Fig 2: All discernable domains of SAV1 are required for MST2 activation.(A) Immunoblots of lysates of 293FT cells co-transfected with the indicated Myc-MST2 and HA-SAV1 or HA-RASSF1A plasmids. (B) Immunoblots of lysates of 293FT cells co-transfected with Myc-SAV1 and the indicated FLAG-MST2 plasmids. (C) Immunoblots of lysates of 293FT cells co-transfected with Myc-MST2 and the indicated FLAG-SAV1 plasmids. (D) Immunoblots of cell lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated Myc-SAV1 plasmids. (E) Total MST2 and pT180 blots of the auto-kinase reactions by the indicated MST2-SAV1 complexes at the indicated time points (left panel). The kinetic profiles of T180 autophosphorylation of MST2-FL/SAV1-∆N198 (red line) and MST2-FL/SAV1-∆N268 (black line) are shown on the right. The relative anti-pT180 intensities were normalized against MST2-FL without PP2A phosphatase treatment (100%). Data are representative of at least two independent experiments. (F) Immunoblots of lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated N-terminal truncation of Myc-SAV1 constructs. (G) 293FT cells were co-transfected with FLAG-MST2 and Myc-SAV1 plasmids. The total cell lysates were blotted with the indicated antibodies. (H) 293FT cells were co-transfected with the indicated plasmids encoding FLAG-SLMAP, HA-MST2 and Myc-SAV1. Anti-FLAG IP and the total cell lysates (input) were blotted with the indicated antibodies.

Fig 3: The N-terminal region of SAV1 directly inhibits PP2A.(A and B) Association of PP2A A-C with the N terminal region of SAV1. 293FT cells were mock transfected or transfected with the indicated FLAG-SAV1 plasmids. The total cell lysates (input) and anti-FLAG IP were blotted with the indicated antibodies. Asterisk designates IgG. (C) UV traces of the PP2A A and SAV11-90 complex (blue line), PP2A A alone (magenta line), SAV11-90 alone (green line), and molecular weight standard (dashed line, corresponding molecular weight indicated in kDa) fractionated on a Superdex 200 gel filtration column, respectively. The underlined fractions were separated on SDS-PAGE and stained with Coomassie. (D) The N-terminal region of SAV1 directly inhibits dephosphorylation of MST2 pT180 by PP2A. Quantitative MST2 pT180 and total MST2 immunoblotting of phosphatase reactions containing human MST2 and SAV11-90 at the indicated concentrations. The relative anti-pT180 intensities were normalized against MST2 without PP2A phosphatase treatment (100%). Data are representative of at least two independent experiments.

Fig 4: CORO7 forms a complex with the components of the Hippo pathway.A–C, schematic representation of domains and truncated constructs of CORO7 (A), LATS1 (B), and SAV1 (C) used in coimmunoprecipitation assays. D–F, HA-LATS1 (D), HA-MST2 (E), or HA-SAV1 (F) was expressed together with wild-type (WT), N-terminal (N), or C-terminal (C) Flag-CORO7 in HEK293T cells. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The whole cell lysate (WCL) samples were loaded for indicating the expression levels. G, HA-CORO7 was transfected with the Flag-tagged truncated forms of LATS1 in HEK293T cells, and the lysates were immunoprecipitated by anti-Flag antibody. Subsequently, they were immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. H, Flag-CORO7 was transfected with the HA-tagged truncated forms of SAV1 in HEK293T cells. The lysates were immunoprecipitated by anti-HA antibody and immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. I, HEK293T cells were transfected with HA-tagged truncated forms of SAV1, HA-MST2, and Flag-CORO7. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. Data information: Flag-tagged vector and HA-tagged vector were transfected as negative controls (−). All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBM, FERM-binding motif; HA, hemagglutinin; LATS1, large tumor suppressor kinase 1/2; MOB1, MOB kinase activator 1; MST2, mammalian sterile 20-like kinase 2; SARAH, Sav/Rassf/Hpo; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate.

Fig 5: SAV1 stimulates MST1/2 activation through antagonizing STRIPAK.(A) SAV1-MST2 forms a complex with STRIPAKSLMAP. 293FT cells were co-transfected with FLAG-SAV1 and the indicated Myc-MST2 plasmids. The total cell lysates (input) and anti-FLAG IP were blotted with the indicated antibodies. (B) Immunoblots with the indicated antibodies of lysates of control MCF10A cells and MCF10A cells with SLMAP, SAV1, or both deleted. Asterisk denotes a non-specific band. (C) Relative expression of YAP target genes CTGF and CYR61 in control, SLMAP KO, SAV1 KO, and SLMAP/SAV1 DKO MCF10A cells. The gene expression was measured by quantitative real-time RT-PCR and normalized to GAPDH. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; **p<0.01; and ***p<0.001). (D) Model for SAV1-dependent MST2 activation during Hippo signaling.