The pFR-Luc plasmid, the pFA2-cJun fusion trans-activator plasmid, and the pFC-MEKK control plasmid were cotransfected into HeLa cells. The pFA2-Jun fusion trans-activator plasmid is specific for the c-Jun N-terminal kinase10,16 (JNK) signaling pathway and the pFC-MEKK control plasmid constitutively expresses MEKK kinase17. The MEKK protein, a known JNK activator, increased the expression of the luciferase gene by more than 100-fold (Figure 2A), indicating activation of the GAL4-cJun fusion protein.5,10 The pFC2-dbd plasmid could not be activated by overexpression of the MEKK protein.
The specificity of the pFA2-CREB fusion activator plasmid is shown in Figure 2B. Cyclic AMP-dependent protein kinase (PKA) is a known activator of the CREB protein, and the pFC-PKA plasmid expresses this active kinase.14,18 When the pFA2-CREB plasmid was cotransfected with the pFC-PKA plasmid and the pFR-Luc reporter plasmid, a 100-fold increase in luciferase expression was seen. As expected, cotransfection of the pFR-Luc and pFC-PKA plasmids with the pFC2-dbd plasmid yielded only background levels of luciferase activity.
In a similar fashion, the specificity of the pFA2-Elk1 fusion activator plasmid
is demonstrated (Figure
2C). Since the MEK1 protein is a known upstream activator of the Elk1