Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation

Independent but closely spaced DNA binding sites for Stat3 and c-Jun are required for maximal enhancer function in a number of genes, including the gene encoding the interleukin-6 (IL-6)-induced acute-phase response protein, α₂-macroglobulin. In addition, a physical interaction of Stat3 with c-Jun, based on yeast two-hybrid interaction experiments, has been reported. Here we confirm the existence of an interaction between Stat3 and c-Jun both in vitro, with recombinant proteins, and in vivo, during transient transfection. Using fragments of both proteins, we mapped the interactive sites to the C- terminal region of c-Jun and to two regions in Stat3, within the coiled-coil domain and in a portion of the DNA binding domain distant from DNA contact sites. In transient-transfection experiments with the α₂-macroglobulin enhancer, Stat3 and c-Jun cooperated to yield maximal enhancer function. Point mutations of Stat3 within the interacting domains blocked both physical interaction of Stat3 with c-Jun and their cooperation in IL-6-induced transcription directed by the α₂-macroglobulin enhancer. While the amino acid sequences and the three-dimensional structures of Stat3 and Stat1 cores are very similar, fragments of Stat1 failed to bind c-Jun in vitro. Although Stat1 binds in vitro to the gamma interferon gene response (GAS) element in the α₂-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the α₂-macroglobulin enhancer.