1. Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation
Changmin Peng, Yue Zhu, Wanjun Zhang, Qinchao Liao, Yali Chen, Xinyuan Zhao, Qiang Guo, Pan Shen, Bei Zhen, Xiaohong Qian, Dong Yang, Jin-San Zhang, Dongguang Xiao, Weijie Qin, Huadong Pei 
Molecular Cell 
Volume 68, Issue 3, Pages e5 (November 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 68, 591-604.e5DOI: (10.1016/j.molcel.2017.10.010)
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3. Figure 1 OGT Regulates YAP Phosphorylation and Activation
(A) YAP O-GlcNAcylation was regulated by glucose starvation. 293T cells were treated with or without glucose for 24 hr, cell lysates were immunoprecipitated with anti-YAP antibody, and YAP phosphorylation and O-GlcNAcylation were checked with the indicated antibodies. Representative experiment result of three biological replicates is shown.
(B) The quantifying analysis of the O-GlcNAcylation band in (A) by ImageJ software.
(C) The HBP directly regulated Hippo-YAP activity. Upper panel: 293T cells were transfected with GFPT1 siRNAs and treated with or without 15 mM GlcNAc for 36 hr, and cell lysates were immunoblotted with the indicated antibodies. Lower panel: cell lysates from upper panel were immunoprecipitated with anti-YAP antibody; YAP O-GlcNAcylation was checked with the indicated antibodies. Representative experiment of three biological replicates is shown.
(D) Knockdown of OGT regulates YAP serine 127 phosphorylation. 293T cells were transfected with the indicated siRNA; cell lysates were immunoblotted with indicated antibodies. Representative experiment of three replicates is shown.
(E) OGT inhibitor (OMSI-1) treatment regulates YAP serine 127 phosphorylation. 293T cells were treated with 15 or 30 μM OSMI-1 for 24 hr, and cell lysates were immunoblotted with indicated antibodies. Representative experiment of three replicates is shown.
(F) Knockdown of OGT decreased YAP target gene expression. Relative expression of ANKRD1, CTGF, CYR61, and GLUT3 mRNA in 293T cells was determined by qRT-PCR. Here YAP and TAZ knockdown is positive control. Transcript levels were determined relative to actin mRNA levels and normalized relative to control cells. The results represent the means (±SEM) of three independent experiments.
(G) OGT inhibitor (OSMI-1) inhibits YAP transcriptional activity. 293T cells were treated with 15 or 30 μM OSMI-1 for 24 hr, and then transcription of YAP target genes was detected as in (F). The results represent the means (±SEM) of three independent experiments.
(H) Knockdown of OGT regulates YAP localization. HeLa cells were transfected with two different OGT siRNAs, and YAP localization was detected by immunostaining with the YAP monoclonal antibody. Left panel: representative images. Right panel: statistical analysis of left panel. Representative experiment of three replicates is shown.
(I) Knockdown of OGT disrupts YAP-TEAD interaction and stimulates YAP θ/LATS1 interaction. 293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with FLAG beads and co-precipitated proteins were detected by western blot. Representative experiment of three replicates is shown.
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4. Figure 2 OGT Interacts with and O-GlcNAcylates YAP
(A) The co-immunoprecipitation of OGT and YAP was examined. Representative blots of three independent experiments are shown.
(B) OGT directly interacts with YAP in vitro. Left: GST pull-down assay of purified His-YAP with the purified GST-OGT from E.coli. Right: GST pull-down assay of the purified His-OGT with the purified GST-YAP from E.coli. Representative blots of three independent experiments are shown.
(C) OGT directly O-GlcNAcylates YAP in vitro. In vitro glycosylation assay was performed with GST-YAP as substrate, which was incubated with purified His-OGT, and the GlcNAcylated YAP was blotted with RL2 antibody and the total GST-YAP protein was detected by Coomassie blue staining. Representative blots of three independent experiments are shown.
(D) Overexpression of OGT increased YAP O-GlcNAcylation level. 293T cells stably expressing FLAG-YAP were transfected with indicated plasmids. Cell lysates were immunoprecipitated with FLAG beads and immunoblotted as indicated. Representative blots of three independent experiments are shown.
(E) Knockdown of OGT decreased YAP O-GlcNAcylation level. 293T cells stably expressing FLAG-YAP were transfected with the indicated shRNA. Cell lysates were immunoprecipitated with FLAG beads and immunoblotted as indicated. Representative blots of three independent experiments are shown.
