O-GlcNAcylation/Phosphorylation Cycling at Ser10 Controls Both Transcriptional Activity and Stability of Δ-Lactoferrin*

Δ-Lactoferrin (ΔLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on ΔLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser10 is O-GlcNAcylated and that this modification is associated with increased ΔLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the 391KSQQSSDPDPNCVD404 sequence as a functional PEST motif responsible for ΔLf degradation and defined Lys379 as the main polyubiquitin acceptor site. We next investigated the control of ΔLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ΔLf response element showed that O-GlcNAcylation at Ser10 negatively regulates ΔLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2–3-fold when phosphorylation was mimicked at Ser10 by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the ΔLf transcriptional complex binds to the ΔLf response element and is phosphorylated and/or ubiquitinated, suggesting that ΔLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser10 by either O-phosphate or O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity.

⌬-Lactoferrin (⌬Lf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on ⌬Lf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser 10 is O-GlcNAcylated and that this modification is associated with increased ⌬Lf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the 391 KSQQSSDPDPNCVD 404 sequence as a functional PEST motif responsible for ⌬Lf degradation and defined Lys 379 as the main polyubiquitin acceptor site. We next investigated the control of ⌬Lf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ⌬Lf response element showed that O-GlcNAcylation at Ser 10 negatively regulates ⌬Lf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2-3-fold when phosphorylation was mimicked at Ser 10 by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the ⌬Lf transcriptional complex binds to the ⌬Lf response element and is phosphorylated and/or ubiquitinated, suggesting that ⌬Lf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser 10 (2,3), and alterations of the O-GlcNAc status are associated with type-2 diabetes, neurological disorders, and cancer (4).
Because numerous proteins, such as transcription factors, signaling components, and metabolic enzymes are modified, O-GlcNAcylation is critical to normal cell homeostasis and gene regulation (5). It notably modulates gene expression, depending on the promoter and its associated transcription initiation complexes. For instance, the C-terminal domain of RNA polymerase II and a subset of general transcription factors are O-GlcNAcylated at transcription initiation (6). Gene silencing may be effected via the recruitment of OGT onto promoters by transcriptional corepressors. It then catalyzes the O-GlcNAcylation of specific transcription actors, modulating their activity. For instance, the association of OGT with the co-repressor mSin3A leads to the recruitment of histone deacetylase, thereby increasing transcriptional down-regulation (7,8). OGA may favor gene transcription, not only by reducing the level of glycosylation but also via its intrinsic histone acetyltransferase domain (9). O-GlcNAcylation may also modulate the activity of transcription factors via the regulation of their trafficking, binding affinity either to protein partners or DNA, and/or turnover (8, 10 -13).
Increasing evidence links O-GlcNAcylation to the proteasome pathway. It has been shown that O-GlcNAcylation is associated with lower proteasomal susceptibility of transcription factors, such as Sp1 (14,15), p53 (16), and the estrogen receptor ␤ (17). Most of these proteins have high PEST scores, and phosphorylation of their PEST (Pro-Glu-Ser-Thr) domain targets them for polyubiquitination (18) and subsequent degradation by the proteasome, whereas O-GlcNAcylation prolongs their half-lives. The proteasome is itself regulated through O-GlcNAcylation of both its regulatory and catalytic subunits (19,20) as well as the ubiquitin (Ub)-activating enzyme E1 (21). Reduced degradation of O-GlcNAcylated proteins might also be due to their specific interaction with chaperones, such as Hsp70 family members that display lectin activity toward the O-GlcNAc motif, protecting them from proteolysis (22).
In many O-GlcNAcylated proteins, a phosphate group can alternatively occupy the same or adjacent sites (16,17,23,24). This O-GlcNAc/P interplay, which leads to a rapid response mechanism and high molecular diversity and fine tunes protein interactions and functions, may also target ⌬-lactoferrin (⌬Lf) and regulate its transcriptional activity and stability. ⌬Lf is a transcription factor that was first discovered as a transcript, the expression of which was observed only in normal cells and tissues (25). Its absence from cancer cells (25,26) is due to genetic and epigenetic alterations (27,28). ⌬Lf messenger is therefore a healthy tissue marker, and we previously showed that its expression level is correlated with a good prognosis in human breast cancer, high concentrations being associated with longer overall survival (26). ⌬Lf is transcribed from the alternative promoter P2 in the lactoferrin (Lf) gene (29), and the use of the first available AUG codon in frame produces an alternative N terminus. Thus, compared with Lf, its secretory counterpart, ⌬Lf is a 73-kDa cytoplasmic protein able to enter the nucleus (30). Potential DNA-binding domains have been suggested for Lf, implicating the strong concentration of positive charges at the C-terminal end of the first helix, which is truncated in ⌬Lf, and at the interlobe region (31,32).
⌬Lf expression provokes anti-proliferative effects and induces cell cycle arrest in S phase (33). It is a transcription factor interacting via a ⌬Lf response element (⌬LfRE) found in the Skp1 and DcpS promoters (30,34). ⌬Lf is also at the crossroads between cell survival and cell death because we recently linked ⌬Lf overexpression to up-regulation of the Bax promoter and apoptosis (35). Because ⌬Lf has several crucial target genes and may act as a tumor suppressor, modifications in its activity or concentration may have marked effects on cell survival, and its transcriptional activity should be strongly controlled. Results of screening ⌬Lf for O-GlcNAcylation and phosphorylation sites showed that the protein potentially undergoes both post-translational modifications. Four putative O-GlcNAc/phosphorylation sites were found at Ser 10 , Ser 227 , Ser 472 , and Thr 559 , and their mutation led to a constitutively active ⌬Lf M4 mutant (34). Here, we map the major O-GlcNAc/P site to Ser 10 , the PEST sequence (amino acids 391-404), and the main poly-Ub site to Lys 379 . We also report that O-GlcNAcylation at Ser 10 down-regulates ⌬Lf transcriptional activity and up-regulates its stability by abrogating Ub-mediated proteolysis, whereas phosphorylation activates both transcription and degradation.
Immunofluorescence and Microscopy-HEK 293 cells were transfected by ⌬Lf C-terminal fused GFP expression vector 24 h prior the 4Ј,6-diamidino-2-phenylindole (Sigma) staining. The p⌬Lf-N-EGFP vector was kindly provided by Dr. C. Teng (National Institutes of Health, Research Triangle Park, NC). Immunofluorescence and microscopy were performed as described (30). Fluorescent microscopy images were obtained at room temperature with a Zeiss Axioplan 2 imaging system (Carl-Zeiss S.A.S., Le Pecq, France) equipped with appropriate filter cubes using a ϫ40 objective lens.
