The leucine zipper domain controls the orientation of AP-1 in the NFAT.AP-1.DNA complex.
BACKGROUND
Heterologous transcription factors bound to adjacent sites in eukaryotic promoters often exhibit cooperative behavior. In most instances, the molecular basis for this cooperativity is poorly understood. Our efforts have been directed toward elucidation of the mechanism of cooperativity between NFAT and AP-1, two proteins that coordinately direct expression of the T-cell growth factor interleukin-2 (IL-2).


RESULTS
We have previously shown that NFAT1 orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions. In the present study, we have constructed cJun-cFos chimeric proteins and determined their orientation using a novel affinity-cleavage technology based on chemical ligation. We find that, in the presence of NFAT, the chimeric heterodimer binds in such a way as to preserve the orientation of the AP-1 leucine zipper, but not that of the basic region.


CONCLUSIONS
Protein-protein interactions between NFAT and the leucine zipper of AP-1 enable the two proteins to bind DNA cooperatively and coordinately regulate the IL-2 promoter. The chemical ligation technology presented here provides a powerful strategy for affinity cleavage studies, including those using recombinant proteins.
Erlanson
D A
DA
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Chytil
M
M
Verdine
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GL
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004492:Edetic Acid / Q000378:metabolism; D004926:Escherichia coli; D004952:Esters / Q000138:chemical synthesis; D015870:Gene Expression / Q000235:genetics; D016350:Leucine Zippers / Q000502:physiology*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004952:Esters; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA; D004492:Edetic Acid