Figure 4.

SHANK2 deregulates Hippo pathway by antagonizing LATS1 activity through sequestration of ARHGEF7. (A) Co-immunoprecipitation of ARHGEF7 and LATS1. (B) Co-immunoprecipitation of ARHGEF7 and SHANK2. (C and C’) SHANK2 overexpression reduced ARHGEF7-LATS1 interaction. For data shown in (A) to (C), Myc-MCL1 was used as negative control and the experiments were done in 293T cells. The relative amount of ARHGEF7-LATS1 interactions were quantitated in (C’) as mean ± SEM from results of three independent experiments. P value was calculated by Student’s t test; **P < 0.01. (D and D’’) The PDZ domain of SHANK2 mediated SHANK2-ARHGEF7 interaction and was crucial for SHANK2’s ability to disrupt ARHGEF-LATS1 interaction. A schematic representation of SHANK protein domains was shown in (D). Deletion of PDZ domain (SHANK2 ΔPDZ) rendered SHANK2 unable to bind ARHGEF7 and abolished SHANK2’s ability to disrupt ARGHEF7-LATS1 interaction (D’). The relative amount of ARHGEF7-LATS1 interactions were quantitated in (D’’) as mean ± SEM from results of three independent experiments. P value was calculated by Student’s t test; *P < 0.05, **P < 0.01. (E and F) Deletion of PDZ domain rendered SHANK2 unable to reduce YAP phosphorylation (E) or promote YAP nuclear localization (F) in high cell density. Experiments were done in 293T cells. Scale bars: 10 μm. (G) CTGF and CYR61 expression level in control, SHANK2 and SHANK2 ΔPDZ groups. Experiments were done in 293T cells. Data represent mean ± SEM from results of three independent experiments. P value was calculated by Student’s t test; ***P < 0.001. (H) Deletion of PDZ domain abolished SHANK2’s ability to promote liver cancer formation in vivo. Vector control or SHANK2, ΔPDZ SHANK2 combined with p53R246S and Myc were hydrodynamically injecting into mouse tail vain to induce liver cancer