Functional analysis of the mutants of hMgcRacGAP.

(A) Suppression of growth of HL-60 cells overexpressing the wild-type or mutated hMgcRacGAPs. Each of the wild-type, the mutant R385A*, and 2 deletion mutants was transduced into HL-60 cells using the retrovirus vector pMX-IRES-EGFP. GFP⁺ HL-60 cells transduced with these viruses were sorted on FACS 2 days after virus infection. GFP⁺ HL-60 cells, transduced with a blank pMX-IRES-EGFP vector, were sorted as a negative control. The cell number of each transfectant was plotted against time. The results shown are the averages ± SD of triplicate cultures. (B) Expression of the Flag-tagged wild-type and mutant hMgcRacGAPs in HL-60 transfectants. Cell lysates from control HL-60 cells (lane 5) and HL-60 transfectants expressing the wild-type (lane 1), ΔCys (lane 2), ΔMyo (lane 2), or R385A* (lane 4) MgcRacGAP (3 × 10⁷ per lane) were examined by Western blotting using the anti-Flag M2 monoclonal antibody. (C) Differentiation of HL-60 cells overexpressing the wild-type or mutated hMgcRacGAPs. Morphologic differentiation of control HL-60 cells and HL-60 cells transduced with the wild-type, R385A*, or deletion mutants of hMgcRacGAP is shown. Cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400  × magnification.