Research ArticleCancer

Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation

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Science Signaling  16 Jun 2015:
Vol. 8, Issue 381, pp. ra58
DOI: 10.1126/scisignal.aaa4998


During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell–derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.


Human telomerase is an RNA-dependent DNA polymerase that contains a catalytic component, human telomerase reverse transcriptase (hTERT), and an internal RNA template, telomerase RNA (TR) (1, 2). Telomerase extends the ends of chromosomes and protects telomeres from replication-dependent attrition, enabling cancer cells to proliferate indefinitely by overcoming the end replication problem (35). Telomerase is overexpressed in >80% of all cancer types (6, 7). Inhibition of telomerase leads to telomere damage, subsequent senescence, and tumor suppression (811). Lamins are key structural components of the nuclear lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane, attaching chromatin domains to the nuclear periphery and localizing some nuclear envelope proteins. Fibroblasts obtained from lamin B1 mutant mouse embryos displayed premature senescence (12). In budding yeast, telomeres are reversibly bound to the nuclear envelope, and small ubiquitin-like modifier protein (SUMO)–dependent association with the nuclear periphery was proposed to restrain bound telomerase (13). Phosphorylation of hTERT increases its stability, and protein phosphatase 2 (PP2A)–dependent dephosphorylation of hTERT inhibits telomerase function (14).

The bioactive sphingolipids ceramide and sphingosine 1-phosphate (S1P) exert opposing functions: ceramide is emerging as a tumor suppressor molecule, whereas S1P promotes tumor growth (1519). Ceramide inhibits hTERT expression by inducing histone deacetylase 1 (HDAC1)–dependent deacetylation of Sp3 (an Sp1 family transcription factor), which represses hTERT promoter function (20). S1P is generated by cytoplasmic sphingosine kinase 1 (SK1) or nuclear SK2 (21, 22). S1P generated by SK1 promotes tumor growth and metastasis (2325). SK1-generated intracellular S1P binds and promotes tumor necrosis factor receptor–associated factor 2 (TRAF2)–dependent nuclear factor κB (NFκB) signaling (21). SK2-generated nuclear S1P directly binds and inhibits HDAC1 and HDAC2 (22). SK2-generated S1P binding also induces prohibitin 2 activity, leading to cytochrome c oxidase assembly and mitochondrial respiration (26). Considering S1P in the context of telomerase, we investigated how the binding of SK2-generated S1P alters hTERT abundance and the function of telomerase.


SK2-generated S1P promotes hTERT stability

To examine the possible roles of S1P in the regulation of hTERT, we determined whether down-regulation of SK1 or SK2 affected hTERT abundance or stability in human lung cancer cells. Small interfering RNA (siRNA)–mediated knockdown of SK2, but not SK1, decreased hTERT protein abundance without affecting that of its mRNA in various human lung cancer cell lines (Fig. 1A and fig. S1, A and B). Compared with controls, stable knockdown of SK2 using one of two short hairpin RNAs (shRNAs) targeting distinct sequences decreased the abundance of hTERT in H1299 and H1650 cells (fig. S1, C and D) and hTERT stability in A549 cells treated with cycloheximide (CHX) (fig. S1, E and F). These data suggested that SK2 promotes hTERT abundance and protein stability.

Fig. 1 SK2-generated S1P regulates hTERT protein abundance and stability.

(A) Endogenous hTERT protein abundance in A549 cells transfected with SK1 or SK2 siRNA. Scr, scrambled. (B) Immunoprecipitation for FLAG and Western blotting for hTERT in lysates from wild-type (WT), SK1-deficient, or SK2-deficient MEFs expressing FLAG-hTERT or vector (Vec) in the absence or presence of CHX (50 μg/ml; 0 to 4 hours). A densitometry from three blots shown below. (C) Quantification of FLAG pull-down and Western blotting for hTERT in lysates from SK2-deficient MEFs cotransfected with hTERTWT-FLAG and either SK2WT or SK2G212E with or without CHX for 4 hours. a.u., arbitrary units. (D) Effects of ABC294640 (ABC) on 17C-S1P formation in the nuclear (Nuc) and cytoplasmic (Cyto) fractions of A549 measured by liquid chromatography and mass spectrometry. Veh, vehicle. (E) Cytoplasmic and nuclear fractionation of A549 cells were performed after treatment with ABC294640 and analyzed by Western blotting with antibodies against lamin B and calnexin nuclear and cytoplasmic markers, respectively. (F and G) Effects of ABC294640 treatment (80 μM) on hTERT abundance in A549 (F) or H157 and H1650 cells (G) at 0 to 8 hours as measured by Western blotting. In all panels, blots are representative of three independent experiments, and data are means ± SD from three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

Like the effects of SK2 knockdown, genetic loss of SK2 promoted the degradation of hTERT protein. In the presence of CHX, ectopically expressed FLAG-tagged hTERT showed decreased protein stability in mouse embryonic fibroblasts (MEFs) from mice lacking SK2 compared to those from the wild type or those lacking SK1 (Fig. 1B). Ectopic expression of V5-tagged wild-type SK2 (V5-SK2WT), but not the catalytically inactive mutant (V5-SK2G212E) (fig. S1G), prevented the degradation of hTERT-FLAG in SK2-deficient MEFs (Fig. 1C). Pharmacological inhibition of SK2 with ABC294640 (27) reduced the nuclear, but not the cytoplasmic, abundance of 17C-S1P, an analog of S1P that contains 17 carbons (28), after labeling cells with 17C-sphingosine, which was used as a substrate in cells (Fig. 1D and fig. S1I). Calnexin and lamin B abundances were measured as cytoplasmic and nuclear markers, respectively (Fig. 1E). Likewise, ABC294640 decreased hTERT abundance in A549, H157, and H1650 cells (Fig. 1, F and G, and fig. S1H). Together, these data so far suggest that knockdown or inhibition of nuclear SK2-generated S1P, but not SK1-generated S1P, results in decreased hTERT stability.

S1P selectively binds hTERT involving the C′3-OH group of S1P

Lipid-protein associations regulate the function and stability of various proteins (22, 29, 30). Thus, we examined whether S1P directly binds and stabilizes hTERT. To determine whether hTERT associates with endogenous S1P, we ectopically expressed wild-type FLAG-tagged hTERT (hTERTWT-FLAG) in A549 cells and measured 17C-S1P binding by immunoprecipitation for FLAG followed by lipid extraction and liquid chromatography and mass spectrometry (fig. S1J). We observed that hTERTWT-FLAG was significantly associated with endogenously generated 17C-S1P compared to the vector control (Fig. 2A and fig. S1J).

Fig. 2 SK2-generated S1P interacts with hTERT by lipid-protein binding.

