Fig 1: YAP interacts with the mTORC2 component Sin1 and is phosphorylated on serine 436. (A) Co-immunoprecipitation of endogenous Sin1 and YAP. U87 cell extracts were immunoprecipitated with either Sin1 or YAP antibodies and immunoprecipitates subjected to immunoblot analysis for the indicated proteins. Lane 1, beads, no antibody; lane 2, immunoprecipitation with an irrelevant antibody (control IgG), lane 3, input cell lysate; lane 4 indicated immunoprecipitate probed with antibodies for the indicated proteins. (B) An in vitro kinase assay was performed with Rictor-immunoprecipitated mTORC2 from U87 cell lysates in the presence of recombinant AKT, native YAP or mutant YAP-5A as indicated in the presence of ?32P-ATP. Reactions were separated by SDS-PAGE followed by autoradiography. As negative controls, immunoprecipitated Mnk1 kinase or Raptor-immunoprecipitated mTORC1 was also used in the presence of YAP1, neither of which displayed detectable phosphorylation. (C) An mTORC2 in vitro kinase assay was performed utilizing Rictor immunoprecipitates isolated from U87 cells and incubated with purified native or S436A mutated recombinant Flag-tagged YAP for the indicated time points. Reactions containing native YAP were performed in the absence or presence of PP242 as shown. Kinase reactions were subsequently immunoblotted using a phospho-specific antibody generated against phospho-Ser436-YAP, Flag or Rictor antibodies for detection. (D) Effects of insulin stimulation in U87 cells. Cells were stimulated with insulin (10 nM, 5 h), lysed and extracts immunoblotted for the indicated proteins. (E) U87 cells were treated with siRNAs targeting Rictor or non-targeting control (siCtrl) for 24 h and cell lysates analyzed by immunoblotting for the indicated proteins. (F) As in (E) except U87 cells were treated with siRNAs targeting Sin1 or non-targeting control (siCtrl).