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Signal Transduction Reporting Systems Using Cis-Acting Enhancer,,,Elements

figure 2

To test the performance of these plasmids (figure 2), we cotransfected each of the individual reporter plasmids into HeLa cells together with an expression vector of a known activator, either MEK kinase (MEKK) or the catalytic subunit of cAMP-dependent protein kinase (PKA). The empty vector without an insert was used as a negative control.5,6 The overexpression of both MEKK and PKA stimulated luciferase expression from pAP1-Luc and pSRE-Luc plasmids. However, pCRE-Luc responded only to PKA and pNFkB-Luc 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.7,8-12 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 pNFkB-Luc.

As true for all transfection experiments, conditions must be optimized to achieve high activation levels. The variables to consider include the choice of cell line, transfection method and protocol. In addition, the amounts of reporter vector and expression vector encoding the gene of interest must be optimized.

Conclusions

Stratagenes PathDetect cis-reporter plasmids were constructed for studying the in vivo activation of signal transd
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