A novel homeo-domain protein, prep 1, forms a regulatory complex with pbx proteins in vivo

Expression of the human urokinase (uPA) gene is regulated by an enhancer containing a combined PEA3/AP-1 site and an AP-1 site, spaced 72bp apart. These two sites have been shown to synergistically cooperate in both basal level and PMA induced activity of the enhancer, dependent on an element, the co-operation mediator (COM) element, positioned between the two sites, and which bind several different nuclear factors. uPA Enhancer Factor 3 (UEF3) has been identified in various cell lines binding specifically to a site within the COM element and in addition, binds to similar regulating motifs found in various other promoters, showing similar organization and activity as the uPA enhancer. We have purified UEF3 from HeLa cell nuclei by standard chromatography. Purification data show that UEF3 is a complex formed by three polypeptides, p64, p50 and p40. Overall, the data further suggest that UEF3 is a heterodimer consiting of p64 complexed with either p50 orp40. We have cloned the cDNA p64, which encodes for a novel protein, termed Prepl, containing a homeodomain similar to those found in mammalian PBX and Drosophila Extradenticle (exd) homeo domain proteins and in yeast Mat2a protein. We find that Prepl exist as a stable complex with Pbx proteins in vivo and that this complex can be made in vitro reconstituting UEF3 activity. Pbx are proteins known to functionally interact with Hox proteins during development. Both its structure and its way of interacting with Pbx, show that Prepl is different from Hox proteins, and our data point to a novel regulatory role for Pbx/Prepl complexes proteins independent on Hox.