Figure 5. YAP is O-GlcNAcylated by OGT at Thr241.

(a) YAP O-GlcNAcylation was measured via HRP-labelled Streptavidin (left) and anti-TAMRA antibodies (middle) in Bel-7402 and SMMC-7721 cells. In vitro O-GlcNAcylation of YAP was measured by incubating purified YAP and OGT proteins with UDP-GlcNAc at 37 °C for 90 min before immunoprecipitation with anti-YAP antibodies, followed by western blot using an anti-O-GlcNAc antibody (right). (b) Enzymatic labelling of O-GlcNAc in WT and T241A YAP-FLAG proteins as analysed by anti-TAMRA antibodies and HRP-labelled Streptavidin in Bel-7402 and SMMC-7721 cells. (c) YAP-FLAG was immunoprecipitated by an anti-FLAG antibody in Bel-7402 and SMMC-7721 cells transfected with WT or T241A YAP-FLAG-expressing plasmids. Phosphorylation of YAP was detected using an anti-p-YAP antibody. (d) Mutation of Thr241 induced LATS1-YAP binding under stimulation of O-GlcNAcylation by combined treatment of GlcNAc (4 mM) and PuGNAc (25 μM). Expression plasmids for YAP-FLAG, WT or T241A were transfected into Bel-7402 and SMMC-7721 cells, and cells were treated as indicated for 24 h before analysis. YAP-FLAG was immunoprecipitated with anti-FLAG antibodies, and the association with LATS1 was measured using anti-LATS1 antibodies. (e) Mutation of Thr241 reduced YAP-promoted transformative phenotypes in vitro. Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with MTT-based assays, soft agar colony formation assays and Caspase 3/7 Glo luciferase assays, respectively, in Bel-7402 and SMMC-7721 cells expressing WT or T241A YAP-FLAG. Scale bar, 500 μm. (f) Mutation of Thr241 diminished YAP-promoted xenograft tumour formation in vivo. The tumour size was measured 72 days after injection of Bel-7402 or SMMC-7721 cells into nude mice. (g) Mutation of Thr241 reduced the half-life of YAP. YAP-FLAG (as indicated) was transfected into Bel-7402 and SMMC-7721 cells, and cells were harvested at the indicated time points after addition of CHX (50 μg ml⁻¹). The expression of YAP-FLAG was normalized to that of GAPDH, and the ‘0 h' point was arbitrarily set to 100%. Representative images are shown, and the data are expressed as the means+s.d. from three independent experiments. **P<0.01 indicates statistical significance. The data from d–f were analysed by Student's t-test.