Fig 2: Graphical demonstration for a putative mechanism how Elmo1 relieves the intermolecular steric inhibition of Ephexin4. Intermolecular association of Ephexin4 mediated by interaction of the SH3 domain with the N20 region of Ephexin4 forms a trimeric complex, whose conformation could be a triangle shape. This conformation prevents RhoG access to the DH domain of Ephexin4. Elmo1 interferes the interaction between the SH3 domain and the N20 region of Ephexin4 and then the geometry of Ephexin4 is disrupted, which facilitates RhoG access to the DH domain of Ephexin4.

Fig 3: Downregulation of Dock1 and Elmo1 reduces Rac1 activity and the expression of migration-associated proteins in MDA-MB-231 cells. (A) A Rac activity assay was performed to evaluate the Rac1 activity of MDA-MB-231 cells. (B) Reverse transcription-quantitative polymerase chain reaction and (C) western blot analysis assays were performed to measure the expression levels of FAK, Talin and Vinculin in MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; Rac1, Ras-related C3 botulinum toxin substrate 1; FAK, focal adhesion kinase; si, small interfering RNA; NC, negative control.

Fig 4: Downregulation of Dock1 and Elmo1 affected the RhoA/Rac1 pathway. Western blot analyses were performed to evaluate the expression levels of (A) RhoA QTPase and RhoA total, and (B) Rac1 GTPase and Rac1 total in MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. **P<0.01 and ***P<0.001 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; RhoA, Ras homolog gene family, member A; Rac1, Ras-related C3 botulinum toxin substrate 1; si, small interfering RNA; NC, negative control.

Fig 5: The activity of Ephexin4 is negatively regulated by its SH3 domain. (a) Schematic diagram of Ephexin4 and Elmo1, and their truncation mutants used in the study. Epx4, Ephexin4; GEF, Guanine nucleotide exchange factor; PH, Pleckstrin homology domain; SH3, SRC homology 3 domain. (b) LR73 cells were transfected with the indicated plasmids, incubated with 2 µm carboxylated beads (left) or apoptotic thymocytes (right) for 2 h, and analyzed by flow cytometry. GFP- and red fluorescence–positive phagocytes were considered to represent phagocytes that had engulfed targets. GFP- indicates the non-transfected population of the cells. (c) LR73 cells were transfected with the indicated plasmids and stained with phalloidin. Images were obtained using confocal microscopy. Arrow heads indicate membrane ruffles. Scale bar, 20 µm. (d,e) 293 T cells were transfected with the indicated plasmids and lysed. The resultant lysates were incubated with GST-Elmo2N-term-bound GST beads for 1 h. Exogenous active RhoG levels were detected with immunoblotting (d) and quantified from four independent experiments (e). (f,g) 293 T cells were transfected with either Ephexin4 or Ephexin4?SH3. Endogenous active RhoG was co-precipitated with GST-Elmo2N-term and detected by immunoblotting (f). Three independent experiments were performed, and the levels of active RhoG were quantitated (g). Data are shown as the mean ± standard deviation and are representative of at least three independent experiments. *P < 0.05, **P < 0.01. Immunoblots are cropped for conciseness and their full-length blots are shown in Supplementary S3. Epx4, Ephexin4; TCL, total cell lysates.