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. 2010 Jan 1;24(1):72–85. doi: 10.1101/gad.1843810

Figure 4.

Figure 4.

The Hippo pathway induces YAP S381 phosphorylation and YAP–β-TRCP interaction. (A) pYAP (S381/384) antibody preferentially recognizes S381/384-phosphorylated YAP. YAP wild type or mutants were coexpressed with Mst2 and Lats2 in HEK293 cells. Flag-YAP was then immunoprecipitated and one set of the immunoprecipitates was treated with λ protein phosphatase as indicated. The immunoprecipitated YAP phosphorylation and protein levels were then detected by anti-pYAP (S381/384) antibody and anti-Flag antibody, respectively. (B) Mst2 and Lats2 induce YAP-S381/384 phosphorylation. Indicated plasmids were cotransfected into HEK293 cells. (KR)Kinase-inactive mutants. YAP phosphorylation was shown by a pYAP (S381/384)-specific antibody. (C) Lats knockdown significantly attenuates YAP-S381/384 phosphorylation. HA-YAP was expressed in HEK293 cells. Lats1 and Lats2 knockdown was achieved by two rounds of siRNA transfection with 24-h intervals. YAP phosphorylation was shown by a pYAP (S381/384)-specific antibody. Anti-HA Western blot indicates YAP protein level. Lats knockdown efficiency was shown by an antibody against both Lats1 and Lats2. (D) Mst2 and Lats2 stimulate YAP–β-TRCP interaction. Indicated plasmids were transfected into HEK293 cells. β-TRCP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated YAP was shown by anti-HA Western blot. (E) Dominant-negative Mst2 and Lats2 inhibit YAP–β-TRCP interaction. Indicated plasmids were transfected into HEK293 cells. (KR) Kinase-inactive mutants. YAP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated β-TRCP was shown by anti-HA Western blot. (F) Lats knockdown inhibits YAP–β-TRCP interaction. Indicated plasmids were cotransfected with or without Lats1/2 siRNA. Lats1/2 siRNA transfection was repeated once after 24 h. β-TRCP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated YAP was shown by anti-HA Western blot. Lats knockdown efficiency was shown by an antibody against both Lats1 and Lats2.