(F) YAP O-GlcNAcylation is reversed by OGA.
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5. Figure 3 YAP O-GlcNAcylation Mainly Occurs at Serine 109 Site
(A–C) YAP was O-GlcNAcylated by OGT at several residues in vivo. YAP was purified from 293T cells and analyzed by MS to identify the O-GlcNAcylation sites. Three different O-GlcNAcylation sites in YAP are shown. More than three independent experiments were performed.
(D) YAP was O-GlcNAcylated at serine 109 in cells. 293T cells were transfected with the indicated plasmids, and YAP O-GlcNAcylation was analyzed by immunoprecipitation with anti-FLAG antibody and western blot with the indicated antibodies. Three independent experiments were performed.
(E) Identification of the O-GlcNAcylation modification sites of YAP by in vitro glycosylation assay. Purified wild-type or mutant GST-YAP was used as substrates. Coomassie blue staining shows the YAP protein used in the assay. Three independent experiments were performed.
(F) The O-GlcNAcylation of wild-type YAP or serine 109A/3SA mutant was reversed by OGA.
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6. Figure 4 YAP O-GlcNAcylation Regulates Its Activation
(A) The serine 109 O-GlcNAcylation of YAP affects serine 127 phosphorylation. L3.6 knockout cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with FLAG beads and western blotted with the indicated antibodies. One representative experiment of three independent experiments is shown.
(B) YAP O-GlcNAcylation influenced its interaction with LATS1, , and TEAD. The indicated plasmids were transfected into 293T cells, and cell lysates were immunoprecipitated with FLAG beads and immunoblotted as indicated. One representative experiment of three independent experiments is shown.
(C) O-GlcNAcylation of YAP regulated its subcellular localization. The indicated plasmids were transfected into L3.6 YAP knockout cells, and YAP localization was detected by immunostaining with the YAP monoclonal antibody. Left panel: representative images. Right panel: statistical analysis of left panel. One representative experiment of three independent experiments is shown.
(D) YAP O-GlcNAcylation-deficient mutant inhibits YAP target gene expression. The indicated plasmids were transfected into L3.6 YAP knockout cells, and mRNA levels of YAP target genes were measured by qRT-PCR (error bars represent mean ± SEM from n = 3 independent experiments).
(E) YAP O-GlcNAcylation-deficient mutant abolishes YAP transcriptional activity. The indicated plasmids were co-transfected with a 5× UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent mean ± SEM from n = 3 biological replicates).
(F) GST-YAP with or without O-GlcNAcylation was generated by in vitro glycosylation assay, LATS1 was purified from E.coli as indicated in STAR Methods, and the direct interaction between YAP (with or without O-GlcNAcylation) and LATS1 was detected by in vitro pull-down assay. One representative experiment of three independent experiments is shown.
(G) The direct interaction between YAP (with or without O-GlcNAcylation) and TEAD was detected as in (F). Three independent experiments were performed.
(H) YAP O-GlcNAcylation directly inhibited YAP serine 127 phosphorylation by LATS1 in vitro. GST-YAP/serine 109A/3SA and LATS1 were purified from E.coli as indicated in STAR Methods, and GST-YAP/serine 109A/3SA was glycosylated by in vitro glycosylation assay, and then in vitro kinase assay was performed using GST-YAP/serine 109A/3SA as substrate and using LATS1 as kinase. Three independent experiments were performed.
(I) The direct interactions between GST-YAP/serine 109A/3SA (with O-GlcNAcylation) and LATS1 were detected as in (F). Three independent experiments were performed.
(J) The direct interactions between GST-YAP/serine 109A/3SA (with O-GlcNAcylation) and TEAD were detected as in (F). Three independent experiments were performed.
(K) OGT itself did not affect the interaction between LATS1 and YAP in vitro. GST-YAP, His-OGT, and LATS1 were purified from E.coli as indicated in STAR Methods, and the interaction between LATS1 and GST-YAP was detected by pull-down assay and western blot.
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7. Figure 5
YAP O-GlcNAcylation Regulates Its Activity in an AMPK-Independent Manner
(A). Knockdown of LATS1/2 interrupted O-GlcNAcylation-induced YAP activation. LATS1/2 siRNAs were transfected into 293T cells. Cells were transfected with OGT siRNAs after 24 hr. Then YAP and its upstream kinase phosphorylation were checked by western blotting. Three independent experiments were performed.