(A) Ectopic expression of hTERTWT-FLAG in A549 cells (right) treated with 17C-sphingosine was measured by immunoprecipitation for the FLAG tag and assessed for bound 17C-S1P (data in fig. S1J). (B) Overlay of the hTERT model with the crystal structure of tcTERT. Distinct domains of TERT are shown in color, and the S1P interacting residue Asp684 located at the interface of the palm and thumb domains is in red stick. TRBD,TERT RBD. (C and D) Binding of biotinylated S1P (B-S1P) to FLAG-tagged WT or mutant hTERT in A549 cells (C) or GM00847 cells (D) assessed by avidin bead pull-down and Western blotting for hTERT. (E) In vitro binding of tcTERT to either S1P [POPC/POPE/S1P (70:20:10)] or LPA [POPC/POPE/LPA (70:20:10)] vesicles relative to the control [POPC/POPE (80:20)]. (F) Kd analysis of tcTERT interaction with S1P vesicles. (G and H) In vitro binding of biotin-tagged S1P to FLAG-tagged WT or D684A mutant hTERT (0.4 mg/ml) partially purified from A549 cells. In all panels, data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

To uncover the structural details of the association between hTERT and S1P, we performed homology modeling and molecular docking studies using the x-ray structure of Tribolium castaneum TERT (tcTERT) for S1P binding (31). The model was refined using amino acid alignment between tcTERT and hTERT (isoform 2, referred herein as hTERT) containing possible putative S1P-binding domains with at least two hydrophobic pockets that do not interfere with DNA binding and telomerase activity (fig. S2A). We then analyzed our results from the hTERT protein docking simulations using S1P as a ligand. The data suggested that the Asp684 residue of hTERT, which is present in hTERT2 but not in hTERT1, might be involved in S1P binding (Fig. 2B and fig. S2B), possibly interacting with the C′3-OH of S1P. To test this model, we incubated lysates from A549 cells overexpressing hTERT with increasing concentrations of biotinylated S1P in the presence or absence of nonlabeled stereoisomers of S1P (d-erythro-S1P, d-erythro-3-O-CH3-S1P, and l-erythro-S1P) (fig. S2C). Biotinylated S1P–bound proteins were captured by avidin-conjugated agarose beads and examined by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with an antibody against hTERT. Biotinylated S1P bound hTERT effectively compared to biotin control, and the presence of nonlabeled d-erythro-S1P, but not l-erythro-S1P or d-erythro-3-O-CH3-S1P, prevented the interaction between hTERT and biotinylated S1P (fig. S2C). Incubation of A549 cell extracts with biotinylated lysophosphatidic acid, which is structurally similar to S1P but lacks the C′3-OH (fig. S2D), did not result in any detectable hTERT binding compared to biotinylated S1P (fig. S2D). Binding between biotinylated S1P and hTERT was also detected in H157 cells (fig. S2E). Thus, these data suggest that hTERT associates with S1P in vitro and in cells with high selectivity for the C3-OH of S1P.

A hydrophobic region of hTERT that includes Asp684 is key for S1P binding

To examine whether the Asp684 residue is involved in S1P binding, we used a mutant hTERT with D684A conversion (hTERTD684A-FLAG), which retains telomerase activity (32) comparable to hTERTWT-FLAG as measured by the polymerase chain reaction (PCR)–based telomeric repeat amplification protocol (TRAP) assay (fig. S2F). Then, we examined the binding of biotinylated S1P to hTERTWT-FLAG or hTERTD684A-FLAG expressed in A549 cells. The D684A mutation prevented the binding of hTERT to biotinylated S1P compared to either hTERTWT-FLAG or another mutant of hTERT (hTERTR669A-FLAG) in which the mutation is in close proximity to Asp684 but was not predicted to interfere with S1P binding (Fig. 2C). To control for endogenous hTERT, we expressed hTERTWT-FLAG or hTERTD684A-FLAG in GM00847 fibroblasts that lack endogenous hTERT. hTERTWT-FLAG, but not hTERTD684A-FLAG, associated with biotinylated-S1P (Fig. 2D). Overall, these data suggest—as predicted by our molecular modeling—that a hydrophobic pocket, localized between the thumb and finger domains and involving the Asp684 residue of hTERT, plays a key role in S1P binding to the C3-OH of S1P.

S1P directly binds tcTERT and hTERT in vitro and in transfected cells

To quantify the interaction between S1P and TERT, we performed surface plasmon resonance (SPR) using S1P- or lysophosphatidic acid (LPA)–containing lipid vesicles and purified recombinant tcTERT protein. Although tcTERT does not contain the Asp684 residue, our modeling studies suggested that both tcTERT and hTERT have similar hydrophobic pockets that might be involved in S1P binding (Fig. 2B). Purified tcTERT was injected at increasing concentrations to detect binding to S1P or LPA vesicles [containing the membrane phospholipids PC (phosphatidylcholine) and PE (phosphatidylethanolamine) and either S1P or LPA at a ratio of 70:20:10] or control vesicles [containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) at a ratio of 80:20], as previously described (29, 33). Purified recombinant tcTERT bound the S1P vesicles in a concentration-dependent manner, whereas tcTERT did not bind the LPA vesicles (Fig. 2E). The dissociation constant (Kd) for tcTERT was ~4.8 μM for the S1P vesicles (Fig. 2F). Thus, these data show the direct binding between S1P and tcTERT in vitro.

To quantify the interaction between S1P and hTERT (which, unlike tcTERT, contains the integral Asp684 residue), we expressed hTERTWT-FLAG and hTERTD684A-FLAG in A549 cells and partially purified hTERT using agarose beads recognizing FLAG. Partially purified hTERT was incubated with biotinylated S1P with or without increasing concentrations of unlabeled S1P. Bound biotinylated S1P was measured using a biotin-sensitive enzyme-linked immunosorbent assay (ELISA). The Kd for hTERTWT-FLAG was ~430 nM, whereas binding between biotinylated S1P and the hTERTD684A-FLAG was undetectable in the presence of 0.1 to 10 μM S1P (Fig. 2, G and H). The stoichiometry was calculated as 1:1 for S1P and hTERT. Collectively, these data suggest that S1P-TERT binding involves the hydrophobic domain between the thumb and finger domains of tcTERT and hTERT and that the Asp684 residue of hTERT is essential for S1P binding. Using SPR, we also predicted that S1P may bind tcTERT in vitro at the conserved hydrophobic domain. However, this binding required much higher concentrations of purified tcTERT because the Asp684 residue is not conserved in tcTERT.

SK2-generated S1P binds hTERT in cells

To determine whether the association between hTERT and S1P is physiologically relevant, we measured their interaction by proximity ligation assay (PLA) using antibodies that recognize S1P (34) and hTERT, respectively. DAPI (4′,6-diamidino-2-phenylindole) staining was used to visualize nuclei. The association between S1P and hTERT was detected in the nuclei of A549 cells transfected with scrambled control siRNA but not in those transfected with siRNA against SK2 (Fig. 3A). Inhibition of SK2 with ABC294640 attenuated the S1P-hTERT association in A549 cells (Fig. 3A). Ectopic expression of hTERTWT-FLAG, but not hTERTD684A-FLAG, resulted in a 4.5-fold increase in S1P binding compared to vector-transfected GM00847 cells (Fig. 3B). Interactions between S1P and other nuclear proteins, such as HDAC3 or SET [Su(var), Enhancer-of-zeste, Trithorax domain–containing oncoprotein], used as negative controls, were undetectable in A549 cells by PLA (Fig. 3C).

