FIG. 2.

Exchange of the PH and Btk domains between GAP1^IP4BP and GAP1^m has no effect on the Ras and Rap GAP activities of either hybrid protein in vivo. (A) Schematic diagram of the PH/Btk domain exchange constructs used in this study. (B, part i) CHO-T cells were transiently cotransfected with 2.5 μg Ras and 1 μg of the relevant GAP1^IP4BP or GAP1^m expression vector. Compared to that in control cells, the amount of Ras-GTP is significantly decreased in cells expressing wild-type GAP1^IP4BP and GAP1^m, as well as in cells expressing the GAP1^IP4BP and GAP1^m PH/Btk domain hybrid proteins. (C, part i) CHO-T cells were transiently cotransfected with 2.5 μg HA-tagged Rap1A and 1 μg of the relevant GAP1^IP4BP or GAP1^m expression vector. Compared to that in control cells, the amount of Rap1-GTP is only significantly decreased in cells expressing wild-type GAP1^IP4BP and in cells expressing GAP1^IP4BP with the PH/Btk domain from GAP1^m. Wild-type GAP1^m and GAP1^m with the PH/Btk domain from GAP1^IP4BP do not display Rap1 GAP activity. Ras-GTP (B, part ii) and Rap1-GTP (C, part ii) levels from CHO-T cells are expressed as percentages of the pulldown level in control cells (average of six separate experiments ± the standard error of the mean).