(B) 293T cells were transfected with the indicated siRNA, and the interaction between YAP and LATS1/14-3-3/TEAD was checked by immunoprecipitation and western blotting. Three independent experiments were performed.
(C) 293T cells were treated as (A), and then the transcription of genes (ANKRD1, CTGF, CYR61, and GLUT3) was measured by qRT-PCR (error bars represent mean ± SEM from n = 3 independent experiments).
(D) YAP O-GlcNAcylation regulated target gene expression dependent on YAP serine 127 site. The YAP serine 127A and serine 109A/127A double-mutant plasmids were transfected into L3.6 YAP knockout cells, and mRNA levels of ANDRD1, CTGF, GLUT3, and CYR61 genes were measured by qRT-PCR (error bars represent ± SEM from n = 3 independent experiments).
(E) The stoichiometry of phosphorylated or O-Glycosylated YAP at serine 109 in serum starvation condition, normal condition, or OGT-overexpressing cells with normal condition. OGT, OGT-overexpressing cells. Three independent experiments were performed (F and G). YAPS109D mutant had the same effects as serine 109A.
(F) The indicated plasmids were transfected into L3.6 YAP knockout cells, and cell lysates were immunoprecipitated with FLAG beads and immunoblotted as indicated antibodies.
(G) mRNA levels of ANKRD1, CTGF, CYR61, and GLUT3 in above cells were measured by qRT-PCR (error bars represent mean ± SEM from n = 3 independent experiments).
(H) OGT regulated YAP phosphorylation in AMPK-depleted cells. OGT was knocked down in AMPK-depleted 293T cells, and YAP and its upstream kinase phosphorylations were checked by western blotting.
(I) OGT regulated the interaction between YAP and LATS1/14-3-3/TEAD in AMPK-depleted cells. 293T cells were transfected with the indicated siRNA. Cell lysates were immunoprecipitated with anti-YAP antibody and the immunoprecipitates were blotted with the indicated antibodies. Three independent experiments were performed.
(J) Knockdown of OGT in AMPK-deleted cells decreased YAP target gene expression. 293T cells were treated as in (H), and then transcription of genes (ANKRD1, CTGF, CYR61, and GLUT3) was detected by qRT-PCR (error bars represent mean ± SEM from n = 3 independent experiments).
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8. Figure 6
OGT-Mediated O-GlcNAcylation of YAP Regulates Cancer Cell Growth and Tumorigenesis
(A) YAP O-GlcNAcylation regulates cancer cell proliferation. YAP knockout L3.6 cells with or without OGT knockdown were transfected with the indicated plasmids, and cell proliferation was analyzed by MTS assay. Three independent experiments were performed.
(B) YAP knockout L3.6 cells and YAP/OGT double-knockout L3.6 cells were transfected with the indicated plasmids; resulting colonies were fixed and stained with crystal violet. One representative experiment of three independent experiments was shown.
(C) OGT-mediated O-GlcNAcylation of YAP affects tumor growth in xenografted mice. Mice (n = 32) were ectopically implanted with 1 × 106 indicated cells on both sides. Here YAP was knocked down by inducible shRNA. Mice were sacrificed 28 days after implantation and tumor weight was measured (error bars represent ± SEM). Scale bar, 1 cm. ∗p < 0.05, two-tailed t test.
(D) YAP regulates OGT transcription. The OGT transcription was confirmed in L3.6 wild-type and L3.6 YAP knockout cells by qRT-PCR.
(E) OGT was identified as a new TEAD/YAP-regulated gene. 293T cells were transfected with the indicated siRNA and plasmids, and the transcription of CTGF, CYR61, and OGT was measured by qRT-PCR (error bars represent ± SEM from n = 3 independent experiments).
(F) YAP O-GlcNAcylation regulates cancer cell growth not only dependent on OGT. L3.6 YAP knockout cells were transfected with the indicated plasmids, and cell proliferation was analyzed by MTS assay. Three independent experiments were performed.
(G) YAP knockout L3.6 cells were treated as in (F); resulting colonies were fixed and stained with crystal violet. One representative experiment of three independent experiments was shown.
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