Fig. 3 Interaction of hTERT with SK2-generated S1P colocalizes with lamin B at the nuclear periphery.

(A) PLA detection of the subcellular localization of the S1P-hTERT interaction in A549 cells transfected with either control (Scr) or SK2 siRNA (upper) or treated with vehicle or ABC294640 (lower). Nuclei were counterstained with DAPI. Scale bars, 20 μm. DMSO, dimethyl sulfoxide. (B) PLA detection of S1P-hTERT binding in GM00847 cells transfected with vector, hTERTWT-FLAG, or hTERTD684A-FLAG. (C) PLA for S1P binding to hTERT compared to HDAC3 or SET (nuclear proteins) in A549 cells. (D and E) Colocalization of S1P (red) and lamin B (green) in the nucleus as assessed by immunofluorescence confocal microscopy (IF-CM) in WT or SK2-deficient MEFs (D) or A549 cells transfected with control (Scr) or SK2 shRNA (E). Scale bars, 100 μm. In all panels, images are representative of three independent experiments, and data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

S1P or hTERT colocalizes with a nuclear periphery protein, lamin B

To demonstrate the nuclear selectivity of the hTERT-S1P interaction, we examined the colocalization of S1P with the nuclear membrane marker lamin B in wild-type or SK2-deficient MEFs or A549 cells using immunofluorescence microscopy. S1P was colocalized with lamin B in wild-type or control-transfected cells, respectively, but S1P was too diminished in SK2-deficient cells to detect abundance at the nuclear periphery (Fig. 3, D and E). Likewise, in transfected A549 cells, hTERTWT-FLAG, but not hTERTD684A-FLAG, colocalized with lamin B in the nucleus (fig. S3). Thus, these data suggest that in the presence of SK2 and its product S1P, wild-type, but not mutant, hTERT localizes to the nuclear periphery.

S1P binding protects hTERT from ubiquitination and proteasomal degradation

To define whether binding by SK2-generated S1P affects hTERT stability by inhibiting ubiquitin-mediated proteasomal degradation, we treated A549 cells with the SK2 inhibitor ABC294640 alone or combined with the proteasome inhibitor lactacystin. Indeed, lactacystin prevented ABC294640-induced suppression of hTERT abundance (Fig. 4A). Likewise, exogenous S1P also prevented the ABC294640-induced loss of hTERT (Fig. 4A). Moreover, in A549 cells, ABC294640 induced the ubiquitination of endogenous hTERT (Fig. 4B and fig. S4A) and increased the amount of hemagglutinin (HA)–tagged ubiquitin that had conjugated to exogenous hTERTWT-FLAG regardless of lactacystin treatment (Fig. 4C and fig. S4B). Likewise, in wild-type MEFs, the stability of ectopically expressed mutant hTERT (hTERTD684A-FLAG) was less than that of the wild-type construct in the presence of CHX, an inhibitor of de novo translation (Fig. 4D). However, inhibition of proteasome activity by MG132 (N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal) stabilized the abundance of hTERTD684A-FLAG (Fig. 4D). Accordingly, although hTERTWT-FLAG was stable in wild-type MEFs (Fig. 4E), its abundance was decreased in SK2-deficient MEFs when treated with CHX, unless simultaneously treated with MG132 (Fig. 4F). Equal transfection efficiency for ectopic expression of wild-type and mutant hTERT without CHX and MG132 exposure in wild-type and SK2-deficient MEFs was measured by Western blotting (fig. S4, C and D). Together, these data suggest that hTERT is degraded through the proteasomal degradation pathway, particularly in the absence of SK2.

Fig. 4 S1P-hTERT binding prevents the ubiquitination and proteasomal degradation of hTERT.

(A) Western blotting for hTERT to assess protein stability in A549 cells pretreated with S1P, lactacystin, or vehicle followed by the SK2 inhibitor ABC294640. (B and C) Pull-down for hTERT and Western blotting for ubiquitin (Ub) showing the effects of ABC294640 on the ubiquitination of endogenous hTERT in A549 cells (B) or of FLAG-hTERT in the presence of the protease inhibitor lactacystin in A549 cells expressing HA-Ub (C). (D) Pull-down for FLAG and Western blotting for hTERT showing the stability of FLAG-tagged WT or mutant (D684A) hTERT in the presence or absence of CHX or MG132 in WT MEFs. (E and F) Pull-down for FLAG and Western blotting for hTERT showing the stability of FLAG-hTERTWT expressed in WT (E) or SK2-deficient MEFs (F) in the presence of CHX alone or with MG132. In all panels, blots are representative of three independent experiments, and data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

S1P-hTERT binding prevents MKRN1-mediated degradation of hTERT

The E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1) interacts with the C terminus (residues 946 to 1132) of hTERT (35), a region that also contains Gly932, Cys931, Phe986, His983, and Ser984 residues and is located within the putative S1P-binding domain of hTERT (Fig. 2B and fig. S2B), suggesting that S1P-hTERT binding may disrupt MKRN1 binding to hTERT. As observed above, knocking down SK2 or inhibiting it with ABC294640 decreased hTERT stability in A549 cells, but simultaneously knocking down MKRN1 (Fig. 5A) almost completely prevented the degradation of hTERT in the presence of CHX (Fig. 5, B and C). Expressing a wild-type (MKRN1WT) but not a catalytically inactive RING domain mutant construct of MKRN1 (MKRN1H307E) increased the ubiquitination of hTERT in the presence of MG132 (Fig. 5D), suggesting that MKRN1 ubiquitinates hTERT. In wild-type MEFs, FLAG-tagged hTERT coimmunoprecipitated with V5-tagged MKRN1 but was decreased in the presence of exogenous S1P (Fig. 5E and fig. S4E). In contrast, the addition of C3-O-CH3-S1P (d-erythro-3-O-methyl S1P, an analog of S1P which lacks C′3-OH), which does not bind hTERT, did not inhibit the interaction between hTERT and MKRN1 (Fig. 5F and fig. S4E). The association between MKRN1 and the S1P-binding mutant hTERTD684A-FLAG was also detectable, and as expected, neither the presence of S1P nor of C3-O-CH3-S1P had any effect (Fig. 5F and fig. S4E). Furthermore, siRNA-mediated reduction of MKRN1 abundance blunted the suppression of A549 cell growth in soft agar that was induced by SK2 knockdown (Fig. 5G). Together, these data indicate that SK2-generated S1P promotes hTERT stability and subsequent cell proliferation by inhibiting the interaction of hTERT with and ubiquitination by MKRN1 and, hence, preventing its subsequent degradation.

Fig. 5 Binding of SK2-generated S1P protects hTERT from MKRN1-mediated degradation.