Purification of DNA, RNA, and Poly(A) ϩ RNA-Genomic DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen), total RNA using the RNeasy minikit (Qiagen), and poly(A) ϩ RNA using the polyATrack mRNA isolation system (Promega). The purity and integrity of each extract were checked using the nanodrop ND-1000 spectrophotometer (Labtech International) and the Bioanalyzer 2100 (Agilent Technologies).
Plasmid Construction and Site-directed Mutagenesis-pGL3-S1 Skp1 -Luc, pcDNA-⌬Lf, and p3xFLAG-CMV10-⌬Lf were constructed as described (30). p3xFLAG-CMV10 (Sigma) and pcDNA3.1 (Invitrogen) were used as null vectors. The Ub-HA expression vector was a gift from Dr. C. Couturier (IBL, Lille, France). The pcDNA-OGT expression vector was constructed using OGT cDNA isolated from the pShuttle-OGT vector (36) (a kind gift of Dr. J. Hart, The John Hopkins University School of Medicine (Baltimore, MD)) and further cloned into the pcDNA3.1 vector. Mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) with pcDNA-⌬Lf as template and primer pairs listed in Table 1. The constructs in which several sites were mutated were done sequentially. Following sequencing, the HindIII-NotI digests were cloned either into pcDNA3.1 for reporter gene assays or into p3xFLAG-CMV10 for protein experiments.
Reporter Gene Assay-Reporter gene assays were performed using the pGL3-S1 Skp1 -Luc reporter vector and the different pcDNA-⌬Lf mutant constructs or a null vector as described (34). Cell lysates were assayed using a luciferase assay kit (Promega) in a Tristar multimode microplate reader LB 941 (Berthold Technologies). Relative luciferase activities were normalized to basal luciferase expression and protein content as described (30) and expressed as a percentage; 100% corresponds to the relative luciferase activity of ⌬Lf WT . Basal luciferase expression was assayed using a null vector and was determined for each condition (OGT, okadaic acid (OA), and glucosamine (GlcNH 2 )) at each concentration.
Each experiment represents at least three sets of independent triplicates.
In Vivo DNA Binding Assays-Chromatin immunoprecipitation (ChIP) and double ChIP (re-ChIP) assays were performed as described (34,37) with some modifications introduced for re-ChIP. Briefly, ChIP complexes (8 ϫ 10 6 cells) were immunoprecipitated with M2, RL2, HA tag, or anti-Ser(P) antibodies all used at 1:250 and twice eluted with 100 l of 1 mM dithiothreitol for 30 min at 37°C. After centrifugation, pooled eluted fractions were diluted 40 times to reduce the dithiothreitol concentration to 25 M with ChIP dilution buffer and then immunoprecipitated with either M2 or rabbit anti-IgG (GE Healthcare) or without antibodies. Ampli-fication conditions of Skp1 and albumin promoters were as described (34). ChIP or re-ChIP results presented in Fig. 5 correspond to one representative experiment among three. qPCR was performed only for the ChIP assay. Amplification was carried out in triplicate (n ϭ 3) in the presence of 2 l of purified DNA, primer pairs used to amplify the Skp1 promoter fragment (34), and Brilliant SYBER Green QPCR Master Mix (Stratagene) according to the manufacturer's instructions. Samples were then submitted to 40 cycles of amplification (denaturation, 30 s at 90°C; hybridation, 30 s at 55°C; elongation, 30 s at 72°C) in a thermocycler Mx4000 (Stratagene). Data presented in Fig. 5D are expressed as a percentage of input.
Proteasomal Degradation Assay-Proteasomal activity assay was performed according to the assay instructions (Chemicon International) on HEK 293 cell lysates. Lactacystin was used as a 20 S proteasome inhibitor. Fluorescence data were collected using a Tristar multimode microplate reader LB 941 (Berthold Technologies) using 380-nm excitation and 460-nm emission filters.
Immunoblotting and Immunoprecipitation-Proteins were extracted from frozen cell pellets in radioimmune precipitation buffer as described (30). For direct immunoblotting, samples mixed with 4ϫ Laemmli buffer were boiled for 5 min. 20 g of protein from each sample were submitted to 7.5% SDS-PAGE and immunoblotted. For immunoprecipitation experiments, 1 mg of total protein was preabsorbed with protein G-Sepharose 4 Fast Flow (GE Healthcare). RL2 (1:250), M2 (1:500), or anti-HA polyclonal (1:100) antibodies were mixed with Protein G-Sepharose beads for 1 h prior to an overnight incubation with the preabsorbed lysate supernatant at 4°C. The beads were then washed five times with lysis buffer. Proteins bound to the beads were eluted with 4ϫ Laemmli buffer and analyzed by immunoblotting as above. Blots were probed at room temperature with primary antibodies (M2, 1:2000; CTD110.6, 1:3000; HA.11, 1:4000; anti-Ser(P), 1:500; RL2, 1:2000; and anti-actin, 1:10,000) for 2 h and with either secondary anti-IgG antibodies conjugated to horseradish peroxidase (GE Healthcare) or secondary anti-IgM antibodies conjugated to horseradish peroxidase (Chemicon International) for 1 h before detection by chemiluminescence (ECL Advance, GE Healthcare). Each result in which immunoblots are presented corresponds to one representative experiment among at least three.
Densitometric and Statistical Analyses-The densitometric analyses were performed using Quantity One version 4.1 software (Bio-Rad), and acquisition was carried out with a GS710-calibrated densitometer (Bio-Rad). M2 densitometric values were normalized to actin and expressed as R ϭ D M2 /Dact. The -fold stability (X) is expressed as this ratio related to the wild type ratio and to the t 0 value as follows for ⌬Lf PEST . X ϭ PEST R t / PEST R t 0 / WT R t / WT R t 0 . All of the statistical analyses were done using Origin 7 software (OriginLab Corp.). Means were statistically analyzed using the t test or analysis of variance, and differences were assessed at p Ͻ 0.05 (*) or p Ͻ 0.01 (**).

Impact of the O-GlcNAc/P Interplay on ⌬Lf Transcriptional
Activity and Stability-Investigation of the O-GlcNAc function has mainly relied on the manipulation of the hexosamine biosynthesis pathway via an increased production of UDP-GlcNAc, the substrate for OGT (38). Thus, cells exposed to increased concentrations of GlcNH 2 or overexpressing OGT exhibit enhanced levels of protein O-GlcNAcylation (39). On the other hand, the use of OA, an inhibitor of PP2A and PP1 phosphatases, is a valuable tool for inducing protein hyperphosphorylation (40,41).
