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Thr435 phosphorylation regulates RelA (p65) NF-kappaB subunit transactivation
John M O'Shea1, Neil D Perkins
1Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
The Biochemical Journal
|December 17, 2009
Summary
Phosphorylation of RelA at Thr435, induced by tumor necrosis factor alpha, enhances CXCL2 gene expression. This modification impacts histone acetylation and HDAC1 recruitment, modulating NF-kappaB transcriptional activity.
Area of Science:
- Molecular Biology
- Cellular Biology
- Immunology
Background:
- Nuclear factor kappaB (NF-kappaB) transcription factors regulate immune responses and cellular processes.
- Post-translational modifications, such as phosphorylation, are critical for modulating NF-kappaB activity.
- The RelA (p65) subunit's C-terminal transactivation domain plays a key role in gene regulation.
Purpose of the Study:
- To investigate the functional consequences of Thr435 phosphorylation in the RelA subunit.
- To determine the impact of Thr435 phosphorylation on NF-kappaB-mediated gene transcription.
- To elucidate the molecular mechanisms underlying RelA Thr435 phosphorylation-induced transcriptional changes.
Main Methods:
- Site-directed mutagenesis to create a T435D phosphomimetic mutant of RelA.
- Quantitative PCR to measure CXC chemokine ligand 2 (CXCL2) mRNA levels.
- Chromatin immunoprecipitation (ChIP) assays to assess histone acetylation and HDAC1 recruitment.
- In vitro interaction assays to study RelA and HDAC1 binding.
Main Results:
- Thr435 phosphorylation of RelA is induced by tumor necrosis factor alpha (TNFα).
- The T435D mutation significantly enhanced CXCL2 mRNA levels in RelA-deficient cells.
- RelA Thr435 phosphorylation led to increased histone acetylation and decreased histone deacetylase 1 (HDAC1) recruitment at target genes.
- Mutation of Thr435 disrupted the interaction between RelA and HDAC1.
Conclusions:
- Thr435 phosphorylation of RelA is a critical regulatory mechanism.
- This phosphorylation event enhances the transcription of specific NF-kappaB target genes, such as CXCL2.
- RelA Thr435 phosphorylation modulates NF-kappaB transcriptional specificity by altering chromatin accessibility through HDAC1 interaction.
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