GTP and GDP binding to normal and D57N recombinant Rac protein and dominant-negative effects of D57N on morphology of NIH/3T3 cells.

(A) WT and D57N Rac2 protein was expressed in E coli and then assayed for binding to ³H-GDP or³⁵S-γGTP as described in “Materials and methods.” Data presented are from 3 experiments (mean ± SEM) P< .001 vs WT. (B-D) Photomicrographs of GFP-positive NIH/3T3 cells after infection with retrovirus vectors expressing only GFP (MIEG3) (B), WT Rac2 (HR2WT) (C), or D57N mutant Rac2 (HR2MU) (D). (E) Fluorescent photomicrographs of the same cells in B-D, stained with rhodamine–phalloidin. Note the extensive ruffling in cells transfected with MIEG or HR2WT and in the untransfected (cell staining only at arrowhead in HR2MU panel). In contrast, note the complete absence of ruffling in 2 cells shown transduced with HR2MU (green nuclei). Bar represents 20 μm.