Prior to investigating whether ⌬Lf transcriptional activity is regulated via O-GlcNAc/P interplay, we first established that HEK 293 cells possess rapid, inducible O-GlcNAc/P mechanisms at the OGT, GlcNH 2 and OA concentrations employed (42). First of all, we verified that cell viability was not perturbed (Fig. 1A). At the concentrations usually used in the literature, such as 40 mM GlcNH 2 and 50 nM OA, cell viability was markedly decreased in HEK 293 cells. For this reason, we used lower concentrations, such as 10 mM GlcNH 2 and 10 nM OA, that did not affect cell viability but at which modulation of the O-GlcNAc/P status was visible (Fig. 1B). Co-transfection of ⌬Lf (1 g DNA/10 6 cells) and OGT (2.5 g of DNA/10 6 cells) expression vectors did not significantly affect cell viability (Fig.  1A). Fig. 1B shows that ⌬Lf is indeed sensitive to OA and GlcNH 2 or OGT but with opposite effects. Treatment with OA led to decreased ⌬Lf glycosylation, whereas treatment with GlcNH 2 or co-transfection with OGT increased it. The same GlcNAcylation pattern was observed using either the RL2 or the CDT110.6 antibody. This result demonstrates clearly that ⌬Lf possesses O-GlcNAc site(s). OA treatment, which favors phosphorylation, decreases the ⌬Lf glycosylation level, suggesting that glycosylation site(s) may exist in balance with phosphorylation site(s). Because RNA polymerase II activity is also controlled by this interplay, we next verified that transcription of ⌬Lf; ribosomal protein, large, P0; or hypoxanthine-guanine phosphoribosyltransferase was indeed not altered under OGT, GlcNH 2 , or OA treatment (Fig. 1C). Our control experiments showed that modulation of the O-GlcNAc content does not impair cell functions at the concentration of OA, GlcNH 2 , or OGT we used.
We then investigated ⌬Lf transcriptional activity using reporter gene assays and a Skp1 promoter fragment containing FIGURE 1. O-GlcNAc/P interplay regulates ⌬Lf transcriptional activity. HEK 293 cells were incubated with GlcNH 2 or OA, or transfected with an OGT construct (OGT) to assess the impact of the O-GlcNAc/P interplay on ⌬Lf. A, cell viability. Cell viability of 10 4 HEK 293 was assayed 24 h after GlcNH 2 or OA treatment or after transfection with pcDNA-OGT at 2.5 or 5 g of DNA/10 6 cells (n ϭ 9). B, ⌬Lf O-GlcNAcylation status. Treated and untreated 3xFLAG-⌬Lfexpressing HEK 293 cell extracts were M2-immunoprecipitated prior to SDS-PAGE and CTD110.6 or RL2 immunodetection. Input was used as the loading control (n ϭ 3). C, gene expression is not altered under GlcNH 2 and OA treatment or OGT overexpression. Poly(A) ϩ RNA was purified from total RNA of ⌬Lf-expressing cells treated with OGT, GlcNH 2 , or OA 24 h after transfection and assayed using real-time PCR. RPLPO and hypoxanthine-guanine phosphoribosyltransferase are internal controls (n ϭ 3). D-F, ⌬Lf transcriptional activity is modulated by OGT, GlcNH 2 , or OA treatment. Cells were co-transfected with pcDNA-⌬Lf and pGL3-S1 Skp1 -Luc and incubated with GlcNH 2 or OA or co-transfected with pcDNA-⌬Lf, pcDNA-OGT, and pGL3-S1 Skp1 -Luc for 24 h prior to lysis. Relative luciferase activities are expressed as described under "Experimental Procedures" (n Ն 9). G, the endoproteolytic activity of the proteasome is not altered under OGT, GlcNH 2 , and OA treatment. The histograms represent the proteasomal activity assayed by following the fluorescence emitted during the degradation of a synthetic fluorescent peptide (69). Lactacystin is a proteasome inhibitor (n ϭ 3). H, Ub-dependent degradation of ⌬Lf is GlcNH 2 -sensitive. Cells were co-transfected with or without 3xFLAG-tagged ⌬Lf (⌬Lf-3xFLAG) and Ub-HA vectors, treated or not with GlcNH 2 , or transfected or not with pcDNA-OGT. Cells were incubated 2 h with 10 M MG132 before lysis in order to inhibit proteasomal degradation. Total cell extracts were immunoprecipitated with anti-HA polyclonal antibodies or used as input. Samples were immunoblotted with M2 (top and bottom) or HA.11 (middle) antibodies. I, ⌬Lf traffic is not affected by GlcNH 2 or OA treatment. Cells were transfected with pEGFP empty or pEGFP-⌬Lf vector and incubated with GlcNH 2 or OA. Fluorescent microscopy was performed after 4Ј,6-diamidino-2-phenylindole (DAPI) staining. Error bars, S.D. IB, immunoblot; IP, immunoprecipitation.
the ⌬LfRE known to be highly transactivated by ⌬Lf (30). ⌬Lf transcriptional activity increased in line with OA concentration (Fig. 1D), whereas it decreased in a dose-dependent manner in the presence of GlcNH 2 (Fig. 1E). Thus, when phosphorylation was augmented, transactivation was increased 6 -7-fold compared with controls, whereas when O-GlcNAcylation was increased in ⌬Lf-expressing cells, transactivation of the Skp1 promoter was strongly reduced. ⌬Lf transcriptional activity also decreased when cells overexpressed OGT but at a lower level (Fig. 1F).
Because, as for many transcription factors, ⌬Lf is rapidly degraded, we next investigated whether its turnover is dependent on both the Ub-proteasome pathway and O-GlcNAc/P interplay. We first verified that treatment with OA, GlcNH 2 , or OGT did not disturb the proteasome pathway. As shown in Fig.  1G, these treatments did not alter or exacerbate proteasomal degradation compared with the untreated and lactacystintreated conditions. Fig. 1H shows that a ladder of polyubiquitinated ⌬Lf forms is visible (top, lanes 3 and 6). We next evaluated whether O-GlcNAcylation regulates ⌬Lf degradation, and the intensity of polyubiquitination was indeed decreased in a dosedependent manner after GlcNH 2 treatment (Fig. 1H, top, lanes 4 and 5) and after OGT overexpression (Fig. 1H, top, lane 7). Equivalent loadings of Ub-HA protein (Fig. 1H, middle) and ⌬Lf (Fig. 1H, bottom) were confirmed by immunoblotting. These data demonstrated that ⌬Lf is more stable in an environment favoring O-GlcNAcylation.
We further investigated whether ⌬Lf traffic might be altered, leading to an exclusive nuclear targeting of the phosphoform. As previously described (29,30), a ⌬Lf-GFP fused protein localizes predominantly to the cytoplasm but also to the nucleus (Fig. 1I, panel 2). Here, we showed that the subcellular localization of ⌬Lf-GFP was not modified with either GlcNH 2 or OA (Fig.  1I, panels 3 and 4, respectively), suggesting that ⌬Lf traffic is not regulated by the O-GlcNAc/P interplay.
