Fig. 3. Mitochondrial stress signal through RhoA, but not MST1/2 and MAP4K to activate YAP.

A Scheme depicting classical Hippo kinase cascade. While LATS1/2 is largely responsible for YAP/TAZ phosphorylation, MST1/2 and MAP4Ks are on the upstream as the core Hippo kinases. B Mitochondrial stress induces LATS1/2 inhibition. HEK293 cells were treated with CCCP for indicated times. Phosphorylation (Thr 1079) of LATS1/2 kinases were determined by immunoblotting. C Deletion of MST1/2 or MAP4K4/6/7 does not affect mitochondrial stress-induced YAP activation. MST1/2 KO, MAP4K4/6/7 KO, MST1/2-MAP4K4/6/7 KO, and wild-type cells were treated CCCP for indicated times. The phosphorylation of YAP was analyzed by immunoblot using Phos-tag gels. D–F Mitochondrial stress requires RhoA to induce YAP activation. RhoA KO and wild-type cells were treated with CCCP for 1 h. D The phosphorylation of YAP was analyzed by immunoblot using Phos-tag gels. E Nuclear localization was determined by fluorescence immunostaining. F YAP/TAZ target gene expression was determined by RT-qPCR. The data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by Student’s t test. G RhoA is activated upon mitochondrial stress. Active RhoA on cells treated with CCCP for indicated times was first immunoprecipitated using antibody specific to GTP-bound form then was immunoblotted for total RhoA. For B–G, HEK293 cells were serum-starved for 12 to 16 h prior to CCCP (50 μM) treatment.