Anrather Josef, Racchumi Gianfranco, Iadecola Costantino
Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021, USA.
J Biol Chem. 2005 Jan 7;280(1):244-52. doi: 10.1074/jbc.M409344200. Epub 2004 Oct 29.
Phosphorylation of nuclear factor-kappa B (NF-kappa B) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-kappa B complexes is regulated in an inhibitor kappa B-independent fashion. As the main transactivator, the p65 subunit of NF-kappa B has an outstanding position in the hierarchy of NF-kappa B proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation domain and the N-terminal Rel homology domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA(-/-) mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic kappa B-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes.
核因子-κB（NF-κB）亚基的磷酸化成为一种机制，通过该机制，核NF-κB复合物的转录活性以一种不依赖抑制蛋白κB的方式受到调控。作为主要的反式激活因子，NF-κB的p65亚基在NF-κB蛋白层级中占据突出地位。p65是一种多重磷酸化蛋白，在C端反式激活结构域和N端Rel同源结构域（RHD）中均有磷酸化位点。在本研究中，我们描述了p65 RHD内两个先前未报道的磷酸化位点。我们表明，RHD内丝氨酸残基的差异磷酸化在顺式作用元件和启动子特异性背景下调节转录活性，从而导致磷酸化状态依赖性的基因表达谱。用野生型p65或p65磷酸化缺陷突变体重构的RelA（-/-）小鼠胚胎成纤维细胞显示出合成κB依赖性报告基因以及内源性基因的独特表达谱。低磷酸化的p65在DNA结合或二聚化行为上未显示顺式作用元件特异性变化。本研究首次表明，位点特异性磷酸化可将转录因子靶向特定的基因子集。