Mapping the Key O-GlcNAc Site to Ser 10 -The low abundance of ⌬Lf, the necessity for producing a 3xFLAG-tagged protein in order to detect it, and the inherent limitation of the sensitivity of tritium labeling render the detection of carbohydrate moieties on ⌬Lf and the subsequent mapping of its glycosylated sites extremely difficult. Therefore, in order to confirm the presence of the O-GlcNAc sites and characterize their roles, we made a series of glycosylation mutants in which only one O-GlcNAc site is preserved, named ⌬Lf S10ϩ , ⌬Lf S227ϩ , ⌬Lf S472ϩ , and ⌬Lf T559ϩ , respectively ( Fig. 2A). ⌬Lf M4 (34) and ⌬Lf WT were used as controls. ⌬Lf and its glycosylation mutants were then expressed in HEK 293 cells, and their levels of expression were compared. Fig. 2B shows that the ⌬Lf S10ϩ mutant was expressed at the same level as ⌬Lf WT (short exposure time) in contrast to the other mutants (long exposure time). ⌬Lf S227ϩ and ⌬Lf S472ϩ were slightly more expressed than were ⌬Lf M4 and ⌬Lf T559ϩ , which were both feebly expressed. These data suggest that the post-translational modifications present on Ser 10 may participate in ⌬Lf stability and that its absence from the other mutants leads to their rapid turnover.
O-GlcNAcylation was then investigated on the ⌬Lf isoforms. Because ⌬Lf mutants are feebly produced, we first immunoprecipitated ⌬Lf-expressing cell lysates with RL2 in order to accumulate enough O-GlcNAcylated material (Fig. 2C). A reverse immunoprecipitation was then performed using the M2 antibody in order to specifically immunoprecipitate ⌬Lf or its glycovariants (Fig. 2D). Fig. 2C shows that ⌬Lf was effectively glycosylated, whereas ⌬Lf M4 was not, confirming that no other O-GlcNAc sites are present on the protein. ⌬Lf S10ϩ , ⌬Lf S227ϩ , and ⌬Lf S472ϩ mutants were glycosylated, whereas ⌬Lf T559ϩ was not (Fig. 2C). The reverse immunoprecipitation of the cell lysates with M2 antibody followed by O-GlcNAc immunodetection with the CTD 110.6 antibody (Fig. 2D) confirmed that ⌬Lf and its ⌬Lf S10ϩ mutant were glycosylated, whereas ⌬Lf M4 and ⌬Lf T559ϩ were not. The O-GlcNAcylated signals corresponding to ⌬Lf S227ϩ and ⌬Lf S472ϩ mutants that were effec- tively visible when RL2 antibody was used, were poorly visible for ⌬Lf S227ϩ and not visible for ⌬Lf S472ϩ when the CTD 110.6 antibody was used. RL2 (43) and CTD110.6 (44) are the two most commonly used antibodies, with CTD110.6 described as being the most specific. Therefore, this divergent result may be due to the fact that both isoforms were too poorly expressed to be detected or that the Ser 472 site was not fully glycosylated. However, we cannot exclude the possibility that performing the immunoprecipitation first with RL2 antibody may favor immunoprecipitation of O-GlcNAcylated ⌬Lf partners. Nevertheless, both immunoprecipitations confirmed without ambiguity that ⌬Lf S10ϩ , ⌬Lf S227ϩ , and ⌬Lf WT mutants were glycosylated, whereas ⌬Lf T559ϩ and ⌬Lf M4 were not.
We next assayed the transcriptional activity of the mutants compared with wild type (WT) (Fig. 2E). The strong transcriptional activity of the glycosylation-null mutant confirmed that O-GlcNAcylation negatively regulates ⌬Lf activity, as previously indicated (Fig. 1, E and F). We compared the activity of mutants in which only one glycosylation site was preserved with that of ⌬Lf M4 in order to evaluate the impact of adding only one regulatory site at a time. The ⌬Lf S227ϩ and ⌬Lf S472ϩ mutants showed transcriptional activities nearly 2-fold greater than WT and close to that of ⌬Lf M4 (Fig. 2E), suggesting that the presence of either O-GlcNAc or phosphate on these two sites does not crucially regulate ⌬Lf transcriptional activity. These sites might be priming sites, as described for phosphorylation, necessary to target OGT or specific kinases to the other sites and may only be transiently O-GlcNAcylated. In contrast, the transcriptional activities of ⌬Lf S10ϩ and ⌬Lf T559ϩ were strongly inhibited compared with ⌬Lf M4 , suggesting that these two sites are primordial for regulation. The absence of response of the ⌬Lf T559ϩ mutant might be due to the feeble expression or rapid degradation of this non-glycosylated and transcriptionally inactive mutant. However, this may not be the only explanation because the removal of all four glycosylation sites in ⌬Lf M4 leads to a constitutively active isoform. Preliminary results on the ⌬Lf T559ϩ mutant showed that it may compete with WT for DNA binding (data not shown), and further work will be necessary to understand the intrinsic role of Thr 559 . O-GlcNAc/P modifications on the Ser 10 site led to a 5-fold inhibition of ⌬Lf transcriptional activity compared with ⌬Lf M4 and 2-fold inhibition compared with ⌬Lf WT (Fig. 2E). This site seems therefore to be a crucial target for the regulation of both ⌬Lf stability and transcriptional activity. We therefore focused our initial attention on Ser 10 and first investigated the impact of O-GlcNAc/P modifications on ⌬Lf turnover by studying their relationship with the proteasome pathway.
⌬Lf Turnover Is Driven through a PEST Motif (Amino Acids 384 -404) and Lys 379 -Short intracellular half-life proteins frequently have a short hydrophilic stretch of amino acids termed a PEST motif. Phosphorylation of the Ser and/or Thr residues and ubiquitination, often of the flanking Lys residues, trigger degradation. Analysis of the ⌬Lf sequence did not allow identification of a PEST motif in the Ser 10 environment but indicated one at the C terminus with three nearly contiguous Ser (Ser 392 , Ser 395 , and Ser 396 ) and two flanking Lys (Lys 379 and Lys 391 ) residues as potential targets either for kinase/OGT or Ub ligase, respectively. Alignment of Lf sequences from other species to this PEST motif shows that the locus is conserved ( Table 2).
