Fig 2: The crystal structure of the MOB1/NDR2 complex reveals phosphorylation-independent interactions of MOB1 with NDR2 through conserved interfaces. a Overall crystal structure of the complex between human MOB1A (residues 33–216) and human NDR2 (residues 25–88). MOB1 and NDR2 are colored in magenta and green, respectively. The Zinc associated with MOB1 is indicated in gray. Secondary structure elements are highlighted. b The negatively charged surface of MOB1 recognizes positively charged residues on NDR2. The electrostatic surface potential of MOB1 is shown (red: negatively charged residues; blue: positively charged residues). NDR2 is shown as a ribbon representation colored in green. c The MOB1/NDR2 binding surfaces are highly conserved. The residues of MOB1 are gradually colored according to conservation scores (dark red: most conserved residues; dark cyan: least conserved residues). NDR2 is shown as a ribbon representation colored in green. d Structure based sequence alignment of MOB1 from indicated species. MOB1 residues mediating interactions with NDR2 are marked with brown (main-chain hydrogen bonds), green (side-chain hydrogen bonds), and magenta circles (van der Waals interactions). α helices are painted with cyan background. Thr12 and Thr35 phosphorylation sites are indicated by red arrows. e, f The interaction interfaces of MOB1 with the α1 (residues 25–56) and α2 (residues 61–84) helices of NDR2. MOB1 and NDR2 and their key interacting residues are shown in pink and green, respectively. Dashed lines represent hydrogen bonds. Side-chain nitrogen, oxygen, and sulfur atoms of MOB1 and NDR2 residues are colored in blue, red and gold, respectively. The hydrogen bond network connecting the α1 and α2 helices of NDR2 is also illustrated in f. For a stereo image of a portion of the electron density map see Supplementary Fig. 18

Fig 3: MOB1 binds differently to the NTR domains of NDR2 and LATS1 through key residues. a Structure based sequence alignment of the conserved N-terminal regulatory domain of NDR/LATS kinases that is required for MOB1 binding. NDR2 residues mediating interactions with MOB1 are marked with red circles. Residues mediating intramolecular interactions of NDR2 are denoted by blue triangles. Residues conserved from yeast to humans are highlighted in green, while residues conserved in at least seven of the conserved NDR/LATS family members are marked in cyan. α helices are indicated with gray rods. b Asp63 of MOB1 is involved in LATS1 binding, but does not contribute to the interaction of MOB1 with NDR2. MOB1 is shown in yellow, while NDR2 and LATS1 are indicated in green and cyan, respectively. Top panel, Phe31 of NDR2 does not interact with Asp63 of MOB1. Bottom panel, His646 of LATS1 forms a hydrogen bond with Asp63 of MOB1. c–f Isothermal titration calorimetry (ITC) assays measuring the dissociation constant (K d) of indicated non-phosphorylated full-length MOB1 or MST2-phosphorylated MOB1 (phospho-MOB1) with wild-type and mutant NDR2 (25–88) or LATS1 (640–703) variants. MOB1/NDR2 complex formation was dramatically increased by prior phosphorylation of MOB1 c. ITC measurements could not detect any interaction between LATS1 wild-type and non-phosphorylated MOB1, while phospho-MOB1 bound to LATS1 d. The NDR2(Y32V) mutant did not associate with non-phosphorylated MOB1, but bound to phospho-MOB1, although with a 20-fold decreased binding affinity compared to wild-type NDR2 (e). The mutation of Val647 to Tyr of LATS1 enabled LATS1 to bind to non-phosphorylated MOB1, and LATS1(V647Y) displayed a 5-fold increased binding affinity for phospho-MOB1 compared to wild-type LATS1 (f)

Fig 4: Human MOB1(wt) or MOB1(K104/K105E), but not MOB1(D63V) or MOB1(D63V/K104E/K105E), expression suppresses elevated Yki activity in mats null mutant wing imaginal disk clones. Analysis of Ex expression in wing imaginal disks of the indicated genotypes (wt wild-type, DV D63V, KE K104E/K105E, DVKE D63V/K104E/K105E). All panels show third instar wing imaginal disks containing mats mutant clones marked by the absence of GFP expression (green in b, g, l, q, v). GFP-negative mats null clones are indicated by dashed lines. Maximum intensity projections of XY confocal stacks are shown in red to visualize Expanded (Ex) labeling (red in a, f, k, p, u). X–Z confocal sections through the same wing imaginal disks were acquired c, h, m, r, w. Nuclei are stained with DAPI in blue. Ex (d, i, n, s, x) and GFP (e, j, o, t, y) protein levels are indicated. All scale bars are 20 µm. As expected, mats null clones displayed increased Yki activity as judged by elevated Ex levels a–e. Expression of MOB1(wt) f–j or MOB1(K104E/K105E) p–t suppresses Yki activity to normal levels in mats mutant clones (compare i, s with d), while expression of MOB1(D63V) k–o or MOB1(D63V/K104E/K105E) u–y does not (compare n, x with d and i)

Fig 5: Acetylation of MOB1 regulates Hippo signaling pathway. (A and B) FLAG-MOB1 plasmids (WT or K11R mutant) were expressed respectively in H1299 KO cells (A) or HEK293T cells (B). Cell lysates were immunoprecipitated with M2-beads (anti-FLAG) and followed by immunoblotting with the p-MOB1 (T12) Ab and FLAG Ab. (C–F) H1299 KO cells (C,E) or HEK293T cells (D,F) were transfected with FLAG-MOB1 (WT or K11R mutant), and the lysates were subjected to immunoblotting with p-YAP (S127) Ab (C,D), p-TAZ (S89) Ab (E,F), p-LATS1 (T1079) Ab, FLAG Ab and Actin Ab (C–F). (G and H) H1299 (G) or HEK293T cells (H) transfected with FLAG-MOB1 (WT or K11R mutant) were coimmunoprecipitated with M2-Beads (anti-FLAG) and then immunoblotted with the indicated antibodies. (I and J) H1299 KO cells (I) or HEK293T cells (J) were transfected with FLAG-MOB1 (WT or K11R mutant), and then nuclear and cytoplasmic proteins were extracted. Nuclear proteins and cytoplasmic proteins were subjected to western blot analysis respectively with YAP, TAZ, FLAG, GAPDH (reference gene for cytoplasm) and YY1 (reference gene for nucleus) antibodies. S.E. means short exposure. L.E. means long exposure. (K) FLAG-MOB1-WT or K11R mutant was expressed respectively in H1299 KO cells, and endogenous ANKRD1, CTGF and CYR61 mRNA expression levels were detected by qPCR. Statistical analyses were performed by GraphPad Prism 6, and error bars represent means ± SD. According to two-tailed Student’s t-test, * is for P< 0.05 and ** is for P< 0.01. Images and statistical results were based on three independent experiments.