Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

作者：Steven M. Ruben, Ramaswamy Narayanan, John F. Klement, Chu-Huang Chen, Craig A. Rosen · 发表于：Molecular and Cellular Biology · 年份：1992 · DOI：10.1128/mcb.12.2.444 · 被引用次数：171 · 研究领域：NF-κB Signaling Pathways、T-cell and Retrovirus Studies、Animal Disease Management and Epidemiology

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests...