ARAP3 Is a PI3K- and Rap-Regulated GAP for RhoA Further titration of the relative levels of ARAP3 andSonja Krugmann,1, * Roger Williams,2

ARAP3 could stimulate the GTPase activity of 10 nM1Inositide Laboratory The Babraham Institute GTP-RhoA approximately 3-fold (not shown). While such a high enzyme to substrate ratio is not unusual in theseCambridge CB2 4AT United Kingdom types of assays, to confirm the ability of ARAP3 to act as a RhoA GAP in vivo, we performed “pull-down ” assays2 MRC Laboratory of Molecular Biology Hills Road (Figure 1B) from Sf9 cells expressing Rac1 or RhoA alone or together with ARAP3. Figure 1B shows thatCambridge CB2 2QH United Kingdom relatively low amounts of ARAP3 were sufficient to cause a significant decrease in the amount of RhoA-GTP in the Sf9 cells lysates, while Rac1-GTP levels were not affected by the presence of larger amounts of ARAP3.Summary We saw a similar substrate specificity when ARAP3 was transiently cotransfected with Rac or Rho in COS-7 cellsRho and Arf family small GTPases [1] are well-known (Figure S1). We conclude that RhoA is a likely physiologi-regulators of cellular actin dynamics. We recently cal substrate of ARAP3.identified ARAP3, a member of the ARAP family of dua