Signal-dependent Phosphorylation and Function of IKKy

IKKy 的信号依赖性磷酸化和功能

• 批准号: 6613532
• 负责人: DEAN BALLARD
• 金额: $ 37.75万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613532
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; enzyme activity; enzyme complex; enzyme linked immunosorbent assay; flow cytometry; gene expression; immunoprecipitation; immunoregulation; inflammation; laboratory mouse; nuclear factor kappa beta; phosphorylation; phosphotransferases; protein kinase; protein structure function; serine; site directed mutagenesis; transcription factor; transfection

DESCRIPTION (provided by applicant): Transcription factor NF-kappaB governs the expression of multiple genes that mediate innate and adaptive immunity. The inappropriate, persistent activation of NF-kappaB underlies acute and chronic inflammatory diseases, rendering it an attractive target for therapeutic intervention. Biologic inducers of NF-kappaB include bacterial lipopolysaccharides, proinflammatory cytokines, antigen receptor agonists, and viral gene products such as the Tax oncoprotein of human T-cell leukemia virus type 1. Each of these signal-dependent responses is regulated by I-kappaB, a cytoplasmic inhibitor of NF-kappaB, and an inducible multicomponent I-kappaB kinase called IKK. Following cellular stimulation, the IKKbeta catalytic subunit of IKK phosphorylates I-kappaB, leading to degradation of the inhibitor and the release of NF-kappaB to its nuclear site of action. This grant application focuses on IKKgamma, a noncatalytic subunit of the same macromolecular enzyme complex. Mutations in the X-linked gene encoding IKKgamma can lead to skin inflammation or humeral immunodeficiencies. Although its mechanism of action remains unknown, IKKgamma is required for signal-dependent activation of IKKbeta. Studies in the applicant's laboratory have revealed that IKKgamma is subject to signal-dependent phosphorylation at multiple serine residues via a mechanism involving IKKbeta. This discovery highlights a previously unrecognized interplay between the two subunits that will be exploited to unravel the biochemical function of IKKgamma phosphorylation. The central hypothesis is that IKKbeta-mediated phosphorylation of IKKgamma affects the regulation of I-kappaB kinase activity, leading to altered gene expression and changes in the cellular responses under NF-kappaB control. To test this hypothesis, experiments are proposed to determine the IKKbeta-responsive sites in IKKgamma that are subject to signal-dependent phosphorylation (Specific Aim 1), the intracellular biochemical function of IKKgamma phosphorylation in NF-kappaB signal transduction (Specific Aim 2), and the in vivo phenotype of mice expressing phosphorylation-defective forms of IKKgamma (Specific Aim 3). The latter experiments will uncover the physiologic basis for IKKgamma phosphorylation in inflammation and immunity. Together, these proposed studies will significantly advance our currently limited knowledge about the molecular mechanism of IKKgamma action and provide new animal models to study NF-kappaB signaling in the immune system.

描述（由申请人提供）：转录因子NF-κ B控制介导先天性和适应性免疫的多个基因的表达。NF-κ B的不适当的、持续的激活是急性和慢性炎症性疾病的基础，使其成为治疗干预的有吸引力的靶点。NF-κ B的生物诱导剂包括细菌脂多糖、促炎细胞因子、抗原受体激动剂和病毒基因产物，如人T细胞白血病病毒1型的Tax癌蛋白。这些信号依赖性反应中的每一种都受到I-κ B（NF-κ B的细胞质抑制剂）和称为IKK的诱导型多组分I-κ B激酶的调节。在细胞刺激后，IKK的IKK β催化亚基磷酸化I-kappaB，导致抑制剂降解和NF-κ B释放到其核作用位点。这项拨款申请的重点是IKK γ，同一大分子酶复合物的非催化亚基。编码IKK γ的X连锁基因突变可导致皮肤炎症或肱骨免疫缺陷。尽管其作用机制尚不清楚，但IKK γ是IKK β信号依赖性激活所必需的。申请人实验室的研究表明，IKK γ通过涉及IKK β的机制在多个丝氨酸残基处发生信号依赖性磷酸化。这一发现突出了以前未被认识到的两个亚基之间的相互作用，将被利用来解开IKK γ磷酸化的生化功能。中心假设是IKK β介导的IKK γ磷酸化影响I-κ B激酶活性的调节，导致基因表达改变和NF-κ B控制下细胞反应的变化。为了验证这一假设，提出了实验来确定IKK γ中受到信号依赖性磷酸化的IKK β反应位点（特异性目的1）、IKK γ磷酸化在NF-κ B信号转导中的细胞内生化功能（特异性目的2）以及表达IKK γ磷酸化缺陷形式的小鼠的体内表型（特异性目的3）。后面的实验将揭示炎症和免疫中IKK γ磷酸化的生理基础。总之，这些拟议的研究将显着推进我们目前有限的知识IKK γ作用的分子机制，并提供新的动物模型，研究NF-κ B信号在免疫系统中。