Fig. 2. Interaction between ARAP2 with Rho family GTP-binding proteins. (A) In vitro tests for RhoGAP activity of ARAPs. The GAP activity of ARAP1 and Flag-tagged [875-1704]ARAP2 using His-tagged Cdc42 as a substrate was measured. (B-D) Effect of ARAP2 on in vivo levels of (B) RhoA-GTP, (C) Cdc42-GTP and (D) Rac1-GTP. ARAP2 or p190RhoGAP were expressed with RhoA-AU5, Cdc42-AU5 or Rac1-AU5, as indicated, in HEK 293 cells. RhoA-GTP, Cdc42-GTP and Rac1-GTP levels were measured by co-precipitation with GST-Rhotekin-RBD-coated beads (for RhoA) or GST-PAK-PBD-coated beads (for Cdc42 and Rac1) followed by immunoblotting. Expression levels of RhoA, Cdc42, Rac1, ARAP2 or p190RhoGAP in the total cell lysates are shown. (E) Quantitation of RhoA-GTP levels in cells expressing ARAP2. The experiment described in B was repeated and signals were quantified with a scanning densitometer. The results presented are normalized to the levels of RhoA-GTP in the absence of RhoGAP or ARAP2 and are the means and range of duplicate determinations. (F) Binding of Rho family proteins to ARAP2 in vivo. HEK 293 cells were co-transfected with ARAP2 and either AU5-tagged RhoA, Rac1, or Cdc42. Proteins were immunoprecipitated using anti-FLAG M2 gel. Co-precipitated Rho family proteins were detected by immunoblotting with an anti-AU5 Ab. Protein expression levels in the total cell lysates is shown. (G) Nucleotide dependence of Rho binding to ARAP2. Lysates of HEK 293 cells transiently expressing ARAP2 were incubated with GSTRhoA loaded with 100 µM GDPßS or GTPS or with GST. (H) Identification of the ARAP2 domain that interacts with RhoA. GST-RhoA–GTPS or GST was incubated with lysates of HEK 293 cells transfected with expression constructs encoding Flag-tagged fragment corresponding to amino acids 467-930 (PH1-ANK domain), 1114-1323 (RhoGAP domain), 1317-1433 (RA domain) of ARAP2. The amount of Flag-tagged protein co-precipitating with GST or GST-RhoA immobilized on beads was determined by immunoblotting. Expression levels of GST or GST-RhoA and the recombinant Flag tagged ARAP2 are shown. (I) Effect of changing arginine 728 to lysine on ARAP2 binding to RhoA. Lysates from cells expressing Flag-ARAP2 or Flag-[R728K]ARAP2 were incubated with GST-RhoA–GTPS or GST as a control, and interaction was determined as described in (F) and (G). A Coomassie-blue-stained acrylamide gel electrophoresis fractionation of GST and GST-RhoA. The levels of recombinant ARAP2 proteins in the cell lysates was determined by immunoblotting. (J) Effect of changing lysine 1190 to proline on ARAP2 binding to RhoA. Lysates from cells expressing Flag-ARAP2 or Flag-[K1190P]ARAP2 were incubated with GST or GST-RhoA–GTPS. The experiment was performed as described in (I). (K) Model of ARAP2 RhoGAP domain and predicted consequences of mutating lysine 1190 to proline. Ribbon image of the structure of the Rho-GDP-AlF^–₄ (purple)–RhoGAP (green) complex. The Mg²⁺ ion is shown in gray and the GDP molecule and AlF^–₄ are shown in ball-and-stick. The Asp side chains from switch II and a conserved lysine residue in RhoGAP protein are shown in the left panel. The lysine to proline mutation residue is highlighted in the right panel. The panel in the center is an enlarged view of salt-bridging interactions between Asp63 from the switch II region with the conserved lysine (proline mutation) residue in RhoGAP protein.