STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.

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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, alpha(2)-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 alpha(2)-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 alpha(2)-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 alpha(2)-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the alpha(2)-macroglobulin enhancer.