(A) MKRN1 knockdown in A549 cells stably transfected with control (Scr) or SK2 shRNA. (B and C) Western blotting for endogenous hTERT stability after MKRN1 knockdown in stable shScr and shSK2 A549 cells treated with CHX (B) or ABC294640 (C) for 4 hours. In (C), bottom blot is actin loading control. (D) Pull-down for FLAG and Western blotting for ubiquitin in WT MEFs coexpressing FLAG-hTERT and either WT or RING mutant (H307E) MKRN1 and pretreated with MG132 for 2 hours. (E) Coimmunoprecipitation of FLAG-hTERT with V5-MKRN1 in A549 cell extracts in the presence of S1P or LPA (5 μM). Samples shown are from the same representative blot but not in contiguous lanes. (F) Coimmunoprecipitation of FLAG-tagged WT or mutant (D684A) hTERT with MKRN1 in A549 cell extracts in the presence of S1P or C3-O-CH3-S1P. (G) Effect of siRNA-mediated MKRN1 knockdown on the anchorage-independent growth on soft agar exhibited by A549 cells stably transfected with SK2 or control (Scr) shRNA. In all panels, blots are representative of three independent experiments, and data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

S1P-hTERT binding regulates endogenous MKRN1-hTERT interaction

We then determined the effects of S1P produced by SK2 on the endogenous hTERT-MKRN1 association. We expressed wild-type or a catalytically inactive mutant of SK2 (SK2G212E) in A549 and H1650 cells and assessed the interaction between hTERT and MKRN1 by immunoprecipitation in the presence or absence of geldanamycin (GA), which induces MKRN1-dependent hTERT degradation (35). Ectopic expression of wild-type, but not mutant, SK2 prevented the GA-induced interaction between endogenous hTERT and MKRN1 (Fig. 6A and fig. S5, A and B). In primary normal human lung fibroblasts (NHLFs), we stably expressed vector or SK2WT and measured hTERT-MKRN1 association using PLA. In vector-transfected controls, there was a high degree of hTERT-MKRN1 association, which was primarily detected in the cytoplasm, and overexpression of SK2WT (Fig. 5, C and D) almost completely abrogated this interaction (fig. S5C). In addition, expression of wild-type, but not mutant, SK2 prevented the GA-induced inhibition of the growth of A549 and H1650 cells on soft agar (Fig. 6B and fig. S5E). Thus, these data demonstrate that SK2-generated S1P inhibits the association between endogenous hTERT and MKRN1.

Fig. 6 SK2-generated S1P prevents hTERT-MKRN1 interaction by mimicking hTERT phosphorylation at Ser921.

(A and B) Effects of WT or catalytically inactive (G212E) SK2 on endogenous hTERT-MKRN1 interaction (A) and proliferation on soft agar (B) in A549 cells, either untreated or treated with GA. (C) Colocalization of FLAG-tagged WT or mutant hTERT (red) with lamin B (green) detected by IF-CM in GM00847 cells. Scale bars, 20 μm. (D) PLA detection of the interaction between MKRN1 and FLAG-tagged WT or mutant hTERT in GM00847 cells. (E) Western blotting to assess the stability of WT or mutant hTERT in the presence or absence of CHX. In all panels, images are representative of three independent experiments, and data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

S1P binding at Asp684 mimics phosphorylation of hTERT at Ser921

To determine how S1P binding protects hTERT from MKRN1-mediated proteasomal degradation, we compared the stabilizing effect of S1P binding to that of the known phosphorylation modification (14). Molecular modeling and simulation data suggested that binding of S1P to hTERT at the Asp684 residue might mimic the phosphorylation of hTERT at Ser921 (fig. S6, A and B). This was tested in experiments in which S921A (a nonphosphorylatable mutation) or S921D (a phosphomimetic mutation) conversions were introduced into the D684A mutant of hTERT to generate hTERTD684A-S921A-FLAG and hTERTD684A-S921D-FLAG double mutants. The colocalization of the double mutants with lamin B at the nuclear periphery, their association with MKRN1, and their stability were assessed in comparison to that of the wild-type construct (hTERTWT-FLAG) in GM00847 cells. hTERTWT-FLAG, but not hTERTD684A-FLAG, was localized to the nuclear periphery, as visualized by confocal microscopy and immunofluorescence. Although hTERTD684A-S921A-FLAG was mainly cytoplasmic, localization of hTERT in the lamin B–containing nuclear periphery was restored in cells expressing the phosphomimetic and S1P binding–defective hTERTD684A-S921D-FLAG (Fig. 6C). Moreover, hTERTS921A-FLAG, which contains intact S1P binding at Asp684, was also localized mainly in the nucleus (Fig. 6C). The MKRN1-hTERT association (measured by PLA) was enhanced in cells expressing hTERTD684A-FLAG but was prevented by expression of hTERTD684A-S921D-FLAG (Fig. 6D). These data are consistent with the restoration of hTERT stability that we observed after the expression of the phosphomimetic mutant, hTERTS921D-FLAG, compared to either hTERTD684A-S921A-FLAG or hTERTD684A-FLAG (Fig. 6E). Collectively, these data suggest that Ser921 phosphorylation mimetic restores hTERT stability in the absence of S1P binding in cells expressing hTERTD684A-FLAG. These studies also suggest that lipid binding of hTERT by S1P allosterically mimics hTERT phosphorylation at its C terminus including Ser921, preventing hTERT-MKRN1 association and stabilizing hTERT at the nuclear periphery.

Nuclear S1P-hTERT binding prevents telomere damage and delays senescence

We then determined whether S1P-hTERT binding has any impact on telomere damage, dysfunction, or senescence—key biological processes counteracted by telomerase. Control NHLFs became positive for β-galactosidase (β-gal), a cell senescence marker, after ~10 passages in culture. Expressing SK2WT delayed the emergence of senescence up to passage 14, but concomitantly knocking down hTERT restored senescence on the basis of β-gal positivity at passage 10 (Fig. 7, A and B, and fig. S7, A to C). We then assessed telomere integrity in these cells by detecting the complex between phosphorylated histone H2AX (γ-H2AX) and telomeric repeat–binding factor 2 (TRF2), which is recruited to damaged or dysfunctional telomeres (36, 37), using a telomere dysfunction–induced foci (TIF) assay. Ectopic SK2 expression prevented telomere dysfunction compared to vector-transfected controls, and TERT knockdown reestablished telomere damage (Fig. 7B and fig. S7D).

Fig. 7 SK2-generated S1P-hTERT plays key roles in the control of senescence, telomere damage, and tumor growth.

