Activated M-Ras protein induces Elk-1–mediated transcriptional activation. (A) HEK293 cells (5 × 105) were transiently cotransfected by lipofection with 500 ng of pFR-Luc reporter plasmid, 500 ng of the indicated experimental plasmids encoding the wild-type or activated mutants M-Ras, and 25 ng of the pathway-specific trans-activator plasmids (Gal4/Elk-1). pFC-MEK1 plasmid encodes constitutively activated MEK1 and was used as a positive control for Elk-1–trans-activation, and pFC-dbd as a negative control (Gal4). Luciferase activity was determined 24 hours after transfection. Data represent the means ± SD from an experiment performed in triplicate. The experiment was repeated 3 times with similar results. (B) HEK293 cells (5 × 105) were cotransfected with the reporter plasmid, pFR-Luc, the trans-activator plasmid pFA-Elk-1 and an expression vector encoding activated M-Ras/71K. After 5 hours of transfection, increasing amounts of PD98059 were added to the cell cultures and luciferase activity was measured after 24 hours. Similar results were obtained with other activated M-Ras mutants (data not shown). As a toxicity control, HEK293 cells were transfected with the reporter plasmid, pFR-Luc, the trans-activator plasmid pFA-Jun, and an expression vector encoding activated MEKK with or without PD98059, and no inhibition was observed (data not shown).
