Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Biochem J. 2010 Feb 24;426(3):345-54. doi: 10.1042/BJ20091630.
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.
RelA（p65）NF-κB（核因子κB）亚基的磷酸化先前已被证明可以调节其诱导或抑制转录的能力。在本研究中，我们研究了 RelA 羧基末端转录激活结构域中 Thr435 磷酸化的后果。我们证实该 Thr435 在细胞中被磷酸化，并被 TNFα（肿瘤坏死因子α）处理诱导。该位点的突变分析显示对转录具有基因特异性影响，T435D 磷酸模拟突变体显著增强了重组 Rela-/- 小鼠胚胎成纤维细胞中 Cxcl2（CXC 趋化因子配体 2）mRNA 水平。染色质免疫沉淀分析显示，这种突变导致与 HDAC1（组蛋白去乙酰化酶 1）募集减少相关的组蛋白乙酰化水平升高。此外，该位点的突变破坏了 RelA 在体外与 HDAC1 的相互作用。在野生型小鼠胚胎成纤维细胞中用 TNFα 处理后，还检测到结合在启动子上的 RelA 的 Thr435 磷酸化。因此，该位点的磷酸化提供了一种额外的机制，通过该机制可以调节细胞中 NF-κB 转录活性的特异性。
