Identification of the nuclear localisation signal of O-GlcNAc transferase and its nuclear import regulation

Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451–453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible β-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.

Scientific RepoRts | 6:34614 | DOI: 10.1038/srep34614 Here, we identified a sequence of three amino acids (DFP) in OGT that act as a NLS. Moreover, we revealed that nuclear import of OGT is mediated by importin α 5. We also elucidated that O-GlcNAcylation of the TPR domain of OGT is required for its direct nuclear translocation. Overall, our data suggest that both the NLS and O-GlcNAc modification of OGT are required for its nuclear localisation.

Results
The DFP motif plays a role in the nuclear localisation of OGT. To identify the NLS of OGT, we generated deletion mutants of OGT and determined their subcellular localisation. HeLa cells were transfected with OGT fused to GFP at the N-terminus and subjected to subcellular fractionation. The first deletion mutant (residues 1-453) contained 13 TPRs, including the sixth and seventh repeats (the local dyad axis of the homodimer) 16 , and localised in the nucleus and cytoplasm. The second deletion mutant lacked the N-terminal 453 amino acids (residues 454-1036) and localised exclusively in the cytoplasm. The third construct contained three more amino acids downstream of the thirteenth TPR (residues 451-1036) and was detected in the nucleus and cytoplasm ( Supplementary Fig. 1). Taken together, these data suggest that the residues 451-453 are important for the nuclear import of OGT. To further demonstrate that these three amino acids are the NLS of OGT, we generated Mono-OGT, which excluded the possibility of an interaction between mutant OGT and endogenous OGT, because the tendency of homodimerisation of OGT is decreased when two hydrophobic residues are replaced with negatively charged residues (W198E and I201D) 16 . The interaction between Mono-OGT and endogenous OGT was significantly decreased compared to WT-OGT ( Supplementary Fig. 2). Furthermore, the three amino Western blotting of aliquots of the fractions was performed with an α -Flag antibody to detect the β -galactosidase proteins, and with α -lamin A/C, α -α -tubulin and α -β -actin antibodies as markers of the nuclear, cytoplasmic and total fractions, respectively. Images of western blot immunoblotted with an α -Flag antibody, was stripped, and then re-immunoblotted with α -lamin A/C, α -α -tubulin and α -β -actin antibodies respectively. Full gel blots for the cropped blots (a) are in the supplementary Fig. 7. (b) Immunofluorescence confirmed the subcellular fractionation results. HeLa cells transiently overexpressing Flag-tagged β -galactosidase constructs were fixed and stained with both an α -Flag antibody (green) and DAPI (blue). Scale bar, 10 μ m. (c) The mean of densitometry readings in five separate locations within the nucleus was obtained and this was compared with the mean measurement of five separate locations within the cytoplasm of each cell. Data were quantified using MetaMorph software. Data show mean ± s.d.; n = 5 locations in the cell. *P < 0.01 (Student's t-test). All data are representative of at least three independent experiments.  (a) HeLa cells were co-transfected with Flag-tagged OGT and Myc-tagged importin α or β . Cell lysates were immunoprecipitated with an α -Flag antibody. Coimmunoprecipitated importin α or β , as well as the loading amounts, were analysed by western blotting with an α -Myc antibody. An α -Flag antibody immunoblotting confirmed that equal amounts of OGT constructs were immunoprecipitated. (b) Cells were transfected as in (b) were immunoprecipitated with an α -Myc antibody. Co-immunoprecipitated OGT was blotted with an α -Flag antibody. An α -Myc antibody immunoblotting confirmed that equal amounts of importin α and β were immunoprecipitated. (c) HeLa cells were transfected with Flag-tagged OGT and immunoprecipitated with an α -Flag antibody. Bound endogenous importin α 5 was detected by an α -importin α 5 antibody. Total lysates were blotted with an α -importin α 5 antibody as a loading control. (d) HeLa cells transiently overexpressing Myc-tagged importin α 5 were immunoprecipitated with an α -Myc antibody. Co-immunoprecipitated endogenous OGT was detected by an α -OGT antibody. Total lysates were blotted with an α -OGT antibody to monitor the amount of OGT. (e) HeLa cells were transfected twice with siRNA targeting importin α 5 or control siRNA. After 3 days, cells were transfected with Flag-tagged OGT.
