Fig 2: Signals activating the Hippo pathway regulate complex formation between CORO7 and the components of the pathway.A, 24 h after seeded to 15% confluency, HEK293T cells transfected with HA-MST2, HA-SAV1, and Flag-CORO7 were deprived of serum (FBS) or treated with 0.25 μg/ml LatB for 1 h. The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels. The WCL samples were also immunoblotted with anti-pYAP antibody. B, HEK293T cells were transfected with Flag-CORO7, Myc-SAV1, and HA-NF2. Three different levels of HA-NF2 were expressed. The cell lysate samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA, anti-Flag, and anti-Myc antibodies. The WCL samples were loaded for indicating the expression levels. C, HEK293T cells expressing Myc-CORO7, HA-NF2, and Flag-SAV1 were deprived of serum (FBS). The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with the same antibodies as in (B). The WCL samples were loaded for indicating the expression levels. All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBS, fetal bovine serum; HA, hemagglutinin; LatB, latrunculin B; MST2, mammalian sterile 20-like kinase 2; NF2, neurofibromatosis type II; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate; YAP, yes-associated protein.

Fig 3: Crystal Structure of the SAV1 WW12ex Homodimer(A) Ribbon diagram showing the overall structure of the SAV1 WW12ex dimer.(B) Surface combined with ribbon-stick model showing the interaction between WW12 and the extension in SAV1. Residues in WW12 that are hydrophobic, negatively charged, and positively charged are colored in yellow, red, and blue, respectively.(C) Superimposition of the structures of the WW tandems from SAV1, KIBRA (PDB: 6J69), and MAGI2 (PDB: 6JJZ), each in complex with their respective targets.(D) Combined surface and ribbon-stick model showing the structural basis for the coupling of the two WW domains by the 12-residue linker in SAV1. The 12-residue linker forms 3 continuous β-turns and is colored in green.(E) Amino acid sequence alignment of the WW tandems from SAV1, KIBRA, and MAGI2 based on the structural alignment shown in (C)Also see Figure S2 and Table S1.

Fig 4: Detailed Interactions between WW12 and the Extension of SAV1(A) Combined ribbon and stick model showing the detailed interactions between WW12 and the extension in the SAV1 WW12ex dimer.(B) Ribbon-stick model showing detailed interactions of the classical type I WW/PPxY motif binding observed in the KIBRA WW tandem in complex with dendrin PY23.(C) Comparisons of the conformation of the “PPPI” (top) and “PRY” (bottom) sequences observed in the SAV1 WW12ex structure with the conformation of a classical “PPSY” sequence bound to a type I WW domain.(D and E) Ribbon-stick model showing that WW1 (D) and WW2 (E) of SAV1 are typical type I WW domains containing all key residues required for binding to PY motif targets.(F and G) ITC-based measurements showing that WW12 (F) and WW12PY (G) of SAV1 can robustly bind to dendrin PY23 due to truncation-induced releases of the autoinhibition of SAV1 WW12ex.Also see Figures S2 and S3.

Fig 5: 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.