Fig 2: Canonical RAS isoforms can form active holophosphatases.a, SPR-derived affinity and cooperativity immobilizing NRAS–GppNHp, NRAS(Q61R)–GTP, KRAS–GppNHp or KRAS(Q61R)–GTP with the indicated analytes. a = KDSHOC2/KDSHOC2–PP1C. Affinity values over 10,000 nM are highlighted in red. b, In vitro dephosphorylation of autoinhibited and active-state BRAF–MEK1–14-3-3 complexes by the SHOC2 holophosphatase. Purified full-length BRAF complexes in the autoinhibited state (left) or active dimeric state (right) were incubated with lambda phosphatase (PPase), PP1Ca or ternary SHOC2 complexes, and were blotted with phospho-specific antibodies for BRAF phosphorylated at Ser365 and Ser729. Equivalent loading of BRAF complexes is shown by Coomassie staining. Phosphorylated Ser365 is selectively dephosphorylated relative to Ser729 in the active dimer, whereas both are relatively protected in the autoinhibited (14-3-3-bound) state. Experiments were conducted twice with similar results. c, The relationship between the knockdown of SHOC2 and MRAS, HRAS, NRAS and KRAS. The dependency scores of each RAS gene and SHOC2 are shown on the x and y axes, respectively. Dashed lines indicate a dependency score of zero. A highly negative dependency score implies that a given cell line is highly dependent on that gene. Cell lines dependent on both SHOC2 and RAS are indicated at the bottom left. Right, the calculated Pearson correlation coefficient (y axis) applied to each mutation group (group of cell lines containing the associated mutation). A higher positive value indicates a stronger positive relationship: the dependency score of SHOC2 decreases/increases in the same lines as the dependency scores of the RAS genes. The n values above each bar show the number of cell lines in each mutation group. d, Immunoblot analysis of MiaPaca2 parental cells (Par), SHOC2-KO (KO) and stable cell lines reconstituted with SHOC2 mutants after 10 nM trametinib (Tram) treatment for 1 h or 24 h (+). Densitometry quantification (percentage variation) of pCRAF/CRAF and pERK/ERK levels from the immunoblot analysis normalized to untreated MiaPaca2 parental cells. The samples were derived from the same experiment and blots were processed in parallel. The images are representative of two independent experiments.

Fig 3: Vemurafenib rescues phosphorylation signals and phenotypes in PGRN-KI mice. a Experimental protocol for administration of B-Raf inhibitor (vemurafenib). The mice were fed vemurafenib (32 mg/kg of BW/day) or mock from 6 to 12 weeks of age. Behavioral tests and western blots were performed at 12 weeks. b In vivo effect of vemurafenib on B-Raf phosphorylation in cortical tissues of PGRN-KI mice. #p < 0.05; ##p<0.01 (N = 6, Tukey’s HSD test). Averages and s.e.m. are shown. c In vivo effect of vemurafenib on MEK phosphorylation in cortical tissues of PGRN-KI mice. #p < 0.05 (N = 6, Tukey’s HSD test). Averages and s.e.m. are shown. d In vivo effect of vemurafenib on ERK phosphorylation in cortical tissues of PGRN-KI mice. #p < 0.05 (N = 6, Tukey’s HSD test). Averages and s.e.m. are shown. e In vivo effect of vemurafenib on phosphorylation of tau in cortical tissues of PGRN-KI mice. Right panels show quantitation of western blot band intensities. ##p < 0.01 (N = 6, Tukey’s HSD test). Averages and s.e.m. are shown. f In vivo effect of vemurafenib on two parameters (% time spent at target region and number of target crosses) in the Morris water maze test. Numbers of mice are shown in the graph. **p < 0.01 (Student’s t-test). Averages and s.e.m. are shown. g Effect of vemurafenib on % total freezing time in the fear-conditioning test. Numbers of mice are shown in the graph. p-values were determined by Student’s t-test. Averages and s.e.m. are shown