(A) β-Gal staining in primary lung fibroblasts (PLFs) [at passages 7 (P7) to P17] that stably coexpress pCDH and control shRNA (shScr) or SK2 and either shScr or shTERT. (B) Telomere damage assessed by the TIF assay (γ-H2AX and TRF2 colocalization) in PLFs at P12, cotransfected as in (A). (C) β-Gal staining in SK2-deficient or WT MEFs at P5. Positive cells were counted from three to four fields. (D and E) Telomere damage as assessed by the TIF assay (D) and senescence by β-gal detection (E) in SK2-deficient MEFs transfected with WT or mutant (D684A) hTERT. (F) Volumes of allograft tumors derived from LLC cells stably transfected with shScr or shSK2 in the flanks of age-matched WT or SK2-deficient mice. (G) Volumes of xenograft tumors derived from A549 cells stably transfected with vector, hTERTWT, or hTERTD684A in mice treated with vehicle or ABC294640 for 21 days. In panels (A) to (E), data are means ± SD from three independent experiments; in (F) and (G), data are means ± SD from four mice each, with each mouse containing two tumors. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. (H) A model of our findings, revealing a nuclear lysophospholipid-mediated mechanism through which S1P binding to a pin-pointed region in hTERT functions as an allosteric phosphomimetic and stabilizes hTERT. Various ways to reduce S1P abundance or binding may induce the degradation of hTERT and, in turn, the acceleration of telomere damage and senescence in tumors.

Accordingly, primary SK2-deficient MEFs became senescent at passage 5, whereas senescence in their age-matched wild-type or SK1-deficient MEFs was not observed until passage 7 (Fig. 7C). Reconstitution of hTERTWT, but not hTERTD684A, prevented telomere damage (Fig. 7D) and delayed senescence (Fig. 7E and fig. S8A) in SK2-deficient MEFs. Telomere restriction fragment (TRF) length measurement by Southern blotting showed no detectable differences in telomere lengths in wild-type or SK2-deficient MEFs at passages 3 and 6 (fig. S8B). As controls, we used genomic DNA isolated from subcutaneous A549 xenografts expressing either vector or hTERTWT isolated from immunocompromised mice after 28 days of growth. Ectopic expression of hTERT lengthened telomeres in the isolated tumor cells (fig. S8C). Collectively, these data suggest that SK2 delays senescence and promotes telomere maintenance through its activity on hTERT.

SK2-generated S1P promotes hTERT stability and tumor growth

Because cellular senescence plays key roles in tumor suppression (3840), we determined the effects of systemic versus cellular S1P produced by SK2 on the regulation of tumor growth. We measured the enlargement of Lewis lung carcinoma (LLC) allograft–derived tumors in wild-type and SK2-deficient mice. Knocking down SK2 in LLC cells before implantation almost completely inhibited tumor growth compared to controls (Fig. 7F). Systemic loss of SK2 through whole-body deletion decreased the growth of control LLC allografts by ~50% compared to wild-type mice (Fig. 7F), without affecting murine TERT abundance. These data indicate that molecular targeting of tumors, rather than systemic SK2, inhibits TERT expression and suppresses tumor growth in vivo, but that systemic SK2 inhibition seems to play a role in the regulation of tumor growth by an independent, unknown mechanism.

Finally, we measured the effects of stable expression of hTERTWT or hTERTD684A on ABC294640-mediated inhibition of A549 cell–derived xenograft tumor growth in immunocompromised mice. hTERTWT conferred resistance to ABC294640-mediated tumor suppression when compared to vector-transfected controls (Fig. 7G). However, expression of hTERTD684A had no effect on ABC294640-mediated tumor suppression compared to controls (Fig. 7G). Overall, these results suggest that pharmacologically targeting SK2 in the tumor or inhibiting S1P-hTERT binding in cancer cells may suppress tumor growth.


Here, our data revealed that SK2-generated nuclear S1P directly binds hTERT and regulates hTERT stability in the lamin B–positive nuclear periphery by inhibiting MKRN1-dependent hTERT ubiquitination and degradation. Binding of hTERT by S1P appears to function as an allosteric phosphomimetic of hTERTS921 at its C terminus including Ser921, which prevents MKRN1-hTERT association, stabilizing hTERT at the nuclear periphery. Nuclear S1P-hTERT binding plays important biological functions regulated by telomerase, such as protection of cells from telomere damage, delaying senescence and preventing tumor suppression. Accordingly, SK2 inhibition or prevention of S1P-hTERT binding accelerated senescence and telomere damage and suppressed tumor growth and proliferation (Fig. 7H).

Protein stability of hTERT is regulated by a direct and selective interaction between the C′3-OH of S1P and the Asp684 residue of hTERT, which is predicted to localize within a hydrophobic region between the thumb and finger domains of hTERT. The binding of S1P to purified recombinant tcTERT (31) revealed that S1P directly binds TERT in vitro. In cells, the binding of nuclear S1P to hTERT prevented MKRN1-hTERT interaction, modulating hTERT ubiquitination and degradation. Many signaling proteins such as oncogenic RAS, hedgehog (HH), and autophagy-related Atg8 family proteins are covalently modified with sphingolipids (30, 41) or phospholipids, which is key for their correct protein localization and function in cells (4244). Recently, a nuclear phospholipid, phosphatidylinositol 5-phosphate (PI5P), was shown to bind and activate UHRF1 (ubiquitin-like with PHD and RING finger domains 1), a nuclear factor that maintains DNA methylation patterns during replication (45). Our data demonstrated that S1P binding prevents the association between MKRN1 and hTERT, thereby promoting the stability of hTERT. Because MKRN1 interacts with the C terminus (residues 946 to 1132) of hTERT (35), lipid binding of hTERT by S1P, mimicking the phosphorylation status at its C terminus, including Ser921, seemed to interfere physically with MKRN1-hTERT association. We believe that this is the initial discovery for a function of protein-lipid binding (hTERT binding by S1P) as an allosteric phosphomimetic to regulate a protein-protein (hTERT-MKRN1) interaction and to control hTERT stability in the nuclear periphery, leading to delayed senescence.

S1P binding–dependent hTERT stabilization altered senescence by protecting against telomere damage in primary human lung fibroblasts and wild-type MEFs, whereas SK2 loss in MEFs increased telomere damage and accelerated senescence. Furthermore, hTERTWT, and not hTERTD684A, protected SK2-deficient MEFs from telomere damage and delayed senescence, which seemed to be independent of telomere length control. These data are intriguing because senescence in MEFs might not be regulated solely by telomere shortening; rather, senescence in MEFs might be controlled at least in part by induction of telomere damage. However, our inability to detect any changes of telomere length in wild-type versus SK2-deficient MEFs might be due to their extra long telomeres (>21 kb), which are difficult to measure accurately using Southern blotting. It is also possible that noncanonical roles for S1P-bound hTERT (isomer 2) might play a role in regulating telomere damage or senescence without affecting telomere length. This, however, is unclear and needs further investigation. SK2-deficient mice do not exhibit any detectable obvious phenotype due to telomerase instability or accelerated senescence. This is consistent with TERT-deficient mice, which do not show any aging phenotype for the first three generations (G1 to G3) and have phenotypic changes starting at G4 through G6 because of the particularly long telomeres in these animals (46). It is known that even partially reduced expression of telomerase has profound effects on telomere maintenance because mice heterozygous for mTERT (mouse TERT) or mTERC (mouse telomerase RNA component) show haploinsufficiency in telomere maintenance (47). These data are consistent with our studies in which mTERT/hTERT instability in response to genetic loss or molecular knockdown of SK2 resulted in accelerated telomere dysfunction, senescence, and tumor suppression.

Overall, these data suggest that lipid binding of hTERT by S1P allosterically mimics hTERT phosphorylation at Ser921, which then prevents hTERT-MKRN1-E3 ubiquitin ligase association, enhancing hTERT stability at the nuclear periphery. Increased hTERT stability at the nuclear periphery by S1P binding has important biological implications for the regulation of telomerase-dependent control of telomere damage and cellular senescence, key processes involved in aging and cancer biology. It is possible that this novel nuclear signaling mechanism mediated by interaction with S1P might regulate not only telomerase but also other proteins with diverse biological functions through protein phosphorylation mimicry.


Cell lines and culture conditions

A549, H157, and H1650 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro) with 10% fetal bovine serum (FBS) (Atlanta Biologics) and 1% penicillin and streptomycin (Cellgro). H1341 small cell lung cancer cells were cultured in RPMI-1640 (American Type Culture Collection) with 10% FBS and 1% penicillin and streptomycin. GM00847 cells were purchased from Coriell Cell Repositories. Wild-type, SK1-deficient, and SK2-deficient MEFs were obtained from K. Argraves (Medical University of South Carolina). GM00847 cells and MEFs were cultured in DMEM and incubated at 37°C with 5% CO2. Primary hNHLFs were purchased from Lonza Group and cultured in fibroblast growth medium as described by the manufacturer.


Plasmids containing hTERTWT and hTERTD684A in pcDNA vectors were obtained from J. Chen (Arizona State University). MKRN1WT and MKRN1H307E plasmids were obtained from M. T. Muller (University of Central Florida). SK2WT and SK2G212E plasmids were obtained from S. Spiegel (Virginia Commonwealth University). pBABE-puro (plasmid #1764), pBABE-puro-hTERT (plasmid #1771), pMD2.G (plasmid #12259), and psPAX2 (plasmid #12260) were purchased from Addgene.

Quantitative PCR

Total RNA isolation was performed using RNeasy (Qiagen), and 1 μg of total RNA was used for complementary DNA (cDNA) synthesis using the iScript cDNA synthesis kit (Bio-Rad). TaqMan probes (hTERT, Hs00162669; SK1, Hs00184211; SK2, Hs00219999; and MKRN1, Hs00831972) were used for quantitative PCR (Life Technologies).


The antibodies used for Western blotting in this study were as follows: hTERT (1531-1, clone Y182, Epitomics), SK2 (ab37977, Abcam), MKRN1 (ab72054, Abcam), rabbit V5 (ab9116, Abcam), ubiquitin (3933S, Cell Signaling Technology), HA (3724, Cell Signaling Technology), calnexin (Sc6465, Santa Cruz Biotechnology), lamin B (Sc6216, Santa Cruz Biotechnology), and mouse V5 (R96025, Invitrogen).

Exogenous S1P treatment

Exogenous S1P (Avanti Polar Lipids) was suspended in fat-free bovine serum albumin (BSA) (4 mg/ml) in 1× phosphate-buffered saline (PBS) (pH 7.4) at 125 μM. S1P was incubated in a sonicator water bath for 5 min followed by incubation at 37° to 55°C for 20 min. d-erythro-C3-O-CH3-S1P and l-erythro-S1P were synthesized at the Lipidomics Shared Resource Facility, Medical University of South Carolina.

Stable shRNA knockdown of SK2

SK2 shRNA (TRCN00000036973)– and nontargeting shRNA–containing plasmids were purchased from Open Biosystems Inc. Cells were cotransfected with pCMV-psPAX2 and pMD2 plasmids in 293T cells using the viral transduction protocol as described by the RNA interference (RNAi) Consortium. The viral supernatants were added to A549 cells, and selection was performed using puromycin (1 μg/ml) for 14 days. The following shRNAs were purchased from Thermo Scientific Inc: shSK2 #1 (TRC0000036973), 5′-CCGGCTACTTCTGCATCTACACCTACTCGAGTAGGTGTAGATGCAGAAGTAGTTTTG-3′; shSK2 #2 (TRCN0000036969), 5′-CCGGGCTTCGTGTCAGATGTGGATACTCGAGTATCCACATCTGACACGAAGCTTTTTG-3′; and shSK2 #3 (TRCN0000036970), 5′-CCGGGTTGCTCAACTGCTCACTGTTCTCGAGAACAGTGAGCAGTTGAGCAACTTTTTG-3′.

Immunoprecipitation and Western blotting

FLAG-hTERT was expressed in cells, which were then treated with CHX (Sigma-Aldrich) (50 μg/ml) for up to 4 hours. The cells were lysed with the FLAG lysis buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100] with protease inhibitor cocktail. Immunoprecipitation was performed using beads conjugated with antibody against FLAG (anti-FLAG M2 affinity gel; A2220, Sigma). Immunoprecipitation and elution of bound proteins with FLAG beads were carried out according to the manufacturer’s instructions. Immunoprecipitation with hTERT antibody (4 μg/ml) was carried out using 250 μg of total protein lysate made up to 500-μl volume with FLAG lysis buffer. Lysates were precleared with 30 μl of protein A/G beads and incubated in a rotary shaker for 1 hour at 4°C. Samples were spun at 1000 rpm for 1 min, and the supernatant was transferred to a new microcentrifuge tube and incubated with an hTERT antibody (4 μg/ml) overnight, followed by the addition of protein A/G beads and incubation at 4°C for 1 hour. Samples were centrifuged at 1000 rpm for 1 min and washed twice with lysis buffer and 1× PBS, followed by the addition of gel loading dye. Samples were boiled using heating block and centrifuged to collect eluted proteins, followed by Western blotting with an antibody against hTERT. A rabbit immunoglobulin G (IgG) control was used alongside hTERT pull-down.

For assays in A549 cells, cells were plated at 200,000 cells per well in a six-well plate for 18 hours before treatment with either DMSO (vehicle control) or ABC294640 (Apogee Inc.) at 80 μM for 8 hours. After treatment, cells were centrifuged at 1300 rpm for 3.5 min and washed with 1× PBS (pH 7.4) (Gibco). Cells were lysed with 1× CHAPS lysis buffer [10 mM tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EDTA, 0.1 mM benzamidine, 5 mM β-mercaptoethanol, 0.5% CHAPS, 10% glycerol] including a protease inhibitor cocktail (Sigma-Aldrich) for 20 min on ice. Cell lysates were centrifuged at 12,000g for 15 min at 4°C, and supernatants were used for Western blotting. SDS-PAGE (4 to 20%) was carried out using the Bio-Rad Criterion apparatus, followed by semi-dry transfer onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk + 0.1% Tween 20 in 1× PBS (pH 7.4). Primary antibodies were used at 1:1000 dilution overnight at 4°C, followed by rabbit or mouse secondary antibodies (Jackson Research Laboratories) conjugated with horseradish peroxidase at room temperature for 1 hour. The rabbit secondary antibody was used at 1:2500 dilution for detecting hTERT and at 1:5000 dilution for the rest of the antibodies. Actin was used as an internal control for Western blotting. The membranes were washed with either 1× PBS with Tween 20 or 1× tris-buffered saline with Tween 20 and developed using ECL Plus chemiluminescence detection kit (GE Healthcare).

