GEF what? Dock180 and related proteins help Rac to polarize cells in new ways

Abstract

Rho GTPase activation, which is mediated by guanine nucleotide exchange factors (GEFs), is tightly regulated in time and space. Although Rho GTPases have a significant role in many biological events, they are best known for their ability to restructure the actin cytoskeleton profoundly through the activation of specific downstream effectors. Two distinct families of GEFs for Rho GTPases have been reported so far, based on the features of their catalytic domains: firstly, the classical GEFs, which contain a Dbl homology-pleckstrin homology domain module with GEF activity, and secondly, the Dock180-related GEFs, which contain a Dock homology region-2 domain that catalyzes guanine nucleotide exchange on Rho GTPases. Recent exciting data suggest key roles for the DHR-2 domain-containing GEFs in a wide variety of fundamentally important biological functions, including cell migration, phagocytosis of apoptotic cells, myoblast fusion and neuronal polarization.

Figure I

Figure I

Figure 1

Schematic representation of the Elmo proteins and their interaction with the Dock180-related proteins. A large portion of the C-terminal domain of Elmo, including part of the PH domain and the proline-rich region (black bar), is required for Elmo to interact with the Dock180-related proteinsc (double-headed arrow). The N-terminal portion of Dock180, in turn, is required for Dock180 to bind to the Elmo proteins. Although the SH3 domain of Dock180 can bind to Elmo in vitro, some studies suggest that this domain is dispensable for the Dock180–Elmo interaction in cells.

Figure 2

The bipartite GEF model. In this model, the GEF activity of Dock180 toward the Rac GTPase becomes detectable, or is greatly enhanced, when Dock180 is bound to Elmo. Mechanistically, it is thought that the PH domain of Elmo interacts in trans with the nucleotide-free (nf) form of Rac bound to the DHR-2 domain of Dock180. This interaction then increases the affinity of Dock180 toward nf-Rac and the subsequent GTP loading of Rac by the DHR-2 domain of Dock180.

Figure 3

Mechanisms to target Dock180 to the membrane through Elmo. (a) Elmo contains a RhoG-binding domain (in yellow), which specifically recognizes the GTP-bound form of the RhoG GTPase. Following cell adhesion to fibronectin, GTP-bound RhoG recruits the Elmo–Dock180 complex to the plasma membrane, promoting the formation of Rac GTP at these sites. Trio is a GEF that can activate RhoG. (b) Following wounding of an epithelial cell monolayer, the Elmo–Dock180 complex localizes and activates the Rac GTPase at the leading edge of the cells migrating to close the wound. In this model, the recruitment of Elmo–Dock180 to the membrane is thought to be mediated by the Arf6 GTPase. Arno is a GEF that can activate Arf6. The exact mechanism by which Arf6 recruits Elmo and/or Dock180 to the membrane remains to be determined. This recruitment could be direct or indirect, through an unidentified bridging protein (depicted as ‘X’). (c) In the C. elegans model, PtdSer, which is exposed on apoptotic cells, binds to the PtdSer receptor on the surface of phagocytes. This interaction leads to a recruitment of Ced-5 (Dock180), Ced-12 (Elmo) and Ced-2 (Crk) to the receptor in the phagocyte and to subsequent activation of Ced-10 (Rac) and engulfment of the apoptotic cell. (d) Shigella injects bacterial proteins into the epithelial cells, to activate Rac-mediated membrane ruffling and subsequent entry of the pathogen to the cells. A key bacterial protein for this function, IpgB1, binds directly to Elmo. The Elmo–Dock180 complex then localizes to the membrane and activates Rac at the sites of Shigella entry. The binding of Elmo to IpgB1 is mediated by the N-terminal Arm repeats in Elmo, which also contain the RhoG binding site [see (a)].

Figure 4

Regulation of Dock180 function. (a) Autoinhibition of Dock180 by intramolecular interactions. The SH3 domain interacts in cis with the DHR-2 domain in the Dock180 molecule, thereby preventing the binding of Rac to the DHR-2 domain. Following Elmo binding to Dock180, the SH3 domain becomes disengaged from the DHR-2 domain, facilitating Rac interaction and subsequent GEF catalysis by the DHR-2 domain. (b) Elmo stabilizes the Dock180 protein in cells. In overexpression models, Dock180 becomes ubiquitylated by hitherto unidentified ubiquitin ligases following its dissociation from Elmo, followed by proteasome-mediated degradation of Dock180. Notably, stimuli that promote Elmo–Dock180 dissociation have not been reported.. Abbreviation: Ub, ubiquitin. (c) Regulation of the activity of the Dock180–Elmo complex by phosphorylation. (i) Elmo interacts with the Src family kinase Hck and becomes phosphorylated on specific tyrosine residues. These phosphorylation events increase the GEF activity of the Elmo–Dock180 complex toward the Rac GTPase by an unknown mechanism. Cellular stimuli that lead to Elmo phosphorylation have not yet been reported. (ii) Integrin engagement on fibroblasts leads to phosphorylation of Dock180 on serine and threonine (S/T) residues. The exact sites of phosphorylation and the consequences of these post-translational modifications on Dock180 are currently unknown.

Figure 5

Regulation of Dock180-related protein signaling by PtdIns(3,4,5)P_3. (a) The DHR-1 and DHR-2 domains of Dock180 and Dock2 integrate PtdIns(3,4,5)P₃ lipids with Rac activation at the leading edge. The DHR-1 domain interacts directly with PtdIns(3,4,5)P₃, and recruits Dock180 and Dock2 to the membrane to activate Rac through their DHR-2 domains. This localized activation of Rac leads to the generation of an actin-rich leading edge, which, in turn, promotes directed movement of the cell toward a migratory attractant (chemokine, cytokine, extracellular matrix gradient). (b) MBC promotes myoblast fusion in a PtdIns(3,4,5)P₃-dependent manner. (i) Impaired myoblast fusion in mbc-null flies. (ii) Transgenic expression of MBC in mbc-null flies rescues myoblast fusion defects. (iii) Transgenic expression of a DHR-1 deletion mutant of MBC in mbc-null flies fails to rescue myoblast fusion. Abbreviations: FC, fusion competent myoblast; F, founder myoblast. (c) Dock7 is required for axon specification in developing hippocampal neurons. In stage 1 neurons, Dock7 is freely distributed at the cell membrane. During budding of multiple dendrites at stage 2 of neuron development, Dock7 accumulates in an asymmetric manner at the base of the dendrite that will become the axon. Interestingly, depletion of Dock7 by siRNA blocks axon specification whereas overexpression of Dock7 promotes the specification of multiple axons. At stage 3, Dock7 is found located along the axon shaft and also at the base of the growth cone. Blocking of the formation of PtdIns(3,4,5)P₃ by pharmacological PI 3-kinase inhibitors prevents the Dock7-mediated axon specification.