We evaluated the functionality of the PEST sequence using a ⌬Lf PEST mutant in which the three Ser residues were replaced by Ala and showed that this mutation leads to a slight increase in ⌬Lf content of about 40% compared with WT (Fig. 3, A and  B). To measure the ⌬Lf turnover rate indirectly, we performed incubations (0 -150 min) with cycloheximide, a potent inhibitor of de novo protein synthesis (45,46). The ⌬Lf content of HEK cells transfected with either ⌬Lf WT or ⌬Lf PEST constructs was analyzed following the addition of cycloheximide (Fig. 3C). Differences in the steady state levels of ⌬Lf were readily apparent after 30 min, which may correspond to the delay necessary for observing the first effects of cycloheximide treatment (Fig.  3C, panel 1). Mutation of the Ser residues in the PEST sequence conferred stability on ⌬Lf (Fig. 3C, panel 5). GlcNH 2 treatment of HEK cells transfected with either ⌬Lf WT or ⌬Lf PEST constructs was also performed (Fig. 3C, panels 3 and 7, respectively), and OGT was coexpressed with ⌬Lf WT (Fig. 3C, panel  9). Actin, which is stable under the same experimental conditions, was used as an internal control (Fig. 3C, panels 2, 4, 6, 8,  and 10). Densitometric data are expressed as -fold stability as described under "Experimental Procedures" (Fig. 3D). Invalidation of the PEST sequence led to a 5-6-fold gain in stability and confirmed that this sequence is determinant for ⌬Lf degradation. GlcNH 2 treatment or OGT overexpression led to a 2-or 3-fold increase in ⌬Lf WT stability, respectively, compared with controls, visible at 90 min, confirming that increasing O-GlcNAcylation protects ⌬Lf from degradation. However, when ⌬Lf PEST -expressing cells were submitted to the GlcNH 2 treatment, the stability of ⌬Lf PEST was not significantly different in the presence or absence of GlcNH 2 suggesting that mutation of the PEST sequence is sufficient to confer stability on ⌬Lf. Moreover, these results also suggest that these two events could be linked because, if they were independent, a greater stability of the ⌬Lf PEST isoform should be visible in the presence of GlcNH 2 . We next studied the invalidation of the PEST sequence on the ⌬Lf M4 mutant. This mutant is detected at low levels in transfected cells, indicating that it is either feebly expressed or rapidly degraded (Fig. 2B). Fig. 3E shows that this invalidation increased ⌬Lf M4 stability and rendered this mutant more resistant to proteasomal proteolysis. We further investigated whether a particular Ser within the PEST motif was involved in this process using a series of single Ser mutants ( Table 2). Whatever the Ser mutated, ⌬Lf expression was identical, suggesting that the three Ser residues were equivalent phosphorylation targets due to their proximity (Fig. 3E). Moreover, prediction results for putative phosphorylation sites using the NetPhos 2.0 server (CBS.DTU; available on the World Wide Web) also emphasized these three Ser residues as kinase targets, albeit with a higher score for Ser 396 (Ser 392 , 0.766; Ser 395 , 0.789; Ser 396 , 0.977).
We then studied whether ⌬Lf-mediated ubiquitination occurs predominantly through Lys 379 -or Lys 391 -linked chains by constructing a series of mutants in which residue 379 or 391 was mutated to Ala either in ⌬Lf or ⌬Lf M4 . The K379A mutation led to a slightly increased expression level of ⌬Lf and completely restored stability to ⌬Lf M4 compared with controls, whereas the K391A mutation had no effect on the ⌬Lf expression level and only slightly increased expression of ⌬Lf M4 (Fig.  3F). This result confirms that the flanking Lys 379 , which is highly conserved among species (Table 2), is involved in ⌬Lf turnover and suggests that it is the major poly-Ub acceptor site. We next verified that ubiquitination of ⌬Lf is indeed Lys 379linked. Fig. 3G shows that polyubiquitination was strongly vis- in which both Lys residues were mutated. Control levels of ubiquitination and ⌬Lf expression are shown in the middle and lower panels, respectively (Fig. 3G). These data confirmed that the Lys 379 residue corresponds to the main Ub ligase target and that Lys 391 corresponds to a minor site. We next investigated which type of relationship may exist between the functional PEST sequence at the C terminus and the O-GlcNAc/P site at the N terminus.
O-GlcNAcylation of Ser 10 Protects ⌬Lf from Polyubiquitination-To determine whether ⌬Lf protein stability was controlled via an O-GlcNAc/P switch on Ser 10 , other mutants were constructed (Fig. 4A), such as the ⌬Lf S10A mutant that has only the Ser 10 residue mutated and the ⌬Lf S10D mutant in which an Asp residue was introduced in place of Ser in order to mimic constitutive phosphorylation as described previously (47,48). Immunoblotting of the different mutants with M2 antibodies is presented in Fig. 4B. ⌬Lf S10A and ⌬Lf S10ϩ had expression levels similar to that of WT, whereas ⌬Lf S10D had an extremely short half-life (Fig. 4C), suggesting that this mutant is an interesting tool for studying degradation of the ⌬Lf phosphoform. Due to the absence of Ser 10 , ⌬Lf S10A was expected, like the ⌬Lf M4 , ⌬Lf T559ϩ , ⌬Lf S227ϩ , and ⌬Lf S472ϩ mutants (Fig.  2B), to be less stable than WT. In ⌬Lf S10A , only Ser 10 is mutated. Therefore, the stability of ⌬Lf S10A might be due to the other sites, which could be used as "protecting sites" in the absence of Ser 10 .
The turnover of these different Ser 10 mutants compared with WT and actin (internal control) is shown in Fig. 4C. Differences in the steady state levels of ⌬Lf and ⌬Lf S10ϩ mutant were readily apparent around 30 -60 min and strongly visible after 90 min (Fig. 4C, panels 1 and 5). Invalidation of the Ser 10 site in ⌬Lf S10A resulted in a markedly prolonged half-life (Fig. 4C,  panel 3). Comparable results were obtained when cells expressing ⌬Lf S10ϩ were cultured in the presence of GlcNH 2 , confirming the crucial role of O-GlcNAcylation in ⌬Lf stability (Fig. 4C,  panel 7). Interestingly, ⌬Lf S10D had a faster turnover rate compared with WT (panel 9), indicating that mimicking phosphorylation at this locus triggers degradation. Fig. 4D summarizes the densitometric data of the ⌬Lf immunoblots expressed as -fold stability, as described under "Experimental Procedures." ⌬Lf S10ϩ , which could be either phosphorylated or glycosylated, was slightly more stable than WT (1.5-fold), whereas the same mutant expressed in hyper-O-GlcNAcylation conditions was 4-fold more stable than WT. Interestingly, the mutation of Ser 10 to Ala also led to ⌬Lf stability (3.5-fold compared with WT), which suggests that stability is not due to the presence of the O-GlcNAc moiety but to the absence of the phosphate group. Mimicking phosphate at Ser 10 in the ⌬Lf S10D mutant shortened its half-life. From these experiments, we conclude that phosphorylation at Ser 10 accelerates ⌬Lf degradation, whereas O-GlcNAcylation at Ser 10 controls its stability, confirming the existence of a strong link between the O-GlcNAc/P interplay and the Ub degradation pathway.
