A Minimal Rac Activation Domain in the Unconventional Guanine Nucleotide Exchange Factor Dock180

Members of the Rho family of GTPases regulate a wide range of cellular activities including cell-cycle progression, gene transcription, cell migration, cell polarity, and vesicular trafficking through their abilities to bind to multiple downstream effectors (1-4). Rho GTPases switch between two states, the GDP-bound inactive state and the GTP-bound active state. Tight regulation of Rho GTPases and their nucleotide-bound state is important for mediating their different cellular functions (5). Three main classes of regulatory proteins for Rho GTPases have been identified and characterized. Guanine nucleotide exchange factors (GEFs) include ~70 mammalian/human proteins that promote the exchange of GDP for GTP on Rho GTPases. GTPase-activating proteins (GAPs) catalyze the hydrolysis of the bound GTP to GDP, and Rho GDP-dissociation inhibitors (GDIs) slow nucleotide exchange while serving to sequester Rho GTPases in the cytoplasm (6-8). Two families of GEFs have been discovered for Rho GTPases, which we refer to here as the Dbl (9) and Dock180 superfamilies (10-12). The Dbl family members all possess two tandem conserved domains, the Dbl Homology (DH) and the Pleckstrin Homology (PH) domains (13-18). The DH domains of Dbl-GEFs are directly responsible for catalyzing GDP-GTP exchange activity while the PH domains are important for protein localization to the plasma membrane. More recently, Dock180-related proteins have been shown to catalyze nucleotide exchange on Rac and/or Cdc42 despite the absence of primary sequence homology to DH domains (19, 20). The functional domain for this unconventional class of GEFs was originally suggested to consist of ~500 residues located within the C-terminal half of Dock-180-related proteins and has been referred to as the Dock180 Homology Region 2 or DHR-2 domain. Dock180 family members have been implicated in multiple biological pathways that include cell phagocytosis (21, 22), cell migration (21, 23-25), tumor suppression (26) and axonal outgrowth (27, 28). Dock180 is the founding member of the family and functions as a Rac-specific GEF. Full-length Dock180 shows much lower GEF activity compared to the isolated DHR-2 domain, apparently due to auto-inhibition (29, 30). Relief of the auto-inhibited state in cells has been suggested to result upon binding to the accessory protein Elmo (19, 31-34). Recently, x-ray crystal structures were reported for the DHR-2 domain of one member of the Dock180 family, Dock9 (Zizimin 1), bound to different nucleotide-bound states of Cdc42 (35). This work has provided a first glimpse of how a Dock180 family member functions in catalyzing GDP-GTP exchange. However, as Dock9 is a Cdc42-specific GEF, we still do not have a detailed picture of how a Rac-specific GEF for the Dock180 family functions and confers specificity for Rac. Moreover, Dock180 exhibits much higher catalytic GEF activity, compared to Dock9, suggesting that the founding member of this GEF family may possess some important distinguishing features with regard to its mechanism of action. In the present study, we set out to define a limit functional domain for Dock180 as an important first step for mechanistic studies and ultimately for high-resolution structural characterizations. During the course of our efforts to obtain an active limit functional domain for Dock180, we modeled the domain structure of the C-terminus of DHR-2 and isolated a stable region from Dock180 that can be expressed in high yield and exhibits full Rac-GEF activity. Here we show that this fragment, designated as Dock180DHR-2c (or sometimes simply DHR-2c), is fully active despite lacking the upstream helical domain that purportedly mediates dimerization of DHR-2 domains in Dock9 and has been suggested to be necessary for the full activation of its GEF activity (35). We provide evidence that this defined region of DHR-2 harbors specific recognition sites that allow for the discrimination between Rac and Cdc42 by Dock subfamilies A (Rac-specific) and D (Cdc42-specific) (20). Moreover, by specifically comparing the activity of the tandem DH-PH domains of the Rac-specific GEF Tiam-1 with that of the Rac-specific Dock180DHR-2c, we define a key contact made by Ala27 in Rac that is necessary together with Trp56 for Dock180DHR-2c- recognition and is absent in the functional coupling of DH-PH domains to Rac.