Signal-dependent Phosphorylation and Function of IKKy

描述(由申请人提供)：转录因子NF-kappaB调控多种基因的表达，这些基因调节先天免疫和获得性免疫。核因子-kappaB的不适当、持续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。核因子-kappaB的生物诱导剂包括细菌脂多糖、促炎性细胞因子、抗原受体激动剂和病毒基因产物，如人类T细胞白血病病毒1型的Tax癌蛋白。这些信号依赖的反应都受核因子-kappaB的细胞质抑制物I-kappaB和可诱导的多组分I-kappaB激酶IKK的调节。在细胞刺激后，IKK的IKKβ催化亚基磷酸化I-kappaB，导致抑制物的降解和核因子-kappaB的释放到其核作用部位。这项赠款申请的重点是IKKGamma，这是同一大分子酶复合体的一个非催化亚单位。编码IKKGamma的X连锁基因突变会导致皮肤炎或体液免疫缺陷。尽管其作用机制尚不清楚，但IKKGamma是依赖信号激活IKKbeta所必需的。申请人实验室的研究表明，IKKGamma通过涉及IKKbeta的机制在多个丝氨酸残基上受到信号依赖的磷酸化作用。这一发现突显了两个亚基之间以前未被认识到的相互作用，这将被用来解开IKKGamma磷酸化的生化功能。中心假说是，IKKbeta介导的IKKGamma的磷酸化影响I-kappaB激酶活性的调节，导致基因表达的改变和核因子-kappaB调控下细胞反应的变化。为了验证这一假设，我们提出了一些实验，以确定IKKGamma中受到信号依赖的磷酸化的IKKbeta反应部位(特异性目标1)，在核因子-kappaB信号转导中IKKGamma磷酸化的细胞内生化功能(特异性目标2)，以及表达磷酸化缺陷形式的IKKGamma的小鼠的体内表型(特异性目标3)。后面的实验将揭示IKKGamma磷酸化在炎症和免疫中的生理学基础。综上所述，这些拟议的研究将极大地提高我们目前对IKKGamma作用的分子机制的有限了解，并为研究免疫系统中的NF-kappaB信号提供新的动物模型。

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.