( each fusion activator plasmid known ac...)In Vivo Signal Transduction Pathway Reporting Systems

each fusion activator plasmid, known activators and nonactivators were cotransfected into HeLa cells with the pFR-Luc reporter plasmid. Cotransfections were performed using standard lipofection methods, and lysates were prepared and assayed for luciferase activity. The pFR-Luc plasmid was cotransfected into HeLa cells together with the fusion activator plasmid specific for the JNK signaling pathway ((pFA-cJun) and the pFC-MEKK control plasmid (figure 3, panel A). The MEKK protein, a known JNK activator, increased the expression of the luciferase gene by more than 100 fold (figure 3, panel A), indicating activation of the c-Jun transactivator fusion protein.^7,15 The pFC-dbd plasmid, which expresses the GAL4 DNA binding domain only and no activation domain, could not be activated by overexpression of the MEKK protein. A mutated form of the c-Jun transactivator fusion protein, which had alanine residues substituted for serine 63 and 73 in c-Jun, could not be activated by MEKK (data not shown).

In a similar fashion, the specificity of the pFA-Elk1 fusion activator plasmid was also demonstrated (figure 3, panel B). The ternary complex factor Elk1 is phosphorylated and activated by MAPK.^10,11 The MEKK and MEK1 proteins are both known activators of the Elk1 protein. Cotransfection of the pFA-Elk1 vector with pFC-MEK1 vector resulted in 50-fold increased expression of luciferase, whereas cotransfection with pFC-MEKK vector resulted in 200-fold increased expression of luciferase. These data are consistent with reports that both MEK1 and MEKK proteins are known activators of the MAPK pathway. ^16-18

The specificity of the pFA-CREB fusion activato
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