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C/EBPα, C/EBPα oncoproteins, or C/EBPβ preferentially bind NF-κB p50 compared with p65, focusing therapeutic

Julia E Dooher1, Ido Paz-Priel, Simone Houng

  • 1Division of Pediatric Oncology, Johns Hopkins University, Baltimore, MD 21231, USA.

Molecular Cancer Research : MCR
|August 5, 2011
PubMed

Insights

CCAAT-enhancer-binding proteins (C/EBPs) interact with p50 more strongly than p65, activating NF-κB target genes independently of canonical NF-κB signals. Targeting C/EBP:p50 interactions may treat inflammatory and malignant conditions.

Area of Science:

  • Molecular Biology
  • Cellular Signaling
  • Transcription Regulation

Background:

  • Canonical nuclear factor kappaB (NF-κB) activation involves p50:p65 complexes binding to chromatin.
  • CCAAT-enhancer-binding proteins (C/EBPs) can interact with NF-κB subunits, potentially influencing gene activation.
  • Previous work showed C/EBPα cooperates with p50, not p65, for Bcl-2 transcription and induces Nfkb1/p50 expression.

Purpose of the Study:

  • To investigate the differential binding affinities of C/EBPs (including isoforms and oncoproteins) to p50 versus p65.
  • To identify specific molecular regions responsible for the interaction specificity between C/EBPs and NF-κB subunits.
  • To elucidate the role of C/EBP:p50 interactions in regulating NF-κB target genes, independent of canonical NF-κB activation.

Main Methods:

  • Utilized FLAG-tagged p50 and p65 variants to assess binding affinities with C/EBPα, C/EBPβ isoforms (LAP1, LAP2, LIP), and myeloid oncoproteins.
  • Employed deletion mutagenesis of the p65 transactivation domain and clustered mutagenesis within the 'p50 insert' region.
  • Performed saturating mutagenesis of the C/EBPα basic region to pinpoint critical residues for p50 interaction.

Main Results:

  • C/EBPα, C/EBPβ isoforms, and myeloid oncoproteins exhibited significantly higher affinity for p50 compared to p65.
  • Specificity for p50 interaction is attributed to unique residues in p50, particularly within the HSDL motif of the 'p50 insert'.
  • Mutagenesis identified R300 and adjacent residues in the C/EBPα basic region as crucial for p50 binding.

Conclusions:

  • C/EBPs can activate NF-κB target genes through interaction with p50, even without canonical NF-κB pathway activation.
  • The interaction specificity is primarily mediated by unique features of p50, not by the presence of the p65 transactivation domain.
  • Targeting C/EBP:p50 interactions offers a potential therapeutic strategy for inflammatory and malignant conditions, possibly synergizing with cytoplasmic NF-κB inhibitors.

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