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. 2012 Mar 27;287(21):17176–17185. doi: 10.1074/jbc.M111.334458

FIGURE 3.

FIGURE 3.

Characterization of the effect of Rho family protein on GAP activity of AGAP1 and AGAP2. A, activation of AGAP1 is shown. GAP assays contained 0.2 μm [α-32P]GTP·myrArf1, 2.2 nm His10AGAP1, 500 μm large unilamellar vesicles, and either 1 mm GDP with 0.5 μm His-RhoA·GDP, His-Cdc42·GDP, and His-Rac1·GDP or 10 μm GTPγS with 0.5 μm His-RhoA·GTPγS, His-Cdc42·GTPγS, and His-Rac1·GTPγS. Activity is expressed as -fold increase over His10AGAP1 only control. Statistical analysis of the data included one way analysis of variance followed by the Dunnett multiple comparison test using AGAP1 only as the control. * p < 0.05; ***, p < 0.001; ns, not significant. B, activation of AGAP2 is shown. Experiment were performed essentially the same as in A, but 1.7 nm AGAP2-His6 was used instead of AGAP1. Statistical analysis was the same as for A. **, p < 0.01 compared with AGAP2 alone. C, heat sensitivity of RhoA effect is shown. The reactions were performed the same as in A, except 0.5 μm his-RhoA·GDP or his-Rac1·GDP was heated at 95 °C for 10 min where indicated. Data were analyzed by analysis of variance followed by Bonferroni's multiple comparison test. **, p < 0.01 compared with AGAP1 alone; ns, not significant compared with AGAP1 alone; #, p < 0.05 compared with AGAP1 + RhoA. D, the effect of RhoA on ArfGAP1 is shown. GAP activity for ArfGAP1 (20 nm) incubated with 0.5 μm RhoA·GDP or RhoA·GTP was determined. The results were analyzed by analysis of variance followed by the Bonferroni's multiple comparison test. ns, not significant compared with ArfGAP1 alone. E, shown is the effect of RhoA·GDP on [ΔGLD]AGAP1. Either 2.2 nm His10AGAP1 or 2.2 nm His10[ΔGLD]AGAP1 was used in the GAP assays. Statistical analysis was the same as for C. ***, p < 0.001 compared with AGAP1 alone; ns, not significant compared with [ΔGLD]AGAP1. F and G, shown is a comparison of substrates for RhoA-dependent AGAP1 catalytic activity. His10AGAP1 was titrated into a reaction containing myrArf1·GTP or myrArf6·GTP either in the absence (F) or presence (G) of 0.5 μm his-RhoA·GDP.