The DFP motif can function as a NLS independently. To validate the importance of the DFP motif of OGT and to examine whether it can act as a NLS independently, we used Flag-tagged β -galactosidase. This protein was too big to freely diffuse into the nucleus and localised exclusively in the cytoplasm when expressed in HeLa cells. However, the addition of DFP to the N-terminus of Flag-tagged β -galactosidase induced its nuclear translocation (Fig. 2a-c), and this was prevented by mutation of DFP to AFP, DAP, DFA or AAA ( Supplementary  Fig. 4a). Then we wanted to test whether the putative NLS of OGT reported in 1997 13 has an actual function as nuclear import. However, fusion of putative NLS (residues 477-493) to the Flag-tagged β -galactosidase did not cause import into the nucleus ( Supplementary Fig. 4b). We also used Flag tagged double-stranded RNA-specific editase 1 (ADARB1) to test the function of DFP motif in ADARB1. ADARB1 mutated DFP (residues 171-173) to AAA showed greatly reduced nuclear import than WT-ADARB1 ( Supplementary Fig. 6). ADARB1 was still localised in the nucleus because ADARB1 interacts with other endogenous ADARB1 in the cells. These results clearly demonstrate that the DFP motif functions as nuclear import independently.
Importin α5 interacts with OGT. Because nuclear transport of proteins commonly requires their interaction with importin α s 34,36 , we determined whether OGT associates with importin α s. The classical nucleocytoplasmic import pathway is mediated by the importin α /β heterodimer 28,29 . While only one importin β isoform exists, six human importin α s have been reported 37 . Although the importin α s differ in terms of their cell-and tissue-specific expression patterns, most are expressed ubiquitously, except for importin α 6, which is only present in testes 24 . Therefore, we tested the association of OGT with the other five importin α s. No binding was observed between OGT and importin β , α 1, α 3, α 4, or α 7 (Fig. 3a,b). However, overexpressed OGT interacted with overexpressed or endogenous importin α 5 ( Fig. 3a-d). To determine whether importin α 5 is also required for the nuclear localisation of OGT, we depleted importin α 5 in HeLa cells by RNA interference (RNAi). In cells treated with small interfering RNA (siRNA) targeting importin α 5, the amounts of overexpressed OGT localised in the nucleus and cytosol were decreased and increased, respectively ( Fig. 3e-g). Thus, our results suggest that importin α 5 is an important karyopherin of OGT.
The DFP motif is required for the interaction of OGT with importin α5. The finding that importin α 5 is involved in the nuclear import of OGT prompted us to examine whether the binding of importin α 5 is dependent on the DFP motif of OGT. We first examined the interaction of the various OGT mutants with importin α 5. Co-immunoprecipitation experiments revealed that WT-OGT interacted more strongly with importin α 5 than Mono-OGT and that Mono-OGT in which the NLS was mutated or deleted showed significantly reduced association with importin α 5 ( Fig. 4a In all experiments, importin α 5 interacted more weakly with Mono-OGT than with WT-OGT. This reduced binding affinity of Mono-OGT for importin α 5 would explain why the nuclear localisation of Mono-OGT was less extensive than that of WT-OGT (Fig. 1b,c). Taken together, these results indicate that importin α 5 has a functional interaction with the NLS of OGT, which affects the nuclear localisation of monomeric OGT.  (Fig. 5a). Next, we created site-specific point mutants of OGT. Mutation of Ser381 and Ser389 with alanine and Thr383, and Thr394 with valine, resulted in a reduction in O-GlcNAc modification (Fig. 5b,c). From this investigation, we determined that After another day, cells were subjected to subcellular fractionation. Western blotting was performed on the cytoplasmic and nuclear fractions. Total lysates were blotted with an α -importin α 5 antibody to monitor the reduction in endogenous importin α 5 and with an α -β -actin antibody as a loading control. *NS; non-specific. (a-e) Full gel blots (a-e) are in the supplementary Fig. 7. (f) Immunofluorescence confirmed the subcellular fractionation results. Cells were prepared as described in (e), fixed, stained with an α -Flag antibody (green) and DAPI (blue). Scale bar, 10 μ m. (g) The mean of densitometry readings in five separate locations within the nucleus was obtained and was compared with the mean readings of five separate locations within the cytoplasm of each cell. Data were quantified using MetaMorph software. Data show mean ± s.d.; n = 5 locations in the cell. *P < 0.01 (Student's t-test). All data are representative of at least three independent experiments.

O-GlcNAc modification of the TPR domain of OGT is important for its nuclear localisation.
