Data from C/EBPα, C/EBPα Oncoproteins, or C/EBPβ Preferentially Bind NF-κB p50 Compared with p65, Focusing Therapeutic Targeting on the C/EBP:p50 Interaction

Abstract

<div>Abstract<p>Canonical nuclear factor kappaB (NF-κB) activation signals stimulate nuclear translocation of p50:p65, replacing inhibitory p50:p50 with activating complexes on chromatin. C/EBP interaction with p50 homodimers provides an alternative pathway for NF-κB target gene activation, and interaction with p50:p65 may enhance gene activation. We previously found that C/EBPα cooperates with p50, but not p65, to induce <i>Bcl-2</i> transcription and that C/EBPα induces <i>Nfkb1</i>/<i>p50</i>, but not <i>RelA</i>/<i>p65</i>, transcription. Using p50 and p65 variants containing the FLAG epitope at their N- or C-termini, we now show that C/EBPα, C/EBPα myeloid oncoproteins, or the LAP1, LAP2, or LIP isoforms of C/EBPβ have markedly higher affinity for p50 than for p65. Deletion of the p65 transactivation domain did not increase p65 affinity for C/EBPs, suggesting that unique residues in p50 account for specificity, and clustered mutation of <i>HSDL</i> in the “p50 insert” lacking in p65 weakens interaction. Also, in contrast to <i>Nfkb1</i> gene deletion, absence of the <i>RelA</i> gene does not reduce <i>Bcl-2</i> or <i>Cebpa</i> RNA in unstimulated cells or prevent interaction of C/EBPα with the <i>Bcl-2</i> promoter. Saturating mutagenesis of the C/EBPα basic region identifies R300 and nearby residues, identical in C/EBPβ, as critical for interaction with p50. These findings support the conclusion that C/EBPs activate NF-κB target genes via contact with p50 even in the absence of canonical NF-κB activation and indicate that targeting C/EBP:p50 rather than C/EBP:p65 interaction in the nucleus will prove effective for inflammatory or malignant conditions, alone or synergistically with agents acting in the cytoplasm to reduce canonical NF-κB activation. <i>Mol Cancer Res; 9(10); 1395–405. ©2011 AACR</i>.</p></div>