Characterization of a nuclear factor that binds juxtaposed with ATF3/Jun on a composite response element specifically...

We have previously identified a 20-bp sequence that mediates induced transcription in response to EGF, Ras, and Raf but not after TPA or UV stimulation. This composite response element, present in a long terminal repeat of a member within the VL30 family of retrotransposons, contains an AP-1-like site that cooperates in function with a juxtaposed sequence unrelated to known transcription factor-binding sites. Using in vitro translated proteins, we here demonstrate that the AP-1-like site preferentially binds ATF3/c-Jun and ATF3/JunD heterodimers. Results from a functional analysis indicate that the ATF3/c-Jun heterodimer, together with factors interacting with the 3' element, are most likely the important mediators of the response because overexpression of JunD, alone or in combination with ATF3, abolishes Ras-induced transcription. Partial purification by phosphocellulose and DNA-affinity chromatography in combination with Southwestern analyses reveals a 52-kDa protein that specifically binds to the sequence juxtaposed to the AP-1-like site. Scatchard analyses show that this sequence, TTAGTTAC, forms two different complexes with K(d)s of 1.9 x 10(-10) and 2.3 x 10(-9) M, respectively. Together, these results suggest that EGF/Ras/Raf induces transcription via combined activation of ATF3/c-Jun and a 52-kDa nuclear factor, whereas JunD acts as a repressor of this response.