We next investigated whether ubiquitination of ⌬Lf is linked to Ser 10 phosphorylation. Fig. 4E shows that polyubiquitination was marked on ⌬Lf WT and ⌬Lf S10ϩ (top, lanes 3 and 4), whereas it was reduced on ⌬Lf S10A (lane 5). Control levels of ubiquitination and ⌬Lf expression are shown in the middle and bottom panels (Fig. 4E). Unfortunately, the high turnover rate of the

. Ub-dependent ⌬Lf degradation is mediated through a PEST sequence at the C terminus and Lys 379 and is inhibited by O-GlcNAcylation.
A and B, deletion of the PEST sequence slightly increases ⌬Lf stability. HEK 293 cells were transfected with either ⌬Lf WT or ⌬Lf PEST constructs for 24 h. Total protein extracts were immunoblotted with M2. C, modulation of ⌬Lf half-life by O-GlcNAcylation. Cells were transfected with either ⌬Lf WT or ⌬Lf PEST and GlcNH 2 -treated or cotransfected with the OGT-construct or not and then incubated with fresh medium supplemented by 10 g/ml cycloheximide for the indicated time 24 h after transfection. Total protein extracts were immunoblotted with either M2 or anti-actin antibodies. D, data are expressed as -fold stability as described under "Experimental Procedures." *, p Ͻ 0.05. E, the three Ser residues of the PEST sequence are equivalent. Mutation of Ser residues was done on the 3xFLAG-⌬Lf M4 construct as template. Cells were transfected by the different constructs, and 24 h after transfection, total protein extracts were immunoblotted with either M2 or anti-actin antibodies. F, mutation of Lys 379 rather than of Lys 391 inhibits degradation. 3xFLAG-⌬Lf and 3xFLAG-⌬Lf M4 constructs were used as template to obtain Lys 379 and Lys 391 mutants. Cells were transfected by the different constructs, and 24 h after transfection, total protein extracts were immunoblotted with either M2 or anti-actin antibodies. G, Lys 379 is the main Ub-ligase target. HEK 293 cells were co-transfected with or without the 3xFLAG-⌬Lf constructs and the Ub-HA-expressing vector for 24 h and then incubated with a 10 M concentration of the proteasomal inhibitor MG132 for 2 h prior to lysis. Total cell extracts were immunoprecipitated with anti-HA polyclonal antibodies or used as input. Samples were immunoblotted with M2 (top and bottom) or with HA.11 (middle) antibodies. Error bars, S.D. IB, immunoblot.
⌬Lf S10D mutant or of ⌬Lf WT under OA treatment precluded the observation of a polyubiquitination signal (data not shown).
In conclusion, our data showed that ⌬Lf turnover is driven through a PEST sequence located at the C terminus with polyubiquitination occurring mainly at Lys 379 . We also demonstrated that the degradation process is regulated via the O-GlcNAc/P interplay, which targets Ser 10 . As a glycoform, ⌬Lf is stable, whereas as a phosphoform, it is sensitive to degradation. Since proteasomal degradation is triggered by phosphorylation, we suggest that phosphorylation of Ser 10 favors phosphorylation of the PEST sequence, whereas O-GlcNAcylation of Ser 10 prevents it.
Phosphorylation at Ser 10 Controls ⌬Lf Transcriptional Activity-Because OA treatment increases ⌬Lf transcriptional activity, we next questioned whether the phosphoform might be responsible for gene transactivation. Using immunoprecipitation with the M2 antibody and probing the resulting blot with an anti-Ser(P) antibody, we studied the phosphorylation status of ⌬Lf (Fig. 5A, left). Phosphatase treatment markedly abrogated the phosphorylation signal, confirming antibody specificity (right). Immunoblotting (Fig. 5A) showed that ⌬Lf and its Ser 10 mutants exist as phosphoforms. The decreased phosphorylation signals observed under GlcNH 2 treatment confirm that phosphorylation and O-GlcNAcylation may alternate on some of the sites. Therefore, the weaker phosphorylation signal observed with the hyperglycosylated ⌬Lf S10ϩ isoform (lane 9) compared with control (lane 5) strongly suggests that the O-GlcNAc/phosphorylation interplay targets the Ser 10 site. However, because ⌬Lf M4 is phosphorylated, ⌬Lf is also phosphorylated on sites different from the O-GlcNAc/P interplay sites.
We next performed gene reporter analyses as described above and investigated whether phosphorylation at Ser 10 controls ⌬Lf transcriptional activity. Fig. 5B shows that, compared with ⌬Lf WT , ⌬Lf S10ϩ transcriptional activity was inhibited 2-fold as in Fig. 2E, whereas the transcriptional activity of ⌬Lf S10A was increased 1.5-2-fold, and that of ⌬Lf S10D was increased 4.5-5-fold. The prevention of glycosylation of Ser 10 favored transcription, suggesting that O-GlcNAcylation at this site inhibits ⌬Lf transcriptional activity. Mimicking phosphorylation at Ser 10 rendered ⌬Lf even more active than ⌬Lf M4 (Fig.  2E) and strongly suggests that the presence of a phosphate group on this site favors transactivation (Fig. 5B). This result reinforces the status of ⌬Lf S10D as a constitutive phosphorylated mutant. Because Ser 10 is present in a basic environment ( 1 MRKVRGPPVSCIKR 14 ) within a putative truncated DNAbinding domain, we constructed a ⌬Lf ⌬1-14 mutant in which the first 14 amino acid residues were deleted. Surprisingly, this deletion did not affect ⌬Lf transcriptional activity (Fig. 5B), suggesting that the ⌬Lf DNA-binding domain must be located at the hinge region (31,32). Because O-GlcNAcylation and phosphorylation might occur on neighboring sites, we screened the vicinity of Ser 10 and identified Ser 16 that might be used as a replacement target by kinases. We therefore constructed a ⌬Lf S16D mutant in order to mimic phosphorylation at this site. Expression of this mutant led to a basal expression level of the reporter gene (Fig. 5B), showing that constitutive phosphorylation at this locus does not lead to increased transactivation as for ⌬Lf S10D and does not take over when the major acceptor site is invalidated, confirming the key role of Ser 10 .
