Extended Data Fig. 5: RAP2 inhibits RhoA GTPase through ARHGAP29.
a, RAP2A expression inhibits endogenous RhoA GTP-binding. HEK293A cells with doxycycline-inducible expression of RAP2A were established and the expression of Flag–RAP2A was induced by doxycycline. RhoA activity was determined by a GST–Rhotekin-RBD pull-down assay. s.e. denotes short exposure of the RhoA western blot. The image is representative of two independent experiments with similar results. b, Western blot confirms CRISPR-mediated RhoA gene editing. HEK293A cells were transfected with CRISPR plasmids targeting RhoA and selected with puromycin for 3 days. Two sgRNAs were used to generate two RhoA knockout pools (sgRhoA #1 and #2)16. The image is representative of two independent experiments with similar results. c, CRISPR-mediated deletion of RhoA leads to increased cytoplasmic localization of YAP and TAZ in HEK293A cells at high stiffness. The localization distribution is presented as mean + s.e.m. n = 6 biologically independent samples. d, Representative immunofluorescence images from c. Scale bars, 25 μm. e, ARHGAP29 induces YAP phosphorylation in a Hippo pathway-dependent manner. MM-5KO, a HEK293A clone with deletion of MST1, MST2, MAP4K4, MAP4K6 and MAP4K7. MM-8KO, deletion of MST1, MST2, MAP4K1, MAP4K2, MAP4K3, MAP4K4, MAP4K6 and MAP4K7. YAP phosphorylation was detected by phos-tag gels. The image is representative of two independent experiments with similar results. f, Immunoblot showing deletion of ARHGAP29 in HEK293A wild-type and MAP4K4/6/7-tKO cells. The image is representative of two independent experiments with similar results. g, Deletion of ARHGAP29 compromises inhibition of RhoA by low matrix stiffness. Wild-type and ARHGAP29-KO HEK293A cells were cultured at the indicated stiffness and then assayed for RhoA activity with a GST–Rhotekin-RBD binding assay. The image is representative of two independent experiments with similar results. h, Combined deletion of ARHGAP29, MAP4K4, MAP4K6 and MAP4K7 abolishes YAP phosphorylation induced by RAP2A. HA–YAP was co-transfected with vector or Flag–RAP2A into HEK293A cells cultured at high stiffness. HA–YAP phosphorylation was detected by phos-tag SDS–PAGE. The image is representative of two independent experiments with similar results. i, Combined deletion of ARHGAP29, MAP4K4, MAP4K6 and MAP4K7 blocks low stiffness-induced cytoplasmic localization of YAP. Quantification of YAP and TAZ localization in HEK293A cells with deletion of ARHGAP29 and/or MAP4K4, MAP4K6 and MAP4K7 at low stiffness in Fig. 3e. The YAP and TAZ localization distribution is presented as mean + s.e.m. n = 3 biologically independent samples.
