An α-Helical Extension of the ELMO1 Pleckstrin Homology Domain Mediates Direct Interaction to DOCK180 and Is Critical in Rac Signaling

Abstract

The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic ␣-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.

The Arf Family GTPase Arl4A Complexes with ELMO Proteins to Promote Actin Cytoskeleton Remodeling and Reveals a Versatile Ras-binding Domain in the ELMO Proteins Family

“…An Arf-related GTPases, Arl4A, Binds the ELMO1 RBD-We previously reported that the formation of an ELMO-DOCK180 complex is essential for Rac GTP-induced cytoskeletal changes but not for Rac GTP-loading per se, the latter being solely dependent on the intrinsic GEF activity of DOCK180 (12). Our recent data also highlighted that the RBD of ELMO is essential for targeting ELMO to the membrane upon integrin activation (14).…”

“…Immunoprecipitation and GST Fusion Protein PulldownsImmunoprecipitation and pulldown experiment protocols have been described previously (12). Briefly, the cells were lysed for 10 min in a buffer consisting of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 1ϫ Complete protease inhibitor (Roche).…”

“…ELMO family members are established binding partners of DOCK180, and genetic analyses in worms and flies suggest that ELMO is crucial for the biological functions of DOCK180 (1,2). Likewise, cell biology studies in mammalian cells suggest that disrupting the ELMO-DOCK180 interaction blocks signaling from this complex (12,13).…”

Rho-GTPase signaling drives melanoma cell plasticity

“…While initial studies suggested that interaction with ELMO was required for exchange factor activity recent structural work on the DOCK180-ELMO interaction indicates that the role of ELMO may be to act as an adaptor protein to couple to Rac effector functions. 11 The N-terminal portion of ELMO is a binding partner for active RhoG 12 and it was shown that the recruitment of the ELMO-DOCK180 complex to the active RhoG molecule, which is mainly located at the plasma membrane, is crucial for efficient Rac-dependent epithelial cell spreading on the matrix protein fibronectin. 12 In our experimental system, depletion of RhoG leads to abrogation of elongated movement in melanoma cells (Sanz-Moreno V, unpublished observations).…”

Elmo2 Is a Regulator of Insulin-dependent Glut4 Membrane Translocation

“…4). However, at present, we are unable to conclude that the regulation of Rac1 activity induced by insulin may also depend on the interaction between Dock180 and Elmo2, which is also dependent on Elmo2 PH domain (31)(32)(33). Further studies are required to clarify this issue.…”