DNA binding of repressor nuclear factor-κB p50/p50 depends on phosphorylation of Ser337 by the protein kinase A catalytic subunit

被引:83
作者
Guan, HC
Hou, SH
Ricciardi, RP [1 ]
机构
[1] Univ Penn, Sch Dent Med, Dept Microbiol, Philadelphia, PA 19104 USA
[2] Univ Penn, Sch Med, Dept Biochem & Biophys, Philadelphia, PA 19104 USA
关键词
D O I
10.1074/jbc.M412180200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The NF-kappa B p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-kappa B p50 Ser(337) is critical for DNA binding. Here, we report that p50 Ser(337) is constitutively phosphorylated by the protein kinase A catalytic subunit (PKAc) in three different cell types, which may account for the constant binding of p50/p50 to DNA in unstimulated cells. This was demonstrated first by showing that treatment of cells with PKAc-specific inhibitors blocked p50/p50 DNA binding. Second, phosphorylation of p50 by PKAc was prevented by substitution of Ser337 to alanine. Third, both p50 and PKAc proteins as well as kinase activity that phosphorylates p50 were found to co-fractionate following gel filtration chromatography. Finally, PKAc and p50 were shown to be able to reciprocally co-immunoprecipitate one another, and their physical association was blocked by a PKA catalytic site inhibitory peptide. This indicates that phosphorylation of p50 Ser337 involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor alpha, DNA binding of p50/p50 homodimers was not greatly altered. Taken together, these findings reveal for the first time that there is a direct interaction between PKAc and p50 that accounts for constitutive phosphorylation of p50 Ser(337) and the existence of DNA bound p50/p50 in the nuclei of most resting cells. This mechanism of DNA binding by p50/p50 following phosphorylation of Ser337 by PKAc may represent an important means for maintaining stable negative regulation of NF-kappa B gene expression in the absence of extracellular stimulation.
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页码:9957 / 9962
页数:6
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