Next, we examined to what extent O-GlcNAc modification of OGT contributes to its nuclear localisation. Because Mono-OGT showed less nuclear import than WT-OGT (Fig. 1b-e) and has lower O-GlcNAc modification level than that of WT-OGT ( Supplementary Fig. 3). Combined with the previous results, we assumed that O-GlcNAcylation of OGT may impact its nuclear localisation. To address this issue, we constructed the Mono-OGT S389A mutant. To increase O-GlcNAc modification of OGT, cells were treated with Thamet-G, a selective inhibitor of O-GlcNAcase 40 . The nuclear localisation of both WT-OGT and Mono-OGT was increased in Thiamet-G-treated cells (Fig. 6a,b). However, the nuclear localisation of Mono-OGT S389A was almost abolished in both Thiamet-G-treated and untreated cells (Fig. 6a). To decrease O-GlcNAc modification of OGT, cells were treated with 5-thio-GlcNAc 41 , an inhibitor of OGT. The nuclear localisation of both WT-OGT and Mono-OGT was decreased in 5-thio-GlcNAc-treated cells, and the nuclear localisation of Mono-OGT S389A was completely prevented (Fig. 6c,d). These findings were further supported by fluorescence microscopy analysis (Fig. 6e,f). We predicted that the nuclear localisation of OGT was decreased by a change in its conformation upon exposure of the NLS (residues 451-453). OGT S389A had the same enzyme activity as WT-OGT ( Supplementary Fig. 5a,b) and the substitution of Alanine for Ser389 in OGT does not affect protein-protein interaction with other proteins (Supplementary Fig. 5c). This indicated that the decrease of the nuclear localisation of OGT was not due to distortion of its structure, but due to exposure of its NLS. Collectively, our data indicate that the nuclear localisation of OGT is mainly regulated by O-GlcNAc modification of Ser389. Our results are summarised in Fig. 7. O-GlcNAc modification at Ser389 probably results in exposure of the hidden NLS of OGT and its association with importin α 5 and β , resulting in its nuclear localisation.  Discussion OGT transfers GlcNAc from uridine diphosphate-N-acetyl glucosamine to the hydroxyl group of a serine or threonine residue on cytoplasmic and nucleus protein substrates 9,12 . The TPR domain of the enzyme crystallises as a dimer with an interface between the two subunits 16 . The catalytic domain has a nucleotide-binding domain and there are hinge regions between the twelfth and thirteenth TPRs, where OGT pivots dramatically 17 . Based on these previous studies, we investigated the molecular changes that allow the nuclear import of OGT, which is not fully understood. We identified the NLS of OGT (DFP at position 451-453). Deletion or alanine substitution of DFP abolished the nuclear localisation of OGT and β -galactosidase localised in the nucleus when fused to DFP, suggesting that this NLS plays an essential role in nuclear import. We used Mono-OGT (W198E and I201D) 16 because endogenous OGT can interact with the exogenously expressed proteins and transport them to the nucleus. Immunoprecipitated Flag-tagged Mono-OGT showed a very weak binding affinity for endogenous OGT ( Supplementary Fig. 2), confirming the weak interaction between Mono-OGT and endogenous OGT. This is why very faint bands of Mono-OGT 451-453 AAA and Mono-OGT Δ 451-453 were detected in the nuclear fraction (Fig. 1b). Moreover, because Mono-OGT weakly interacted with endogenous OGT, we predicted that the extent of O-GlcNAc modification of Mono-OGT is much lower than that of WT-OGT. To test this possibility, we immunoprecipitated Flag from lysates of cells transiently overexpressing Flag-tagged WT-OGT and Mono-OGT. The level of O-GlcNAcylated Mono-OGT was considerably lower than that of O-GlcNAcylated WT-OGT ( Supplementary Fig. 3). This reduced O-GlcNAc modification of Mono-OGT decreased its interaction with importin α 5 (Fig. 4a-c). Together, these experiments explain why the nuclear importation rate of Mono-OGT was lower than that of WT-OGT. On the other hand, the nuclear localisation of transfected Mono-OGT Δ 451-453 and Mono-OGT 451-453 AAA was significantly impaired compared with that of WT-OGT and Mono-OGT ( Fig. 1b-e). The identified DFP motif of OGT is not a classical NLS and many proteins that have the same motif localise in the nucleus or remain in the cytoplasm. Surprisingly, the nuclear import of exogenously expressed double-stranded RNA-specific editase 1 (ADARB1) was decreased when its DFP residues at positions 171-173 were mutated to AAA (Supplementary Fig. 6). In these experiments, endogenous ADARB1 was knocked down using RNAi because this protein reportedly forms a homodimer 42,43 . Regulation of the nuclear import of other proteins that have a DFP motif should be studied. We postulate that both the NLS and O-GlcNAcylation of Ser389 contribute to the nuclear localisation of OGT. O-GlcNAc modification might induce a conformational change to facilitate nuclear translocation, similar to other nucleocytoplasmic proteins, such as phosphorylation of extracellular signal-regulated kinase 5 and human telomerase reverse transcriptase 44,45 . Additionally, a recent publication showed that phosphorylation of Thr444 of OGT is important for its nuclear localisation 19 . We assume that Thr444 is in close proximity to the DFP motif at positions 451-453 and acts the same as Ser389. In summary, we identified a unique NLS that is responsible for the nuclear localisation of OGT. This NLS controls the nuclear localisation of overexpressed β -galactosidase containing the DFP motif. We also showed that OGT is imported into the nucleus using the DFP motif mediated by importin α 5. Our data indicate that O-GlcNAcylation of the TPR domain of OGT (Ser389) is required for its nuclear localisation. These findings establish a foundation for how nucleocytoplasmic proteins are regulated and exist in the nucleus and cytosol simultaneously without any other sequestering proteins.

