Phosphorylation of serine 337 of NF-kappaB p50 is critical for DNA binding.
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.
Hou
Shihe
S
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Guan
Hancheng
H
Ricciardi
Robert P
RP
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014162:Transfection
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D013912:Threonine; D012694:Serine; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases