IL 1 INDUCED KINASES & TRANSCRIPTIONAL FACTORS

IL 1 诱导的激酶

• 批准号: 3304479
• 负责人: KAROL BOMSZTYK
• 金额: $ 15.43万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304479
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; cell nucleus; confocal scanning microscopy; cytoplasm; enzyme substrate complex; gel mobility shift assay; gene expression; genetic library; genetic regulatory element; immunofluorescence technique; immunoglobulin genes; interleukin 1; interleukin 2; molecular cloning; monoclonal antibody; nucleic acid probes; phosphorylation; protein kinase; protein purification; protein sequence; protein structure function; tissue /cell culture; transcription factor; western blottings

The intracellular pathways by which interleukin-1 (IL-1) signals gene expression are not well understood. We have been interested in defining the IL-1 signal transduction pathways that trigger the kappa light chain immunoglobulin and the IL-2 gene expression in lymphocytes. IL-1 activates multiple membrane and cytoplasmic events including activation of protein kinases. At the nuclear level IL-1-induced kappa light chain immunoglobulin and IL-2 gene expression is regulated by the EkappaB transcriptional element which recognizes a number of different complexes including NF-kappaB. We have shown that IL-1 induced gene expression is associated with the activation of a nuclear kinase that is contained within a novel DNA-binding complex specifically recognized by the EkappaB transcriptional element. The EkappaB-associated complex that we have identified contains a number of subunits including a 65kD protein which is a substrate for the IL-1 responsive EkappaB-associated kinase. The objective of this proposal is to test a hypothesis that this IL-1 responsive protein kinase serves to link IL-1 induced cytoplasmic events with the kappa light chain immunoglobulin and the IL-2 gene expression. We will first attempt to identify which of the EkappaB-associated proteins contains the IL-1 responsive kinase. Sequential chromatography will be used to synthesize degenerate oligonucleotides for screening of lymphocytes cDNA libraries and the cloning of the IL-1-responsive EkappaB-associated protein kinase. Similar approach will be used for partial amino acid sequencing of the 65kD EkappaB-associated substrate. We will attempt to clone the 65kD EkappaB-associated protein only if the amino acid sequence is that of a novel protein and if it is itself a kinase. The IL-1-responsive kinase might regulate either the DNA-binding or the transcriptional activity or both of the EkappaB-binding complexes. Gel shift analysis and methylation interference will be sued to test whether phosphorylation of the EkappaB- associated proteins by the IL-1 responsive kinase alters the DNA-binding activity of factors recognized by the EkappaB transcriptional element. In vitro transcription assays will be attempted to assess the role of the IL- 1-responsive kinase in modulating the transcriptional activity of the EkappaB-binding proteins. The IL-1-responsive EkappaB-associated kinase is present in the nucleus but it also might be present in the cytoplasm. The entire pool or only a fraction of the IL-1-responsive kinase might be bound to the EkappaB-associated complex. To determine the cellular distribution of the IL-1-responsive kinase in relation to the other EkappaB-associated proteins we will attempt to produce antibodies to the EkappaB-associated proteins and use immunofluorescence for localization. The activation of the IL-1-responsive EkappaB-associated kinase might be mediated by a more proximal kinase of the IL-1-initiated "phosphorylation cascade". In vitro phosphorylation assay will be attempted to determine whether the EkappaB- associated kinase is activated by another IL-1-responsive kinase. Our hypothesis would be supported if we show that a) the IL-1-responsive EkappaB-associated kinase stimulates gene expression by modifying the EkappaB-associated proteins, and b) that this kinase is itself stimulated by a more proximal IL-1-responsive kinase.

细胞介素1（IL-1）信号基因的细胞内途径 表达不太了解。 我们对定义感兴趣 触发Kappa轻链的IL-1信号转导途径 免疫球蛋白和淋巴细胞中的IL-2基因表达。 IL-1激活 多个膜和细胞质事件，包括蛋白质的激活 激酶。 在核水平IL-1诱导的Kappa轻链 免疫球蛋白和IL-2基因表达受Ekappab调节 识别许多不同复合物的转录元素 包括NF-kappab。 我们已经表明IL-1诱导的基因表达是 与包含在内的核激活相关的激活 Ekappab专门识别的一种新型DNA结合复合物 转录元素。 我们拥有的Ekappab相关复合物 确定包含许多亚基，包括65kD蛋白 IL-1响应性Ekappab相关激酶的底物。 这 该提议的目的是检验一个IL-1的假设 响应蛋白激酶可连接IL-1诱导的细胞质事件 带有Kappa轻链免疫球蛋白和IL-2基因表达。 我们将首先尝试确定哪些Ekappab相关蛋白 包含IL-1响应激酶。顺序色谱法将使用 合成退化的寡核苷酸以筛选淋巴细胞cDNA 图书馆和IL-1响应性Ekappab相关蛋白的克隆 激酶。 类似的方法将用于部分氨基酸测序 65KD Ekappab相关的底物。 我们将尝试克隆65KD 仅当氨基酸序列是A的蛋白 新型蛋白质，如果它本身是一种激酶。 IL-1响应激酶 可能调节DNA结合或转录活性或 两种ekappab结合络合物。 凝胶移位分析和甲基化 将起诉干涉以测试Ekappab-的磷酸化是否 IL-1反应激酶相关的蛋白质会改变DNA结合 ekappab转录元件识别的因素的活性。 在 体外转录测定将尝试评估IL-的作用 1响应性激酶调节转录活性 ekappab结合蛋白。 IL-1响应性Ekappab相关激酶是 存在于细胞核中，但也可能存在于细胞质中。 这 整个池或仅一小部分IL-1响应激酶可能是绑定的 到Ekappab相关的复合物。 确定细胞分布 与其他Ekappab相关的IL-1响应激酶 蛋白质我们将尝试生产与Ekappab相关的抗体 蛋白质并使用免疫荧光进行定位。 激活 IL-1响应性Ekappab相关激酶可能由A更多介导 IL-1引发的“磷酸化级联反应”的近端激酶。 体外 磷酸化测定将尝试确定Ekappab-是否是否 相关的激酶被另一种IL-1响应性激酶激活。 如果我们证明a）IL-1响应性，我们的假设将得到支持 ekappab相关激酶通过修饰刺激基因表达 ekappab相关的蛋白质，b）该激酶本身是刺激的 通过更近端的IL-1响应激酶。