DNA Binding of Repressor Nuclear Factor-κB p50/p50 Depends on Phosphorylation of Ser337 by the Protein Kinase A Catalytic Subunit

Abstract

The NF-B p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-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 Ser 337 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 Ser 337 involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor ␣, 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 Ser 337 by PKAc may represent an important means for maintaining stable negative regulation of NF-B gene expression in the absence of extracellular stimulation.The nuclear factor NF-B is a transcription factor identified by Sen and Baltimore nearly 20 years ago (1). NF-B plays a critical role in transcription regulation of genes involved in immune response, inflammation, cell proliferation, differentiation, apoptosis, and oncogenesis (2-6). In vertebrates, five members of NF-B family have been identified. These include RelA (p65), RelB, c-Rel, NF-B1 (p50/p105), and NF-B2 (p52/ p100). All of these proteins share a highly conserved Rel homology domain termed RHD within the N-terminal 300 amino acid region. The RHD is responsible for DNA binding, dimerization, nuclear translocation, and interaction with IB inhibitory proteins. Although all NF-B family members can bind to DNA, only p65, RelB, and c-Rel contain a transactivation domain in their C-terminal regions.The NF-B family members can form various homodimers and heterodimers among which the p50/p65 heterodimer is the most abundant and studied species. In most resting or unstimulated cells, p50/p65 heterodimers are confined in the cytoplasm in an inactive form by forming a complex with IB proteins. Treatment of cells with NF-B stimuli such as cytokines, mitogens, and bacterial lipopolysaccharide leads to phosphorylation of IB by IB kinase and subsequent ubiquitination and degradation of IB by the 26 S proteasome (7,8). This allows NF-B to ...

Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway

“…Similarly, as discussed above, p50 is phosphorylated at the RelA Ser-276 equivalent, that is, Ser-337 (Hou et al, 2003;Guan et al, 2005). Thus, it is likely that the equivalents of RelA Ser-276 and Ser-281, as well as other conserved sites, can be modified in all NF-kB subunits.…”

“…p50 and c-Rel are also modified by PKAc at Ser-337 and Ser-267, respectively, the equivalent sites to RelA Ser-276 (Mosialos et al, 1991;Hou et al, 2003;Yu et al, 2004;Guan et al, 2005) (Figure 3; Table 3). Since this serine is also conserved in RelB (but not p52), it is probable that similar modification occurs with this subunit.…”

NF-κB in Renal Inflammation

“…[32][33][34][35][36] Homo-or heterodimers of p50 and p52 repress B site-dependent transcription possibly as a result of competition for DNA binding with other transcriptionally active dimers, such as p50/RelA. 37 Suppressors of cytokine signaling 1 forms part of a nuclear protein complex that promotes the ubiquitylation and proteasomal degradation of RelA-containing dimers, thus quenching NF-B responses. 38 Recently, suppressors of cytokine signaling 1 overexpression was shown to decrease inflammation in experimental diabetic nephropathy.…”

Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A

“…A possible intermediary transcriptional repressor is NF-κB p50/p50 homodimer. DNA binding by NF-κB p50/p50 was found to require phosphorylation of serine-337 by the catalytic subunit of PKA in HeLa cells, COS-7 cells and adenovirally transformed murine kidney cells [22]. Four NF-κB binding sites were identified in the rat IGFBP-2 promoter and were involved in hyperoxia-induced NF-κB activity in rat lung alveolar epithelial cells [23].…”

“…Four NF-κB binding sites were identified in the rat IGFBP-2 promoter and were involved in hyperoxia-induced NF-κB activity in rat lung alveolar epithelial cells [23]. The fore-mentioned phosphorylation of NF-κB p50/p50 by PKA's catalytic subunit, and consequent transcriptional repression, were found to be constitutive and cAMP-independent in cells at rest [22]. Such a mechanism may explain why forskolin failed to further decrease IGFBP-2 levels in the H295-R cells.…”