DOCK8 is a Cdc42-specific GEF. (A) Extracts from human embryonic kidney 293T cells expressing HA-tagged DOCK8, DOCK8 ΔDHR-2, or DOCK2 were pulled down with GST-fusion proteins encoding Cdc42, Rac1, or RhoA. Assays were done in Tris-buffered saline/Tween-20 supplemented with 10mM EDTA (E) or 10mM MgCl2 plus 30μM GTPγS (M). Data are representative of 2 independent experiments. (B) Recombinant DOCK8 DHR-2 and DOCK2 DHR-2 proteins were analyzed for their GEF activities toward Cdc42 (top panels) or Rac1 (bottom panels) using mant-GTP. RFU indicates relative fluorescence units. Data are representative of 2 independent experiments. (C) Overall structure of the complex between DOCK8 DHR-2 and Cdc42. DOCK8 and Cdc42 are colored salmon and green, respectively; the switch I region of Cdc42 is shown in black. (D-E) Rac1 bound to DOCK2 DHR-2 (Protein Data Bank accession code 3B13) and Cdc42 bound to DOCK9 DHR-2 (Protein Data Bank accession code 2WM9) are superposed onto the Cdc42-binding site of DOCK8 DHR-2. Rac1 is colored yellow; Cdc42 bound to DOCK8 is light green; Cdc42 bound to DOCK9 is dark green; and DOCK8, DOCK9, and DOCK2 are salmon, purple, and blue, respectively. The arrow in panel E indicates the different location of the DHR-2 lobe B in DOCK2 compared with those in DOCK8 and DOCK9.
