( r plasmid is shown in figure ...)In Vivo Signal Transduction Pathway Reporting Systems

r plasmid is shown in figure 3, panel C. The PKA protein is a known activator of the CREB protein. When the pFA-CREB plasmid was cotransfected with the pFC-PKA plasmid and the reporter plasmid, a 100-fold increase in luciferase expression was seen. Further experiments (data not shown) using a construct with a mutation in the PKA phosphorylation site of the pFA-CREB plasmid showed that the mutated fusion activator could not be activated by the PKA protein.

Activation of PathDetect Reporting Systems by Extracellular Stimuli

Figure 4

In addition to activation by uncharacterized gene products, the PathDetect trans-reporting systems can be used to study the effects of physiological stimuli and drug candidates. Figure 4 shows the activation of the pFA-Elk1 vector by 10% serum, indicating activation of the MAPK signal transduction pathway. The amount of pFA-Elk1 vector used in this experiment seems limiting because the activation obtained from 10 ng of pFA-Elk1 is 10-fold greater than the activation obtained with 1 ng. Similarly, the PathDetect c-Jun trans-reporting system can be activated by ultraviolet irradiation, and the PathDetect CREB system was activated by known PKA-activating agents such as the adenylate cyclase toxin from Bordetella pertussis and forskolin (data not shown).

Applications of the PathDetect Systems

The PathDetect c-Jun, Elk1 and CREB trans-reporting systems give specific assessment of JNK, MAPK or PKA activity. Such specificity allows the effects of uncharacterized proteins on the JNK, MAPK and PKA pathways to be determined. These reporting systems show minimal background activity because mammalian transcripti
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