Characterization of human E4BP4, a phosphorylated bZIP factor

摘要

In this report we described the isolation of the transcription factor E4BP4 by λgt11 expression cloning using a probe containing the CRE/ATF-like sequence located between ?2764 by and ?2753 by in the upstream regulatory region for the human IL-1β gene. DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 β promoter sequences. By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by gel shift assay indicated that E4BP4 bound specifically to CRE/ATF-like site, not NFκ B-like site. In cotransfection studies, E4BP4 repressed promoter activity and this repression was mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggested that the DNA binding as well as repression activities required leucine heptad repeat domain. Analysis of E4BP4 produced in Escherichia coli and SO cells infected with recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E. coli-produced protein. Analysis of posttranslational modifications indicated that E4BP4 produced in Sf9 cells was phosphorylated and this phosphorylation was important for the DNA binding activity of E4BP4.