ARAP 3 Is a PI 3 K - and Rap - Regulated GAP for RhoA Further titration of the relative levels of ARAP

family GTPases [2, 3], in a screen for PtdIns(3,4,5)P3 Rap Binds to ARAP3 and Activates Its Rho binding proteins. PtdIns(3,4,5)P3 is the lipid product of GAP Activity In Vitro class I phosphoinositide 3OH-kinases (PI3Ks) [4] and Just C-terminal to ARAP3’s Rho GAP domain lies a is a signaling molecule used by growth factor recepweakly conserved Ras binding domain (RBD; Figure 2A). tors and integrins in the regulation of cell dynamics To investigate whether ARAP3 binds to Ras family pro[5]. We report here that as a Rho GAP, ARAP3 prefers teins, we immobilized ARAP3 protein on nitrocellulose RhoA as a substrate and that it can be activated in and overlayed with [ -P]GTP preloaded GST-fusion vitro by the direct binding of Rap [6] proteins to a proteins of several Ras family members. We detected neighbouring Ras binding domain (RBD). This activaan interaction with Rap1B and 2B and Rheb but not with tion by Rap is GTP dependent and specific for Rap H-Ras, R-Ras, TC21, M-Ras, or RalA (Figure 2B). All versus other Ras family members. We found no evianalyzed Ras family members interacted with the Raf dence for direct regulation of ARAP3’s Rho GAP ackinase RBD but not with the Rac/Cdc42 binding domain tivity by PtdIns(3,4,5)P3 in vitro, but PI3K activity was of PAK (PAK CRIB). To map binding of Rap-GTP to required for activation by Rap in a cellular context, ARAP3’s putative RBD, a three-dimensional model of suggesting that PtdIns(3,4,5)P3-dependent translothe ARAP3 RBD was constructed manually based on cation of ARAP3 to the plasma membrane may be the structures of the complex of p110 PI3K with Ras required for further activation by Rap. Our results indi[7] and the complex of the RalGDS RBD with Ras [8]. cate that ARAP3 is a Rap-effector that plays an imporThis suggested that Arg1155 would be central to the Rap tant role in mediating PI3K-dependent crosstalk bebinding interface of ARAP3. A R1155E point mutation tween Ras, Rho, and Arf family small GTPases. indeed abolished the interaction between ARAP3 and