( Ras-responsive. 11 The pFA-cFos ...)New Fusion Trans-Activator Plasmids for Studying Signal Transduction,,,Pathways

Ras-responsive.¹¹ The pFA-cFos plasmid was constructed by inserting the DNA fragment encoding residues 208 to 313 of rat c-Fos¹² into the BamH I and EcoR I sites of pFA-CMV. Our results (figure 3A) in transient transfection assays demonstrate that the MEKK protein activates the GAL4-c-Fos fusion protein expressed from the pFA-cFos plasmid. The mechanism of this activation is not yet characterized. PKA, MEK1 and other extracellular stimuli failed to activate the GAL4-c-Fos fusion protein (data not shown).

figure 3B

ATF2, or activating transcription factor 2, belongs to a large family of transcription factors that recognize the ATF2/CRE elements identified in the adenovirus early gene promoters.^8-10,13 ATF2 forms homodimers as well as heterodimers with other members of the ATF, Jun and Fos transcription factors. The regulation of ATF2 is not fully characterized. However, phosphorylation of Thr-69 and Thr-81 in ATF2 by JNK is known to activate the ATF2 transcription factor.¹³ In order to construct pFA-ATF2 (1-96), the DNA fragment encoding amino acid residues 1-96 of human ATF2^14,15 was inserted into the BamH I and EcoR I sites of the pFA-CMV vector. Consistent with previous reports, the GAL4-ATF2 fusion protein expressed from the pFA-ATF2 plasmid was strongly activated by the MEKK protein in transient transfection assays (figure 3A).

Conclusions

Stratagene has constructed three new fusion trans-activator plasmids for use with the PathDetect in vivo signal transduction pathway reporting systems. All three of these plasmids can be used in a variety of mammalian cell lin
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