Fig. 4: The PLCε E2α′ helix is needed for RhoA-dependent activation.

a Schematic showing the boundaries for internal deletions in the PLCε EF3/4 module. b (Left) Basal and RhoA^G14V-stimulated activities of PLCε variants lacking the E2α′ helix (Δ1275–1289), the loop connecting it to the F3α helix (Δ1287–1298) in the EF3/4 module, or both (Δ1275–1298). Deletion of any of these regions largely eliminates RhoA-dependent activation. At least three independent experiments from independent transfections were performed for each variant. Data shown represents the average of triplicate measurements ± SD, and analyzed using unpaired, one-tailed t-test with Welch’s correction to compare the basal and RhoA-stimulated activities of each variant. ****p < 0.0001, ***p < 0.0003, **p < 0.0080, *p < 0.0112. (Left) The change in maximal activity ± SD was calculated by subtracting the RhoA-stimulated activity from the basal activity of each variant. Data were analyzed using a one-way ANOVA and Kruskal–Wallis test comparing each variant to PLCε, followed by a Dunn’s multiple comparisons test. For Δ1275–1289, **p < 0.0041, for Δ1287–1298, *p < 0.0134, and for Δ1275–1298, **p < 0.0097. c The PLCε E2α′ helix (red) binds to the switch regions of RhoA (light blue). Additional contacts with RhoA are made by residues in the EF1/2 module and the loop linking E2α′ to the F3α helix. Labeled residues were subjected to site-directed mutagenesis, and their impact on RhoA-dependent activation was quantified. d (Left) Mutations in the G protein–PLCε interface decrease RhoA-dependent activation. At least three independent experiments from independent transfections were carried out for each variant. Data shown represents the average of triplicate measurements ± SD, and was analyzed using unpaired, one-tailed t-test with Welch’s correction to compare the basal and RhoA-stimulated activities of each variant. ****p < 0.0001, ***p < 0.0003, **p < 0.0080, *p < 0.0112. (Right) Mutation of PLCε Trp1051 in EF1/2, residues Ala1282, Ile1283, and Ala1286 in E2α′, and Ile1295 in the E2α′-F3α loop significantly decreases maximum RhoA-dependent activation. Data was analyzed using a one-way ANOVA and Kruskal–Wallis test comparing each variant to PLCε, followed by a Dunn’s multiple comparisons test. Representative Western blots are shown below, with empty pCMV vector (EV) and β-actin used as loading controls. Differences in expression were not found to be statistically significant but may still contribute to variation in activities. PLCε variants express a C-terminal FLAG tag and are detected with an anti-FLAG antibody, while RhoA contains an N-terminal HA tag and is detected using an anti-HA antibody.