Constitutive and interleukin-1-inducible phosphorylation of p65 NF-κB at serine 536 is mediated by multiple protein kinases including IκB kinase (IKK)-α, IKKβ, IKKε, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription
Constitutive and interleukin-1-inducible phosphorylation of p65 NF-κB at serine 536 is mediated by multiple protein kinases including IκB kinase (IKK)-α, IKKβ, IKKε, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription
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DOI：
10.1074/jbc.m409825200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Kracht, M
中科院分区：
文献类型：
--
作者：
Buss, H;Dörrie, A;Kracht, M
核因子-kappaB p65(RelA)丝氨酸536的磷酸化在多种促炎刺激下被生理性地诱导，但相关的通路尚未最终揭开，这种磷酸化的功能在很大程度上是难以捉摸的。与以前的研究不同，我们没有发现c-jun氨基末端激酶、p38激酶、细胞外信号调节激酶或磷脂酰肌醇3-激酶在白介素1或肿瘤坏死因子诱导的Ser-536磷酸化中的作用的证据，如药物抑制剂所揭示的那样。我们不能通过针对IkappaB激酶(IKK)-α/β(迄今为止特征最好的Ser-536激酶)的RNA干扰或IKKβ抑制剂SC-514或任何一种IKK的显性负突变体来抑制Ser-536的磷酸化。在没有IKK激活的情况下，绿色荧光蛋白P65融合蛋白在Ser-536处被磷酸化，这表明存在不依赖于IKKalpha/β的Ser-536激酶。细胞提取物的层析分离可以鉴定出两种不同的磷酸化丝氨酸-536的酶活性。峰1代表一个未知的激酶，而峰2含有IKKalpha、IKKbeta、IKKepsilon和TBK1。在体内和体外过表达的IKKepsilon和TBK1磷酸化Ser-536。在模拟磷酸化(S536D)或保留Ser-536和Asp-533之间预测的氢键(S536N)的p65缺乏的成纤维细胞中，突变的p65蛋白的重组表明，Ser-536的磷酸化有利于TFIID的一个成分TATA结合蛋白相关因子II31介导的IL-8转录。在没有磷酸化的情况下，氢键有利于Split的辅阻遏子氨基末端增强子与p65末端反式激活结构域的结合。总而言之，我们的结果提供了证据，证明至少有五种激酶聚集在p65的Ser-536上，并且这个磷酸化位点在基础转录机制的组成部分向IL-8启动子募集方面具有新的功能。
Phosphorylation of NF-kappaB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IkappaB kinase (IKK)-alpha/beta (the best characterized Ser-536 kinases so far) or the IKKbeta inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKalpha/beta-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKalpha, IKKbeta, IKKepsilon, and TBK1. Overexpressed IKKepsilon and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter.