Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity

Abstract

Mammalian DOCK180 protein and its orthologues Myoblast City (MBC) and CED-5 in Drosophila and Caenorhabditis elegans, respectively,function as critical regulators of the small GTPase Rac during several fundamentally important biological processes, such as cell motility and phagocytosis. The mechanism by which DOCK180 and its orthologues regulate Rac has remained elusive. We report here the identification of a domain within DOCK180 named DHR-2 (Dock Homology Region-2)that specifically binds to nucleotide-free Rac and activates Rac in vitro. Our studies further demonstrate that the DHR-2 domain is both necessary and sufficient for DOCK180-mediated Rac activation in vivo. Importantly, we have identified several novel homologues of DOCK180 that possess this domain and found that many of them directly bind to and exchange GDP for GTP both in vitro and in vivo on either Rac or another Rho-family member, Cdc42. Our studies therefore identify a novel protein domain that interacts with and activates GTPases and suggest the presence of an evolutionarily conserved DOCK180-related superfamily of exchange factors.

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

“…Previous in vitro observations suggested that Dock5 could activate the GTPase Rac1. (26) To confirm this on endogenous Rac1, we expressed in HEK-293T cells the DHR2 domain of mouse Dock5, lying between amino acids M1132 and Y1661 and corresponding to the equivalent domain defined in Dock1 (11,27) (Fig. 1D).…”

“…To further study the physiologic role of Dock5 in OCs, we differentiated OCs from primary BMMs isolated from Dock5 þ/þ (11) Black background highlights residues conserved between the two sequences. (E) Total cell extracts were prepared from 293T cells expressing GFP-fused Dock5-DHR2 or GFP and submitted to GTP-trapping using the GST-fused CRIB domain of PAK1.…”

“…The Dock family of GEFs consists of 11 Dock1-related proteins (also known as Dock180-related proteins) in mammals. (11,12) Docks are characterized by a unique DHR-2 (or CZH-2) domain that catalyzes nucleotide exchange on Rac1 or Cdc42 and a DHR-1 domain that localizes the GEFs to phosphatidylinositol (3,4,5)phosphate-enriched membranes. Dock1, Dock2, and Dock5 also contain an SH3 domain at the N terminus and a polyproline domain at the C terminus.…”

DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

“…A guanine nucleotide exchange factor (GEF), DOCK2, which is a hematopoietic cell-specific homolog of the CDM family protein [1], plays an essential role in the regulation of cell motility and cytokine production through the activation of Rac in human hematopoietic cells [2; 3; 4]. A knockout study against DOCK2 demonstrated the alternative role of DOCK2, regarding murine T-and B-cell motility [5] and control of the cardiac transplant rejection in mice [6].…”

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

“…8 As with other GTPases, Rac functions as a binary switch by cycling between an inactive guanine diphosphate (GDP)-bound and an active GTP-bound form. Dock180 and its homologues contain a Docker domain that can interact directly with nucleotide-free Rac and mediate Rac GDP/GTP exchange in vitro; 8,9 Rac activation mediated via the Dock180 : ELMO complex has been linked to cytoskeletal changes during engulfment and cell migration. 10,11 Two additional players, MIG-2/RHOG and UNC-73/Trio, play a role upstream of ELMO : Dock180.…”