Biochemical Studies On The Unconventional Rac Specific Gef, Dock180

Abstract

The Rho family of GTPases comprises of approximately twenty family members, which regulate multiple cell activities including cellular migration, cell polarity, vesicle trafficking and a variety of enzymatic activities. Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by removal of bound GDP and the subsequent of loading of GTP, which leads to downstream effector recognition. Two families of GEFs have been described: The classical Dbl-GEF family members share conserved DH-PH domains, where the DH domain harbors the GEF activity for the Dbl family members while the PH domain is thought to help localize the GEF protein to the plasma membrane. In contrast to Dbl-GEFs, the more recently described Dock180 GEF-family members do not share primary sequence homology with DH-PH domains, although they exhibit robust nucleotide exchange activity for Rho GTPases. Here we describe the biochemical characterization of the conserved limit DHR-2 domain of Dock180 and its activation of the Rac GTPase. We delineate a limit functional sub-domain of DHR-2 which is composed of approximately 300 residues in the C-terminal portion of DHR-2 (referred to below as DHR-2c). Our data show this region is both necessary and sufficient for robust GEF activity as the DHR-2 domain specifically activated Rac both in vitro and in vivo. Scanning mutagenesis of Rac also revealed that DHR-2c binds to Rac in a manner distinct from the classical Dbl-family Rac-GEFs. Specifically, both alanine 27 and tryptophan 56 of Rac are demonstrated to provide a bipartite recognition site for DHR-2c GEF-specific recognition, whereas, for Dbl-family GEFs, tryptophan 56 of Rac is the primary determinant of GTPase specificity. We also identified the corresponding residue (methionine 1524) on Dock180 to specifically recognize tryptophan 56 of Rac. These results define the core residues for Dock180's guanine nucleotide exchange activity while highlighting recognition sites that underline GTPase specificity for Dock180-family members.