Because ⌬Lf transcriptional activity is altered by O-GlcNAcylation at Ser 10 and an OGT⅐OGA complex has been described in the vicinity of transcription factors bound to their response elements (8,9), we next considered whether glycosylated ⌬Lf binds DNA. Using a ChIP assay we investigated the binding of the different Ser 10 mutants compared with WT. As shown in Fig. 5C, specific ChIP PCR products were detected for each mutant. It is interesting to note that the PCR product signals for ⌬Lf WT and ⌬Lf S10ϩ were equivalent, whereas treatment with GlcNH 2 led to a weaker signal for both, suggesting that fewer promoter sites were occupied. Because ⌬Lf WT and ⌬Lf S10ϩ were equivalently expressed (Fig. 4B) even under GlcNH 2 treatment (Fig. 1, B and C, respectively), we suggest that glycosylation inhibits binding to DNA and that among the ⌬Lf intracellular pool, only the Ser 10 phosphoforms bind ⌬LfRE. These results were confirmed by the detection of a PCR product signal comparable with that of WT for ⌬Lf S10D , which was poorly expressed but extremely active (Fig. 4B), suggesting that a large proportion of ⌬Lf S10D binds ⌬LfRE (Fig. 5C). The detection of a weaker signal for ⌬Lf S10A , which was expressed similarly to WT, shows that without phosphorylation and glycosylation at Ser 10 , ⌬Lf still binds DNA, but its capacity to occupy promoter sites is reduced. Real time PCR was next performed to quantify promoter site occupancy (Fig. 5D). The qPCR data confirmed the PCR results except that promoter site occupancy for ⌬Lf S10ϩ and ⌬Lf S10D was twice as high as that of WT. Treatment with GlcNH 2 led to a 0.5-fold promoter site occupancy compared with WT, confirming that favoring GlcNAcylation prevents DNA binding.  ). B, relative luciferase activity of ⌬Lf and its O-GlcNAc mutants. HEK 293 cells were co-transfected with pGL3-S1 Skp1 -Luc vector and pcDNA3.1-⌬Lf WT or Ser 10 mutant constructs. Relative luciferase activities are expressed as described under "Experimental Procedures" (n Ն 9; **, p Ͻ 0.01). C and D, O-GlcNAcylation inhibits DNA binding. The in vivo binding of ⌬Lf and its Ser 10 mutants to the Skp1 promoter fragment was examined in HEK 293 cells treated or not with GlcNH 2 (n ϭ 3). Cross-linked DNA-⌬Lf complexes were immunoprecipitated, and precipitated DNA fragments were PCR-amplified (C) or real time PCR-amplified (D) with specific primers covering the ⌬LfRE present in the Skp1 promoter. The PCR-amplified DNA purified from the sonicated chromatin was used as input and loading control. ChIP assays were performed using M2, anti-rabbit IgG antibodies as a nonspecific control (irrelevant; IR) and without antibody (NIP). Amplification of the albumin promoter region was used as a negative control. E, ⌬Lf transactivation complex is not O-GlcNAcylated. Re-ChIP was performed as above for the ChIP assay with some modifications. The first immunoprecipitation was performed using M2, RL2, anti-Ser(P) or anti-HA antibodies. Then, prior to reversal of protein-DNA cross-linking, the chromatin fragments were subjected to reprecipitation using M2, irrelevant antibody, or no antibodies (n ϭ 3). Error bars, S.D. IB, immunoblot; IP, immunoprecipitation.
In addition, we performed a re-ChIP assay to investigate whether ⌬Lf or a ⌬Lf-associated transcriptional complex binds to the endogenous human Skp1 promoter in vivo as a phosphoform. Moreover, since the half-life of ⌬Lf is short as a phosphoform, we studied the possibility that ⌬Lf also exists as a ubiquitinated isoform on DNA. Using a re-ChIP assay, we showed that phosphorylated and ubiquitinated but not O-GlcNAc ⌬Lf complexes were specifically co-localized on the Skp1 promoter fragment (Fig. 5E). The slight amplification observed in panel 1 (NIP and IR) might be due to the fact that the two immunoprecipitations were performed with the same antibody, increasing the background level. Our results clearly demonstrate that phosphorylated and/or ubiquitinated ⌬Lf or ⌬Lf associated with phosphorylated and/or ubiquitinated proteins specifically binds the Skp1 promoter segment with close proximity in vivo, whereas glycosylated ⌬Lf or ⌬Lf associated with glycosylated proteins does not. Because ⌬Lf is ubiquitinated at Lys 379 and phosphorylated at Ser 10 , we suggest that these two post-translational modifications might be concomitantly present on ⌬Lf bound to DNA and may both be determinant in its activity. Further work will have to be done to demonstrate such a partnership, and for that, specific antibodies against the phospho-Ser 10 or the Ub-Lys 379 or poly-Ub-Lys 379 will be obtained.

DISCUSSION
O-GlcNAc/P modification of transcription factors modulates their transcriptional activity by regulating their turnover, traffic, binding to DNA, or cofactor recruitment. ⌬Lf is a transcription factor controlling the expression of key molecular actors and as such should be highly regulated. In this study, we demonstrated that it is alternatively O-GlcNAcylated or O-phosphorylated at Ser 10 and that these two alternative modifications play distinct roles in modulating its turnover and transcriptional activity.
The concentration of transcription activators and the rate of their degradation are under the control of the proteasome, and there is direct evidence that a switch between O-GlcNAcylation and phosphorylation regulates the process. Phosphorylation drives proteins to degradation via the capping of PEST hydroxyl groups, whereas O-GlcNAcylation hinders it mainly by competing for and masking these hydroxyl groups from kinases. Numerous proteins, such as the transcription factor Sp1 (14), the estrogen receptor (49), the eukaryotic initiation factor eIF2a-p67 (50), or p53 (16), are protected from proteasomal degradation by O-GlcNAcylation. Here, we show that ⌬Lf has a short half-life compatible with its function and is stabilized when Ser 10 is O-GlcNAcylated. Moreover, we showed that Ser 10 is not present within a phosphodegron, which is a recognition signal for Ub ligases. The ⌬Lf degradation motif ( 391 KSQQSSDPDPNCVD 404 ) is conserved in Lf from different species, and the mutation of all three Ser residues led to increased stability of the protein, clearly confirming the functionality of this motif. Mutation of each Ser separately indicated that they behave similarly, suggesting that they are equivalent targets of kinases due to their proximity, but we do not know whether they are also substituted with GlcNAc moieties. YinOYang l.2 server predictions indicated Ser 292 and Ser 395 as OGT targets but with low scores. The ⌬Lf M4 isoform is not glycosylated, suggesting that no further glycosylation sites are present, but we cannot exclude glycosylation of the PEST motif only when Ser 10 is glycosylated or the possibility that the ⌬Lf M4 isoform, which is extremely unstable, exists only as a phosphorylated PEST isoform.