Online Methods
Cell culture, DNA transfection and plasmids. HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT) supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 100 μ g/ml streptomycin at 37 °C in 5% CO 2 . DNA was transfected using polyethylenimine (Sigma-Aldrich, St Louis, MO) as described previously 46 . siRNA was transfected by lipofection (Lipofectamine Plus; Invitrogen, Carlsbad, CA). Human WT-OGT, mutant OGT, WT-β -galactosidase and mutant β -galactosidase were cloned Immunofluorescence microscopy. Cells were grown on precision coverslips (0.17 ± 0.01 mM thickness; Glaswarenfabrik Karl Hecht GmbH & Co KG, Sondheim, Germany) and preparation of the cells were described previously 47 . For immunofluorescence analysis, mouse monoclonal anti-Flag antibodies (1:5000) were applied for 2 h, followed by rinses (2 × 5 min) in PBS containing 1% bovine serum albumin and incubation with the appropriate fluorescent secondary antibodies for 1 h. DAPI was used to stain nuclei. After rinsing, coverslips were mounted on glass slides with Mowiol. Immunofluorescence was recorded with a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany) using a Plan-Apochromat 63× objective (1.4 NA). Meta Imaging Series ® MetaMorph software (Meta Series Software 7.7.0; Molecular Devices) was used to quantify the data by taking densitometry readings of five separate locations within the nucleus and cytoplasm of each cell.

SDS-PAGE and in-gel digestion.
The eluted OGT sample was loaded onto a 4-12% Bis-Tris NuPAGE gel (NOVEX, San Diego, CA) for electrophoresis and stained with Coomassie Brilliant Blue (Sigma-Aldrich). The gel bands corresponding to OGT were excised and subjected to in-gel tryptic digestion following a general protocol 48,49 . Briefly, OGT bands were destained with 50% (v/v) acetonitrile (ACN) prepared in 25 mM ammonium bicarbonate and 100 mM ammonium bicarbonate for 15 min. Proteins were reduced with 20 mM DTT at 60 °C for 1 h and then alkylated with 55 mM iodoacetamide at room temperature for 45 min in the dark. After dehydration, the proteins were digested with 12.5 ng/μ l analytical grade porcine trypsin (Thermo Scientific Pierce, Rockford, IL) prepared in 50 mM ammonium bicarbonate overnight at 37 °C. Peptides were then extracted from the gel pieces with 50% (v/v) ACN prepared in 5% formic acid, dried under a Centrivap concentrator (Labconco, Kansas City, MO) and stored at − 20 °C until use.
Mass spectrometry. The peptide samples extracted by in-gel digestion were suspended in 40 μ l of solvent A (0.1% formic acid prepared in water, Optima LC/MS grade, Fisher Scientific, Pittsburgh, PA). Thereafter, 2 μ l of the sample was loaded onto a house-packed 75 μ m (inner diameter of microcapillary) × 15 cm C18 (5 μ m, 100 Å) column and separated with a 5-30% gradient of solvent B (0.1% formic acid prepared in ACN) for 90 min at a flow rate of 300 nL/min. Mass spectra were recorded on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) interfaced with a nanoAcquity UPLC (Waters, Milford, MA). The Orbitrap Fusion Tribrid mass spectrometer was operated in several modes all in Orbitrap, namely, full scan MS1, data-dependent acquisition high-energy collision dissociation scan, product ion-triggered MS3 ETD scan and product ion-triggered MS3 EThcD scan. The raw data were processed using the Trans-Proteomic Pipeline (v4.8.0 PHILAE) and compared with a database composed of human OGT1 (O15294-3, UniProt ID), about 500 decoy proteins and common contaminants. Carbamidomethyl of cysteine was considered the fixed modification, and variable modification was set for oxidation of methionine and O-GlcNAcylation of serine and threonine. Statistical analysis. Each experiment was repeated three to four times with consistent results. Data are representative of the mean values obtained. Differences between groups were evaluated using the two-tailed unpaired Student's t-test. P values < 0.05 were considered to indicate statistical significance for all statistical evaluations. (*P < 0.01 and **P < 0.05).