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Thr435 phosphorylation regulates RelA (p65) NF-kappaB subunit transactivation.

ABSTRACT: Phosphorylation of the RelA (p65) NF-kappaB (nuclear factor kappaB) subunit has been previously shown to modulate its ability to induce or repress transcription. In the present study we have investigated the consequences of Thr435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr435 is phosphorylated in cells and is induced by TNFalpha (tumour necrosis factor alpha) treatment. Mutational analysis of this site revealed gene-specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 (CXC chemokine ligand 2) mRNA levels in reconstituted Rela-/- mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation associated with decreased recruitment of HDAC1 (histone deacetylase 1). Moreover, mutation of this site disrupted RelA interaction with HDAC1 in vitro. Thr435 phosphorylation of promoter-bound RelA was also detected at NF-kappaB target genes following TNFalpha treatment in wild-type mouse embryonic fibroblasts. Phosphorylation at this site therefore provides an additional mechanism through which the specificity of NF-kappaB transcriptional activity can be modulated in cells.

SUBMITTER: O'Shea JM

PROVIDER: S-EPMC2829238 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Thr435 phosphorylation regulates RelA (p65) NF-kappaB subunit transactivation.

O'Shea John M JM Perkins Neil D ND

The Biochemical journal 20100224 3

Phosphorylation of the RelA (p65) NF-kappaB (nuclear factor kappaB) subunit has been previously shown to modulate its ability to induce or repress transcription. In the present study we have investigated the consequences of Thr435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr435 is phosphorylated in cells and is induced by TNFalpha (tumour necrosis factor alpha) treatment. Mutational analysis of this site revealed gene-specific effects on transcription ...[more]

PMID: 20001970

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Project description:Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis, and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because nuclear factor-kappaB (NF-kappaB) activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-kappaB-mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated tumor necrosis factor (TNF)-induced activation of NF-kappaB. The glycoside also inhibited NF-kappaB activated by carcinogenic and inflammatory agents, such as cigarette smoke condensate, phorbol 12-myristate 13-acetate, okadaic acid, hydrogen peroxide, lipopolysaccharide, and epidermal growth factor. When examined for the mechanism of action, we found that picroliv inhibited activation of IkappaBalpha kinase, leading to inhibition of phosphorylation and degradation of IkappaBalpha. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of Cys(38) in p65 to serine abolished this effect of picroliv. NF-kappaB inhibition by picroliv leads to suppression of NF-kappaB-regulated proteins, including those linked with cell survival (inhibitor of apoptosis protein 1, Bcl-2, Bcl-xL, survivin, and TNF receptor-associated factor 2), proliferation (cyclin D1 and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (intercellular adhesion molecule-1 and matrix metalloproteinase-9). Suppression of these proteins enhanced apoptosis induced by TNF. Overall, our results show that picroliv inhibits the NF-kappaB activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects.

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Thr435 phosphorylation regulates RelA (p65) NF-kappaB subunit transactivation.

Publications

Thr435 phosphorylation regulates RelA (p65) NF-kappaB subunit transactivation.

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