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In Vivo Signal Transduction Pathway Reporting Systems

r activities by inducing intracellular signal transduction pathways that converge at a protein kinase.1-4 Thus, the in vivo effects of a newly discovered gene, growth factor or drug candidate on the signaling molecules along these pathways can be assessed by measuring the activation or inactivation of key kinases. Many of these protein kinases, such as JNK, MAPK and PKA, can in turn phosphorylate and activate specific transcription factors. As a result, the in vivo activities of these kinases can be measured by their effect on specific transcription factors whose activity can be determined by reporter enzyme assays.

figure 1

The PathDetect trans-reporting systems have been designed for rapid, easy and reliable in vivo assessment of the activation of certain signal transduction pathways. The PathDetect systems include a fusion trans-activator expression vector (figure 1, panel A) specific for the designated pathway, a reporter vector (figure 1, panel B), and plasmids that contain genes that encode proteins known to activate the trans-activator protein as a positive control (figure 1, panel C). All three fusion trans-activator plasmids share the GAL4 DNA binding domain5,6 but have different activation domains derived from c-Jun,7-9 Elk110-12 or CREB13 transcription factors, which are specifically phosphorylated and activated by JNK, MAPK or PKA, respectively. This specificity allows a protein or chemical to be assigned to a pathway. Since no transcription factor in mammalian cells can bind the GAL4 binding element efficiently, the use of GAL4 binding elements in the reporter vectors minimizes backgrou
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