FIG. 3.

EGF-induced stimulation of TBP promoter activity requires the activation of Ras and the three classes of MAPKs. (A) Expression of a dominant negative mutant Ras (RasAla15) inhibits EGF induction of TBP promoter activity. JB6 cells were transiently cotransfected with the TBP promoter construct p−4500/+66hTBP-luc (as described in the legend for Fig. 2A) and 2 μg of the RasA15 expression plasmid or empty vector. The change in TBP promoter activity was determined relative to that with no EGF treatment and in the absence of RasA15 expression. (B) Inhibitors of p38, JNK, and ERK1/2 MAPKs each block EGF stimulation of TBP promoter activity. Cells transfected with p−4500/+66hTBP-luc, as described in the legend for Fig. 2A, were pretreated with U0126 (10 μM), SB202190 (1 μM), or SP600125 (2.5 μM) for 1 h and then incubated with EGF (12.5 ng/ml) for 30 min. The values shown are the means ± the standard errors of the means of at least four independent experiments. (C) Kinase inhibitors are selective for blocking the activation of each of the MAPKs. Cells were pretreated with inhibitors as described for panel B and then incubated with or without EGF (12.5 ng/ml) for 30 min as indicated. Immunoblot analysis of the resultant cell lysates was carried out using specific anti-phospho-MAPKs, as indicated.