immunoprecipitation followed by western blot analysis was performed. Download figure Download PowerPoint These results prompted us to examine further the functional significance of the NES-like motif in hTERT. We introduced the nes mutation (Figure 5A) into hTERT-HA and examined the intracellular localization of the resulting mutant, hTERTnes-HA. In contrast to the wild-type hTERT-HA, 14-3-3Θ-ΔN failed to redistribute hTERTnes-HA into the cytoplasm of 293T cells. Nuclear localization of hTERTnes-HA was observed in 87% (26 of