C‐terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac‐GEF

Abstract

Genetic studies in Caenorhabditis elegans identified an evolutionarily conserved CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), CED-10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain-independent Rac guanine nucleotide exchange factor (Rac-GEF); but it is still unclear how CrkII functions in Rac-GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C-terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C-terminal SH3 domain of CrkII (CrkII-SH3-C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac-GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3-N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N-terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C-terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac-GEF.

Activation of Rac1 by RhoG regulates cell migration

“…However, we could not exclude the possibility that CrkII is required for the regulation of cell migration by RhoG, because Dock180⌬N-ISP, which is a large protein with multiple regions, might function as a dominantnegative protein towards CrkII for other reasons. The relationship between the p130Cas-Crk-Dock180 and the RhoG-ELMO-Dock180 pathways in the regulation of cell migration remains unclear, but a recent study reported that the C-terminal second SH3 domain of CrkII regulates the interaction between RhoG and ELMO (Akakura et al, 2005). Further studies are required for elucidating the precise mechanisms for the regulation of cell migration mediated by Dock180-dependent activation of Rac.…”

CZH proteins: a new family of Rho-GEFs

“…This pathway may mediate integrin-induced Rac activation, cell migration, phagocytosis and nerve growth factor (NGF)-induced neurite outgrowth Grimsley et al, 2004;Katoh and Negishi, 2003). The RhoG and Crk pathways might be connected, since Crk, Dock180 and ELMO can form a ternary complex (Gumienny et al, 2001;Wu et al, 2001), and CRK might regulate interactions between Dock180, ELMO, Rac and RhoG (Akakura et al, 2005).…”

The phosphoinositide phosphatase MTM-1 regulates apoptotic cell corpse clearance through CED-5–CED-12 in C. elegans

“…GTP-loaded) MIG-2 regulates corpse removal by modulating plasma membrane recruitment of the unconventional bipartite CED-5 (Dock180)-CED-12 (Elmo) GEF complex (Gumienny et al, 2001;Wu and Horvitz, 1998a). The GEF complex is stabilized further by the adaptor molecule CED-2 (CrkII), which also promotes the activation of CED-10 (Akakura et al, 2005;Gumienny et al, 2001). GTP-bound CED-10 initiates extensive cytoskeletal rearrangements, a requirement for the engulfment of cell corpses (Kiyokawa et al, 1998;Reddien and Horvitz, 2000).…”