17C-Sphingosine labeling and measurement of S1P

A549 cells were treated with 17C-sphingosine (5 μM) for 30 min, and the cytoplasmic and nuclear fractions were analyzed for 17C-S1P formation using liquid chromatography and mass spectrometry, as previously described (28).

Homology modeling of hTERT

Currently, there is no experimental structural information available for hTERT. A model of a complete hTERT-RNA-DNA complex has been published (PMID: 21606328) and was optimized for mechanistic interactions with the RNA template and DNA. The goal of our modeling and simulation studies was to better understand the topology and chemistry of potential TERT lipid interaction sites, not to predict the overall structure or catalytic mechanism of the full-length enzyme complex. Basic Local Alignment Search Tool (BLAST) searching of hTERT (accession number O14746) amino acid sequence against the Protein Data Bank (PDB) database indicated a highly similar sequence from tcTERT containing the RNA-binding domain (RBD) and reverse transcriptase (RT) domain. The hTERT amino acid sequence has 41% coverage and 27% identity to the tcTERT structure. To begin homology modeling, the amino acid sequence from hTERT was aligned with the amino acid sequence of the x-ray crystal structure of tcTERT. Pairwise sequence alignment was performed with BioEdit v7.0 using standard Clustal alignment parameters and the BLOSUM62 matrix. Alignment of tcTERT and hTERT indicated 127 identities and 104 similarities, giving an overall similarity of 31% (231 of 744). Before homology modeling, the tcTERT structure was protonated at pH 7.5 and the structure was energy-minimized, with heavy atoms constrained.

Alignment homology modeling was performed using the Molecular Operating Environment (MOE) Homology Model. Ten models were generated, and the best model was selected using fine-grain intermediates, generalized born/volume integral (GB/VI) scoring, and all atom optimized potential for liquid simulations (OPLS-AA) force field. OPLS was chosen because it tolerates small molecules like S1P better than most other energy fields [Assisted Model Building with Energy Refinement (AMBER), Chemistry at Harvard Macromolecular Mechanics (CHARMM), and Merck Molecular Force Field (MMFF)]. There is a series of short inserts in hTERT that are not present in tcTERT. A large insertion is located right before the RT domain. The insert is a region of low confidence in the predicted model. Using MOE, we refined this region using a loop library. Comparison of the homology models of hTERT with tcTERT revealed that the overall root mean square deviation divergence of the crystal structures from the homology model was 1.27 Å. As expected, the highest structural divergence was centered on the largest insert of hTERT between the RBD and the RT domain.

Molecular docking of hTERT with S1P

To further understand the potential regulatory mechanism of TERT, S1P was docked to hTERT. Before the simulation, S1P chirality was formalized and the molecule was protonated at pH 7.5. Docking was set to probe the entire protein surface. Initial placement calculated 500 poses for S1P using Triangle Matching with London dG scoring. The top 250 poses were then refined using force field–based refinement and alpha sphere and excluded volume (ASE) scoring. Four of the top five poses showed Asp684 within 20 Å of S1P. The large number of poses and the two-stage docking were done to fully explore the entire surface and to place scoring emphasis on the shape and hydrophobicity of the interaction. The best pose was then subjected to bimolecular energy minimization of the S1P-hTERT complex.

Detection of S1P-hTERT binding

A549 cells were lysed in 1× CHAPS lysis buffer. One milligram of total protein was incubated with biotinylated S1P (5 μM). For cold competition assays, nonbiotinylated sphingolipids were preincubated with cell lysates before binding reactions were carried out using biotinylated S1P. Streptavidin beads were added to the reaction mixture, and the assay was performed as described by the manufacturer (Miltenyi Biotec) (29, 30).

A549 cells transfected with the hTERTWT-FLAG or hTERTD684A-FLAG (or empty vector) were lysed by freeze-thawing in a lysis buffer containing 50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40 with a protease inhibitor cocktail (1:500). Cell lysates (400 μg of protein) were incubated with 150 μl of beads conjugated with an antibody against FLAG (Sigma-Aldrich) for 18 hours at 4°C with agitation. Then, the beads were washed and incubated with or without unlabeled S1P in the presence of increasing concentrations of biotinylated S1P in 150 μl of binding buffer containing 50 mM tris (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 15 mM NaF, and 0.5 mM NaV3O4 for 30 min at 30°C. Biotinylated S1P–bound hTERT proteins were eluted with 40 μl of FLAG peptide (250 μg/ml) and quantified using biotin-ELISA (Eagle Biosciences). The results were analyzed using GraphPad Prism 4 software.

Binding of S1P with tcTERT using SPR

SPR was used to measure the kinetics of S1P-tcTERT binding in vitro. Lipid vesicles containing S1P (PC/PE/S1P, 70:20:10), LPA (PC/PE/LPA, 70:20:10), or control vesicles (POPC/POPE, 80:20) were injected with purified tcTERT (250 nM to 6 μM) at a flow rate of 30 μl/min in 10 mM Hepes (pH 7.4) containing 0.16 M KCl. All SPR measurements were performed at 25°C as previously described (29). For each experiment, POPC/POPE (80:20), used as a control surface, and S1P or LPA (10 mol% S1P or LPA) vesicles, used as active surfaces, were injected with tcTERT. Each data set was repeated three times to verify the binding of tcTERT to control or active surfaces.

Quantitative detection of S1P-hTERT binding

A549 cells transfected with FLAG-tagged hTERTWT and hTERTD684A vectors or an empty vector were lysed by freeze-thawing in buffer containing 50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1:500 protease inhibitor cocktail. Lysates (400 μg of protein) were incubated with 150 μl of agarose-conjugated antibodies specific for FLAG (Sigma-Aldrich) at 4°C for 18 hours with agitation. Then, the beads were extensively washed and incubated without or with unlabeled S1P in the presence of increasing concentrations of biotinylated S1P (starting at 250 nM) in 150 μl of binding buffer. Bound hTERT proteins were eluted with 40 μl of FLAG peptide (250 μg/ml). Biotinylated S1P bound to the eluted proteins was quantified using biotin-ELISA (Eagle Biosciences). The results were analyzed using GraphPad Prism 4 software. The stoichiometry was determined using the following equation: n = NKd/(L + N), in which N = L/M (L, free ligand concentration; M, protein concentration).


A549 cells (50,000 per well) were plated on glass coverslips in a six-well plate for 18 hours. Cells were fixed and permeabilized using 4% paraformaldehyde (20 min) and 0.1% Triton X-100 in 1× PBS (pH 7.4) for 10 min. The cells were then blocked with 1% BSA and dissolved in 1× PBS (pH 7.4) for 1 hour. Cells were incubated for 18 hours at 4°C with antibodies specific for S1P (Sphingomab, LT1002; 20 μg/ml), mouse IgG, lamin B, or hTERT (1:1000) in blocking solution followed by Alexa Fluor 488–, Alexa Fluor 594–, or Cy5-conjugated secondary antibodies (1:500) for 1 hour. Immunofluorescence was performed using a Leica TSC SP2 AOBS TCS confocal microscope or an Olympus FV10i microscope with 543- and 488-nm channels for visualizing red and green fluorescence. Images were taken at ×63 magnification. At least three random fields were selected for images.

