Regulation of Ephexin4 GEF Activity by Elmo

Abstract

Ephexin4, a member of the Ephexin family of guanine nucleotide exchange factors (GEFs), has a GEF activity toward RhoG and is involved in diverse RhoG-related cellular processes. Especially, a recent study has revealed that phagocytosis of apoptotic cells is synergistically promoted by Ephexin4 and Elmo1 in a RhoG-dependent manner. The removal of apoptotic cells by phagocytosis is called efferocytosis. Efferocytosis is essential for development and tissue homeostasis in that a huge number of dying or dead cells are efficiently cleared through it. Therefore, it is important to understand how efferocytosis is regulated. Although the cooperation between Elmo1 and Epheixn4 during engulfment of apoptotic cells was observed, the underlying mechanism remains elusive. Here, we report a mechanism by which Elmo1 interacts with Ephexin4 to activate RhoG. We found that RhoG activation, phagocytosis of apoptotic cells and membrane ruffle formation were induced more effectively by Ephexin4ΔSH3, a mutant lacking its SH3 domain, than by wild-type Ephexin4. Based on co-immunoprecipitation assays, the SH3 domain mediated the intermolecular interaction of Ephexin4 by binding to the N20 region and this interaction prevented RhoG from accessing the GEF domain of Ephexin4. Furthermore, we found that the association between Elmo1 and Ephexin4 was mediated by both the N20 region and the SH3 domain of Ephexin4. Specifically, C-terminus of Elmo1 competed with the N20 region of Ephexin4 for binding to the SH3 domain, facilitating the binding of RhoG to Ephexin4. Consequently, the presence of Elmo1 augmented the activity of Ephexin4 and thus the level of active RhoG, apoptotic cell clearance, and membrane ruffle formation in cells expressing both Ephexin4 and Elmo1 were similar to those in cells with Ephexin4ΔSH3 expression. In addition, the importance of the intermolecular interaction in the autoinhibition of Ephexin4 was further supported by experiments using a point-mutated Ephexin4. We found that a substitution of the highly conserved glutamate residue at position 295 with an alanine disrupted the intermolecular interaction of Ephexin4, leading to an increased binding of RhoG. Therefore, Ephexin4E295A promoted RhoG activation, clearance of apoptotic cells, and formation of membrane ruffles more than wild-type Ephexin4. Taken together, these data suggest that the SH3-domain-mediated intermolecular interaction of Ephexin4 autoinhibits its GEF activity by impeding binding of RhoG, whereas the association of Elmo1 relieves this steric hindrance.