CHARACTERIZATION OF HUMAN E4BP4, A PHOSPHORYLATED BZIP FACTOR

In this report we described the isolation of transcription factor E4BP 4 by lambda gt11 expression cloning using a probe containing the CRE/A TF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-I beta gene. DNaseI protection, ge l mobility shift analysis, and cotransfection studies were performed t o investigate the binding and functional properties of E4BP4 using IL- 1 beta promoter sequences. By DNaseI footprinting, a protection patter n was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by,eel shift ass ay indicated that E4BP4 bound specifically to CRE/ATF-like site, not N F kappa B-like site. In cotransfection studies, E4BP4 repressed promot er activity and this repression was mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggested that the DNA binding as w ell as repression activities required leucine heptad repeat domain. An alysis of E4BP4 produced in Escherichia coli and Sf9 cells infected wi th recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E. c oli-produced protein. Analysis of posttranslational modifications indi cated that E4BP4 produced in Sf9 cells was phosphorylated and this pho sphorylation was important for the DNA binding activity of E4BP4.