Role of NF-kappaB phosphorylation in inflammatory signaling

描述（由申请人提供）：转录因子NF-κ B在诱导对促炎信号的细胞应答中起核心作用，并控制参与免疫应答、应激应答、癌症和程序性细胞死亡的大量基因的表达。NF-KappaB在两个水平上受到调节：除了抑制分子（IkappaB）的细胞质保留外，还有第二个调节网络控制核NF-KappaB复合物的转录活性。这种调节涉及细胞蛋白激酶，并通过NF-κ B亚基的翻译后修饰起作用。具体而言，NF-κ B p65亚基的磷酸化已被认为是通过促进启动子从其失活状态转变为其活性状态来实现靶基因表达的重要步骤。本研究的目的是阐明p65位点特异性磷酸化如何调节核NF-κ B活性。我们提供的证据表明，p65在Rel同源结构域（RHD）内被多重磷酸化，并且独特的磷酸化调节其转录活性。此外，我们发现，p65磷酸化控制基因表达的c/s元件依赖的方式。指导这一提议的中心假设是，NF-κ B介导的基因表达的特异性是通过p65亚基的差异磷酸化实现的。具体目标#1将解决p65是否受“磷酸化密码”的影响，该密码将p65反式激活靶向选定的基因子集。我们将采用分子和药理学的方法来研究如何启动子含有不同的c/s作用的调控元件是响应p65磷酸化。具体目标#2是研究p65磷酸化和乙酰化之间的关系。具体目标#3是探索p65 RHD磷酸化控制NF-κ B转录活性的机制。研究将采用分子和生物化学的方法来研究差异磷酸化的p65蛋白如何影响三个模型基因的启动子复合体组装和染色质重塑。在具体目标#4中，检验了p65差异磷酸化调节整体基因表达的假设。我们将使用高密度基因阵列来分析差异磷酸化p65蛋白对细胞NF-κ B依赖性基因表达的影响。总之，这些研究将确立p65磷酸化在实现信号特异性中的作用，并为NF-κ B依赖性促炎基因调控机制提供新的见解。

DESCRIPTION (provided by applicant): Transcription factor NF-KappaB plays a central role in inducing cellular responses to pro-inflammatory signals and controls the expression of a vast array of genes involved in immune response, stress response, cancer and programmed cell death. NF-KappaB is regulated on two levels: Beside cytoplasmic retention by inhibitory molecules (IkappaBs), there is a second regulatory network that controls transcriptional activity of nuclear NF-KappaB complexes. This regulation involves cellular protein kinases and acts through post-translational modifications of NF-KappaB subunits. Specifically, phosphorylation of the NF-KappaB p65 subunit has been implicated as an important step in achieving target gene expression by facilitating promoter transition from its inactive to its active state. The goal of this research program is to elucidate how site specific phosphorylation of p65 regulates nuclear NF-KappaB activity. We provide evidence, that p65 is multiply phosphorylated within the Rel Homology Domain (RHD) and that distinctive phosphorylation regulates its transcriptional activity. Furthermore, we show that p65 phosphorylation controls gene expression in a c/s-element dependent manner. The central hypothesis guiding this proposal is that specificity in NF-KappaB mediated gene expression is achieved through differential phosphorylation of the p65 subunit. Specific Aim #1 will address whether p65 is subject to a "phosphorylation code" that targets p65 transactivation to selected subsets of genes. We will employ molecular and pharmacologic approaches to investigate how promoters containing distinct c/s-acting regulatory elements are responsive to p65 phosphorylation. Specific Aim #2 is to study the relationship between p65 phosphorylation and acetylation. Specific Aim #3 is to explore the mechanism by which p65 RHD phosphorylation controls NF-KappaB transcriptional activity. Studies will employ molecular and biochemical approaches to investigate how differentially phosphorylated p65 proteins affect promoter complex assembly and chromatin remodeling on three model genes. In Specific Aim #4, the hypothesis is tested that p65 differential phosphorylation modulates global gene expression. We will use high-density gene arrays to analyze the impact of differentially phosphorylated p65 proteins on cellular NF-KappaB dependent gene expression. In summary, these studies will establish the role of p65 phosphorylation in achieving signal specificity and provide new insights into the mechanism of NF-KappaB dependent pro-inflammatory gene regulation.