Figure 4.

TRAP6 inhibits Lats kinase activity. (A) HEK293A cells were treated with 2 μM TRAP6 for 1 h. The activity of immunoprecipitated MST1 was determined by in vitro kinase assay using GST-Mob as a substrate. P³² incoporation into GST-Mob was detected by autoradiograph. (B) HEK293A cells were cotransfected with the indicated plasmids. After transfection, cells were treated with 2 μM TRAP6 for 1 h. YAP phosphorylation was determined. (C) HEK293A cells were pretreated with 2 μg/mL C3 for 4 h and then incubated with TRAP6 for the indicated times. The presence of FBS is indicated. Lats1 immunoprecipitated from the cell lysates was subjected to in vitro kinase assays using GST-YAP as a substrate. YAP phosphorylation was detected by phospho-YAP (S127) antibody. (D) Stimulation conditions were similar to those in C. Lats1 was immunoprecipitated with anti-Lats1 antibody from the cell lysates and then subjected to immunoblotting as indicated. (E) Flag-YAP was cotransfected with wild-type or a kinase-dead (KR) mutant of HA-Lats2. After transfection, cells were treated with 2 μM TRAP6 for 1 h. (B,E) Phosphorylation of Flag-YAP was determined by phos-tag gel.