FIGURE 7.

Rab7 CMT2B mutants show lower Elk-1-driven gene activation and c-fos and Egr-1 expression on EGF stimulation. A, HeLa cells stably expressing GFP-tagged WT Rab7 and CMT2B mutants were transfected with plasmids pFR Luc and pFA2 Elk-1 to study Elk-1-driven gene activity. Cells were starved in DMEM for 14 h, followed by EGF stimulation (100 ng/ml) in a time-dependent manner. Following stimulation, cells were kept in serum-free medium for 6 h and lysed, and luciferase activity was determined in a luminometer. Each time point was performed twice and measured in triplicates. Relative light units are normalized to the protein amounts. Disease mutants showed lower Elk-1-driven gene activity. Rab7 CMT2B mutants were compared with the wild-type Rab7 at respective time points. Differences are represented as ±S.E. (error bars). B, HeLa cells stably expressing GFP-Rab7 wild type and CMT2B mutants were serum-starved, EGF-stimulated for 1 h, and processed for RT-PCR as detailed under “Experimental Procedures.” The bar graphs show the lower expression of c-fos upon EGF stimulation in cells expressing GFP-Rab7 CMT2B mutants compared with the wild type. Differences are represented as ±S.E. C, bar graphs showing the lower expression of Egr-1 upon 1-h EGF stimulation in cells expressing GFP-Rab7 CMT2B mutants compared with the wild type. Differences are represented as ±S.E. D and E, bar graphs showing no difference in expression levels of SMN1 and Mcl-1 upon 1-h EGF stimulation in cells expressing GFP-Rab7 CMT2B mutants compared with the wild type. *, p < 0.05 based on one-way ANOVA with Dunnett's post-test and relative to Rab7 wild-type control.