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. 2020 Apr 9;217(6):e20191817. doi: 10.1084/jem.20191817

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© 2020 An et al.

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Figure 4. — Interplay between pYAP^T83 and pYAP^S127 in response to serum starvation. (A) Validation of the homemade pYAP^T83 antibody. HEK293FT cells were cotransfected with Flag-tagged YAP (WT or T83A mutant) and Myc-MST4 for 48 h. Cell lysates were processed for Flag immunoprecipitation and were analyzed using indicated antibodies (n = 4). (B) Serum starvation triggers concomitant phosphorylation of YAP at Thr83 and Ser127. 293A cells cultured in medium with or without serum for 12 h were harvested and analyzed by Western blotting using indicated antibodies (n = 3, quantification values represent the mean from three repeats). (C) MST4 promotes YAP phosphorylation at Thr83 and Ser127 in response to serum starvation. 293A cells (WT and MST4 KO) were subjected to serum starvation for 12 h and analyzed by Western blotting using indicated antibodies (n = 3, quantification values represent the mean from three repeats). (D) LATS1/2 promotes YAP phosphorylation at Thr83 and Ser127 in response to serum starvation. 293A cells (WT and LATS1/2 KO) were subjected to serum starvation treatment and analyzed by Western blotting (n = 3, quantification values represent the mean from three repeats). (E) Cell lysates derived from HEK293FT cells expressing indicated Flag-tagged YAP proteins were incubated with Flag beads. Immunoprecipitates were subjected to Western blotting analysis using antibodies (n = 2, quantification values represent the mean from two repeats). (F and G) YAP Thr83 phosphorylation constrains YAP^S127A-induced transcriptional activation and CTGF expression. 293A YAP KO cells reconstituted with indicated YAP constructs were subjected to either luciferase assay (F; n = 3, ***, P < 0.001, unpaired t test) or qPCR analysis of mRNA expression of CTGF (G; n = 3, ***, P < 0.001, unpaired t test). (H) YAP Thr83 phosphorylation limits YAP^S127A-induced tumor formation in vivo. Nude mice injected with HGC-27 cells were further infected with lentivirus harboring indicated YAP mutants. Representative images showed the tumors induced by indicated YAP mutants (left panel). The tumor weights were measured and analyzed 3 wk after injection (three mice/group, n = 2 biological repeats, n.s., not significant, **, P < 0.01; ***, P < 0.001, unpaired t test; and one-way ANOVA with Dunnett’s post hoc analysis as compared with vector control). (I) Schematic illustration of the concomitant pYAP^T83 and Ser127 in response to serum starvation. Data are presented as the mean ± SEM. See also Fig. S3.