We next investigated Ub targets by mutating lysine residues neighboring the PEST motif and demonstrated that ⌬Lf ubiquitination occurs on Lys 379 and Lys 391 with Lys 379 as the main target. The ⌬Lf KK double mutant was devoid of Ub, confirming that only these two residues are involved. The formation of Ub ladders observed with ⌬Lf K379 and ⌬Lf K391 also revealed that, despite the possibility of its multimonoubiquitination, ⌬Lf undergoes polyubiquitination. Unexpectedly, the ⌬Lf PEST mutant was still ubiquitinated, suggesting the existence of other degradation motifs. ⌬Lf is involved in S phase control and should be ubiquitinated via the SCF complex, but it is possible that another complex, such as anaphase promoting complex/ cyclosome, might be involved. Interestingly, ⌬Lf possesses a 475 RSNLCAL 491 sequence, which may behave as a potential RXXLXX(L/I/V/M) D-box motif (ELM D-box entry), the target of anaphase promoting complex/cyclosome (51). The presence of two degradation motifs suggests that ⌬Lf may be degraded throughout the cell cycle. Nevertheless, further work has to be done in order to prove the functionality of this D-box.
The relationship linking O-GlcNAcylation and the Ub pathway has not yet been elucidated. Although Yang et al. (16) demonstrated that O-GlcNAcylation inhibits ubiquitination of p53, a recent study by Guinez et al. (21) shows that O-GlcNAc and Ub can coexist on the same protein and suggests that the Ub/O-GlcNAc ratio may send proteins either to destruction or repair. Here, we demonstrated that enhancement of the O-GlcNAc status within the cells inhibited ⌬Lf ubiquitination, and the absence of Ser 10 as in the ⌬Lf S10A mutant was accompanied by a decrease in polyubiquitination, suggesting that this modification of ⌬Lf S10ϩ and ⌬Lf WT only occurs on the phosphoforms. Phosphorylation at Ser 10 , by acting through the creation of a negatively charged region and/or the triggering of transient conformational changes, may lead to phosphorylation at the PEST locus, conferring a priming site role on Ser 10 .
The O-GlcNAc/P interplay also modulates transcriptional activity. O-GlcNAcylation directly activates FoxO1 (52), p53 (53,54), and NF-B (55) and Sp1 indirectly via cofactors (14,15), whereas it inhibits c-Myc (24) and mouse estrogen receptor ␤ (49). In this work, we have demonstrated that GlcNAcylation inhibits ⌬Lf transcriptional activity, whereas phosphorylation activates it, and that Ser 10 is central to this regulation. An absence of modification at Ser 10 leads to gene transactivation, whereas phosphomimetism increases it, confirming the inhibitory role of glycosylation. Because the expression of ⌬Lf S10ϩ is much greater than that of ⌬Lf S10D , we suggest that ⌬Lf exists normally in the cell as a pool of stable but inactive glycoforms that, under appropriate stimuli, become activated by phosphorylation and sensitive to degradation. However, another explanation is that only the phosphoform is present in the nucleus. Nucleocytoplasmic traffic may be regulated via O-GlcNAcylation because the modification of the O-GlcNAc status leads to a change in the cellular distribution of Tau (56), Alpha4, and Sp1 (57) but does not influence Stat5a traffic (58).
Here, we showed that ⌬Lf-GFP traffic was not affected by GlcNH 2 or OA treatment. But even if the nucleocytoplasmic traffic is not governed by the O-GlcNAc/P interplay, because the OGT⅐OGA complex and kinases are present within both compartments (59,60), nuclear ⌬Lf might exist only as a phosphoform.
Phosphorylated transcription factors are usually more competent to bind DNA and activate transcription than their non-phosphorylated counterparts, but there is direct evidence for the involvement of O-GlcNAcylation. PDX-1 O-GlcNAcylation increases its ability to bind DNA (61) and enhances p53 DNA binding by hiding an inhibitory domain at the N terminus (54). O-GlcNAcylation of HIC1 does not affect its specific DNA binding (62), and whatever the modifications present on Stat5, it binds its response element similarly (58). However, O-GlcNAcylation at the C terminus of Sp1 abolishes homopolymerization and dramatically affects its function (15).
In this study, we demonstrated that in vivo ⌬Lf binding to ⌬LfRE occurred with the unmodified or the phosphomimetic Ser 10 isoform but decreased when O-GlcNAcylation was increased, suggesting that the glycoform is unable to bind DNA. ⌬Lf S10A bound DNA and transactivated transcription at a basal level, but given the dynamic nature of the O-GlcNAc/P interplay, it is doubtful whether an unmodified ⌬Lf isoform exists. Nevertheless, we can infer that transactivation by ⌬Lf is a twostep process, starting at a basal level and increasing with phosphorylation, as depicted in Fig. 6. Moreover, using the re-ChIP assay, we were able to show that the ⌬Lf transcriptional complex linked to ⌬LfRE is phosphorylated. Our data demonstrate that O-GlcNAcylation at Ser 10 inhibits DNA binding, whereas phosphorylation favors it and promotes transactivation.
The O-GlcNAc/P content fluctuates during cell cycle progression. A recent study showed that increasing O-GlcNAc levels induces a slowing down of both S and G 2 /M phases, whereas a reduced O-GlcNAc level impairs the G 1 /S checkpoint transi-tion (63). Because temporal control of Ub-proteasome-mediated protein degradation is critical for normal G 1 and S phase progression, ⌬Lf modifications may switch between glycosylation and phosphorylation, depending on the cell cycle phase. Progression of the cell cycle requires degradation of cyclins and cyclin inhibitors. At the G 1 /S check point, Skp1, one of the targets of ⌬Lf, is involved in the process when associated with the SCF complex (64,65). Thus, O-GlcNAcylation of ⌬Lf, by down-regulating Skp1 expression, may alter SCF activity, whereas phosphorylation of ⌬Lf may increase it. Regulation of the transcriptional activity of ⌬Lf by the O-GlcNAc/P interplay may therefore modulate the Ub-proteasome-mediated degradation of cell cycle regulators. Furthermore, we demonstrated that ⌬Lf is itself ubiquitinated; thus, its turnover could be regulated by feedback control via overexpression of Skp1. On the other hand, ubiquitination also occurs on the ⌬Lf⅐DNA complex. Modification by Ub is not only a destruction signal but also determines membrane receptor internalization, sorting at the endosomal compartment, activation of DNA repair, or transactivation of transcription factors, such as c-Myc and SRC-3 (66 -68). As an example, SRC-3 is first activated by multi-(mono)ubiquitination and then polyubiquitinated prior to degradation. Therefore, ⌬Lf might require concomitant preubiquitination and phosphorylation as a transcriptional activation signal before being degraded as a polyubiquitinated isoform.