FIG 5.

E4orf4 induces Lats kinase-independent hyperphosphorylation of YAP. (A) H1299 lung carcinoma cells were infected with adenoviral vectors expressing either GFP (AdGFP) or HA-E4orf4 (AdHA-E4orf4) at a multiplicity of infection (MOI) of 35 and harvested, and whole-cell extracts (WCE) were analyzed by SDS-PAGE and Western blotting using the indicated antibodies. WCEs also were treated with λ phosphatase as described in Materials and Methods. (B) Analysis similar to that described for panel A, with MDA-MB-231 breast cancer cells infected with adenoviral vectors expressing either GFP (AdGFP) or HA-E4orf4 (AdHA-E4orf4) at an MOI of 50. (C) Analysis of YAP phosphorylation was determined in cells expressing wild-type HA-E4orf4, class I mutant HA-R81A/F84A, or class II mutant K88A expressed from transfected plasmid cDNAs. Immunoprecipitates obtained using anti-HA antibodies were blotted using anti-YAP phosphoserine-127, anti-FLAG, or anti-HA antibodies as indicated. An asterisk indicates an unspecific band.