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Assess the In Vivo Activation of Signal Transduction Pathways with,,,PathDetect Reporting Systems

of cAMP-dependent protein kinase (PKA). The vector without an insert was used as a negative control.17,18 Transfection procedure, maintenance of transfected cells, cell lysate preparation, and luciferase assay are previously described.5

Expression of both MEKK and PKA increased the luciferase expression from the pAP1-Luc and pSRE-Luc plasmids. However, the pCRE-Luc plasmid responded only to PKA and the pNFkB-Luc plasmid only to MEKK. These results are consistent with previous reports that both MEKK and PKA are capable of activating the expression of genes containing SRE and AP-1 or NF-kB binding elements in their promoter, while the CRE element found in somatostatin promoter is very specific to PKA.19-21 MEKK, a protein kinase known to activate the stress signaling pathways and the MAP kinase pathway, also activates signaling molecules converging at NF-kB, as measured with the pNFkB-Luc plasmid.

Conclusions

The PathDetect reporting systems offer a unique way to study signal transduction, a cellular process that is key for understanding mechanisms of both normal cellular function and disease. With the PathDetect trans-reporting systems, assess the involvement of gene products, extracellular stimuli, or drug candidates in a particular pathway using c-Jun, Elk1, CREB, ATF2, CHOP, and c-Fos activation domains. Any transcription factor of interest can be cloned and subsequently studied using the pFA-CMV fusion trans-activator cloning vector. The PathDetect cis-reporting systems provide a simple format for studying the interaction of a gene product or extracellular stimulus with the CRE, SRE, AP-1, NF-kB, p53, or SRF enhancer elements. Activation of a signaling pathway or interaction with an enhancer element is readily measured by the luciferase assay,
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