Fig 4: Suppression of Tyro3 signal rescues tau mislocalization and cognitive impairment in mutant PGRN-KI mice. a Co-staining of phosphorylated tau (Ser203 or Thr220) and PSD-95. Confocal microscopic analysis of M2 regions of background (B6, C57BL/6J), PGRN-R504X-KI (PGRN-KI), vemurafenib-treated PGRN-KI, Gö6976-treated PGRN-KI, and lentivirus-tau-shRNA–infected PGRN-KI mice. All images were acquired by confocal microscopy (LSM510META, Carl Zeiss, Germany). Z-stack images were acquired with the following parameters; objective: ×63, average: line 2; filter (Cy3 ChS1: 550–679 nm; FITC Ch1: 505–530 nm; DAPI Ch2: 420–480 nm); master gain (Cy3 ChS1: 760; FITC Ch1: 741; DAPI Ch2: 615). b Quantitative analyses of PSD95-positive dots and p-tau/PSD95 double-positive spots. All bar graphs indicate averages and s.e.m. Three mice were used for each group. The number of spots was counted in 10 image fields (100 × 100 µm) for each mouse, and the average was used for calculation of mean with s.e.m. for each group. The number of p-tau/PSD95 double-positive spots increased in PGRN-KI mice. #p < 0.05, ##p < 0.01 (N = 6, Dunnett’s test). Averages and s.e.m. are shown. c In vivo effects of KD of B-Raf, PKCa, Tyro3, Gas6, and tau on two parameters (% time spent at target region and number of target crosses) in the Morris water maze test, performed with PGRN-KI mice (N = 6 in each experiment) at 12 weeks. #p < 0.05, ##p < 0.01 (N = 6, Dunnett’s test). Averages and s.e.m. are shown. d In vivo effect of KD of B-Raf, PKCa, Tyro3, Gas6, and tau on % total freezing time in the fear-conditioning test, performed with PGRN-KI mice (N = 6 in each experiment) at 12 weeks. #p < 0.05, ##p < 0.01 (N = 6, Dunnett’s test). Averages and s.e.m. are shown. e Tau-KD with or without vemurafenib/Gö6976 had similar effects on % time spent at target region and number of target crosses in the Morris water maze test and on % total freezing time in fear-conditioning test. Both tests were performed at 12 weeks. *p < 0.05 (N = 6, Dunnett’s test). Averages and s.e.m. are shown. f Western blot analysis confirming shRNA-mediated knockdown of B-Raf, PKCa, and tau. Lower graphs show quantitation. P-values were determined by Tukey’s HSD test (N = 6). Averages and s.e.m. are shown. p-values are shown in Supplementary data 1

Fig 5: Suppression of Tyro3 signal rescues decrease in spine abundance and tau mislocalization in mutant PGRN-KI mice. a Experimental protocol for knockdown vectors. AAV-Syn-EGFP was injected into the area adjacent to M2 at 6 weeks of age. Lentivirus for expression of Tau-shRNA, scrambled shRNA, B-Raf-shRNA, or Tyro3-siRNA, or plasmid vectors for expression of PKCa-shRNA, scrambled shRNA, or Gas6-shRNA dissolved in in vivo-jetPEI, were continuously injected into the subarachnoid space of the right M2 via osmotic pump from 8 to 12 weeks of age. Dendritic spines in layer 1 of M2 were observed by two-photon microscopy with three mice at 12 weeks of age. 8 to 10 images were obtained from one mice and the average of spine parameters were used for quantitative analyses (N = 3). The protocols for vemurafenib and Gö6976 were similar to those shown in Fig. 6 7, and two-photon microscopy was performed at 12 weeks of age. b Static spine morphology was observed by two-photon microscopy. Spine protrusion number, length, head diameter, and volume were analyzed. *p < 0.05; **p < 0.01 (N = 3, Student’s t-test). Averages and s.e.m. are shown. c Images were obtained by two-photon microscopy at 0, 8, and 24 h on the last day of injection. Dynamic changes in spines were analyzed by serial observation. Spine formation and elimination were counted. *p < 0.05; **p < 0.01 (N = 3, Student’s t-test). Averages and s.e.m. are shown. d Lentivirus for expression of shRNA-Tau was injected according to the protocol. **p < 0.01 (N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. e Lentivirus for expression of shRNA-B-Raf was injected according to the protocol. **p < 0.01 (N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. f Plasmid for expression of shRNA-PKCa was injected according to the protocol. *p < 0.05; **p < 0.01 (N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. g Lentivirus for expression of siRNA-Tyro3 was injected according to the protocol. Scrambled RNA was used as a control. **p < 0.01 (N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. h Plasmid for expression of shRNA-Gas6 was injected according to the protocol. Scrambled RNA was used as a control. **p < 0.01 (N = 3, Tukey’s HSD test). Scrambled RNA was used as a control. Averages and s.e.m. are shown. i Mice received oral administration of vemurafenib (32 mg/kg of BW/day) from 6 to 12 weeks of age, and two-photon microscopic analysis was performed at 12 weeks. **p < 0.01 (N = 3, Tukey’s HSD test). Averages and s.e.m. are shown. j Gö6976 (4.4 µM) or PBS was injected via osmotic pump into the subarachnoid space of PGRN-KI and C57BL/6J mice from 10 to 12 weeks of age, and two-photon microscopic analysis was performed at 12 weeks. **p < 0.01 (N = 3, Tukey’s HSD test). Averages and s.e.m. are shown