Visualization of S1P in nuclear membranes using PLA

A549 cells or wild-type and SK2-deficient MEFs were grown in DMEM growth medium. Cells were then fixed with formalin. Fixed and permeabilized cells were incubated with an antibody specific for S1P (Sphingomab, LT1002; 20 μg/ml) and an antibody that recognizes hTERT at 4°C for 18 hours. PLA was then performed and visualized by IF-CM, using the Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

Stable expression of vector, hTERTWT, and hTERTD684A in A549 cells

pBabe-puro, pBabe-WT, and pBabe-hTERTD684A plasmids were obtained from Addgene. Plat-A amphotropic cells were plated at 70% confluence and transfected with the plasmids using the Effectene transfection reagent for 48 hours. After centrifugation of viral supernatant at 1250 rpm for 5 min, it was filtered through a 0.45-μm filter, and viral transduction of A549 cells were performed using polybrene (8 μg/ml). The selection of transfected cells was performed using puromycin (1 μg/ml) for 14 days. Western blotting was performed to assess the expression of hTERT.

Detection of MKRN1-hTERT interaction

Colocalization of MKRN1 and hTERT in primary human lung fibroblasts was carried out by PLA using the Duolink in situ hybridization kit (Olink Biosciences). Antibodies against MKRN1 (ab119096; 1:50) and hTERT (ab32020; 1:50) were used for 18 hours at 4°C, and PLA signals were detected using IF-CM and the Duolink ImageTool (Olink Biosciences). Association of MKRN1 and hTERT was also detected by immunoprecipitation and Western blotting of A549 and H1650 cells expressing vector, SK2WT, and SK2G212E in the absence or presence of 5 μM GA for 4 hours.

TIF assay

A549 cells and primary human lung fibroblasts were fixed using 4% formaldehyde for 20 min and blocked with 1% goat serum in 0.3 M glycine, 1% BSA, and 0.1% Tween 20 in 1× PBS (pH 7.4) for 2 hours. The fixed cells were then incubated with primary antibodies that recognize γ-H2AX (5 μg/ml; ab2893, Abcam) and TRF2 (5 μg/ml; IMG-124A, Imgenex) for 18 hours. They were then incubated with secondary antibodies containing red (Alexa 568) and blue (Cy5) fluorophores against γ-H2AX and TRF2, respectively (colocalization resulted in purple images). Images were captured using IF-CM, and colocalization was quantified using ImageJ Fiji software (48, 49).

TRAP assay

Cells were lysed in 1× CHAPS buffer, and total protein was quantified using Bradford assay. One microgram of total protein was used per assay reaction. Assay conditions were followed as per the manufacturer’s instructions (Millipore), and the reaction products were separated on a 12.5% acrylamide gel under nonreducing conditions. The TRAP products on gels were then stained using SYBR Green (Invitrogen).

TRF length measurement

TRF length analysis using genomic DNAs was performed using the TRF assay kit (Roche) and the Telo TAGGG probe by Southern blotting as described by the manufacturer.

Detection of senescence by senescence-associated β-gal staining

Primary human lung fibroblasts and MEFs were used up to 17 and 6 passages, respectively, for the detection of senescence using the senescence-associated β-gal assay kit as described by the manufacturer (Cell Signaling Technology).

Anchorage-independent growth on soft agar

Cells were grown in 2× DMEM and 5% agar at a ratio of 9:1. Around 20,000 cells per well were grown in agar medium mix and incubated at 37°C. DMEM was changed for cells every 3 days for 14 to 21 days.

Animal xenograft studies

Severe combined immunodeficient (SCID) mice were purchased from Harlan Laboratories. Age- and sex-matched mice were used. All animal protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. A549 cells (2 × 106) stably transfected with control shRNA or shRNA targeting SK2 were implanted into the flanks of SCID mice (n = 4 mice, each containing two tumors on both flanks).When the tumors were palpable, the mice were treated daily with either vehicle control or ABC294640 by oral gavage at 100 mg/kg for 21 days. Tumor volume was measured using digital calipers. At the end of the 21-day treatment, the mice were euthanized and tumor tissues were collected. Similarly, LLC cells stably transfected with shRNA against SK2 or control (scrambled) shRNA were injected subcutaneously into either wild-type or SK2-deficient C57/BL6 mice (n = 4 mice, each containing two tumors on both flanks). Allografted tumor volumes were measured using digital calipers every 3 days for 14 days.

Statistical analysis

All data are presented as means ± SD, and group comparisons were performed with a two-tailed Student’s t test. P < 0.05 was considered statistically significant.


Fig. S1. Roles of SK1 versus SK2 on hTERT abundance and 17C-S1P binding.

Fig. S2. S1P-TERT binding is mediated by the C′3-OH of S1P and the hydrophobic region of TERT between the thumb and finger domains.

Fig. S3. hTERT colocalizes with lamin B at the nuclear periphery.

Fig. S4. Detection of stably expressed hTERT in MEFs.

Fig. S5. Effects of S1P-hTERT binding on hTERT-MKRN1 interaction and growth inhibition in response to GA treatment.

Fig. S6. S1P binding might mimic protein phosphorylation of hTERT at Ser921.

Fig. S7. Effects of SK2-generated S1P on hTERT-dependent senescence.

Fig. S8. Regulation of senescence by S1P-hTERT binding in wild-type or SK2-deficient MEFs.


Acknowledgments: Sphingomab was provided to us by R. Sabbadini (Lpath Inc.). We thank J. J. Chen (Arizona State University) and S. Spiegel (Virginia Commonwealth University) for providing us with hTERT and SK2 constructs, respectively. We also thank Z. M. Szulc (Lipidomics Shared Resource Facility, Medical University of South Carolina) for providing us with S1P analogs. Funding: This work was supported by research funding from the NIH (CA088932, CA173687, and DE016572 to B.O.), as was the construction of the core facilities used in this study (C06 RR015455). Additional funding was provided by the Biostatistics Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313 to E.G.-M.). Author contributions: S.P.S. performed experiments and helped with study design, data analysis, preparation of figures, and writing of the manuscript; R.M.D.P., J.J.O., N.O., and S.P. performed experiments; Y.K.P and E.S. performed molecular modeling and docking studies; R.V.S. conducted SPR studies; E.G.-M. performed statistical analyses; and C.D.S. provided ABC294640 and contributed to the design of the experiments. B.O. developed concepts and hypotheses, designed experiments, analyzed data, and wrote the manuscript. Competing interests: C.D.S. is the founder of Apogee Inc, which developed ABC294640. The other authors declare that they have no competing financial interests. Data and materials availability: A materials transfer agreement is required for the Sphingomab (S1P) antibody.

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