Abstract
It has been demonstrated that phosphorylation of the p50 subunit of NF-kappaB is required for efficient DNA binding, yet the specific phospho-residues of p50 have not been determined. In this study, we substituted all of the serine and conserved threonine residues in the p50 Rel homology domain and identified three serine residues, Ser65, Ser337, and Ser342, as critical for DNA binding without affecting dimerization. Although substitution with negatively charged aspartic acid at each of these positions failed to restore DNA binding, substitution with threonine, a potential phospho-acceptor, retained DNA binding for residues 65 and 337. In particular, Ser337, in a consensus site for protein kinase A (PKA) and other kinases, was shown to be phosphorylated both in vitro and in vivo. Importantly, phosphorylation of Ser337 by PKA in vitro dramatically increased DNA binding of p50. This study shows for the first time that the DNA binding ability of NF-kappaB p50 subunit is regulated through phosphorylation of residue Ser337, which has implications for both positive and negative control of NF-kappaB transcription.
Highlights
The transcription factor NF-B acts as a central regulator of inflammatory, immune, and stress responses by controlling gene expression of cytokines, chemokines, immunoreceptors, antigen-presenting proteins, growth factors, transcription factors, cell adhesion molecules, stress response proteins, and apoptotic regulators [1, 2]
When serine 276 (Ser276) in the Rel homology domain is phosphorylated by the protein kinase A (PKA) catalytic subunit, p65 is able to associate with coactivator CBP/p300, whereas unphosphorylated p65 associates with corepressor histone deacetylase [10, 30, 31]
We selected the most highly conserved threonine residues in p50 based on the homology among p50, p65, and Dorsal proteins and replaced them with alanine to test their effects on p50 DNA binding
Summary
The transcription factor NF-B acts as a central regulator of inflammatory, immune, and stress responses by controlling gene expression of cytokines, chemokines, immunoreceptors, antigen-presenting proteins, growth factors, transcription factors, cell adhesion molecules, stress response proteins, and apoptotic regulators [1, 2]. This study shows for the first time that the DNA binding ability of NF-B p50 subunit is regulated through phosphorylation of residue Ser337, which has implications for both positive and negative control of NF-B transcription. To determine the potential phosphoresidues that are critical for p50 DNA binding in vivo, we tested the DNA binding abilities of wild type and point mutant p50 proteins in a mammalian cellular environment.
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