IKKa-Dependent Negative Feedback Control of Non-Canonical NF-kB Activation

描述(由申请人提供)：二聚体核因子-：B转录因子在多种免疫过程中发挥关键作用。在静息细胞中，核因子-：B二聚体被释放，通过两条基本信号通路启动基因转录，这两条基本信号通路被称为正则信号通路和非正则信号通路。这两条途径导致核因子-：B二聚体在不同的时间尺度上释放，并且受到不同的调控。规范的NF-：B信号在几分钟内被多种细胞类型上的大量受体激活，而非规范的NF-：B信号在几个小时内被一组选定的受体激活，如BAFF、LT2R、CD40，并在有限数量的细胞类型上排名，如B细胞、成纤维细胞和巨噬细胞。然而，这两条途径在B细胞、破骨细胞和次级淋巴组织的产生和存活中都扮演着关键的、非多余的角色。现已认识到，典型和非典型核因子-B信号的过度激活可导致多种自身免疫和增殖性疾病，包括系统性红斑狼疮、多发性骨髓瘤和弥漫性B细胞淋巴瘤。尽管在典型的NF-：B信号中描述了多种负反馈机制，用于终止信号并防止病理性过度激活，但对于非典型的NF-：B信号通路，还没有描述过负反馈机制。我们的初步研究表明，被Nik激活的IKK1不仅可以诱导P100的磷酸化和加工，而且还可以诱导Nik的磷酸化和失稳。我们进一步定位了Nik中的IKK1磷酸化位点，并表明破坏依赖于IKK1的Nik磷酸化可以在受体激活后显著增加Nik的水平。基于这些初步结果，我们推测，虽然先前报道的TRAF-CIAP复合体是未刺激细胞中NIK降解的原因，但这种依赖IKK1的新的NIK磷酸化和失稳机制在受体激活后调节非规范的NF-：B活性方面发挥了重要的负反馈作用。这项R21拨款的目标是解开这种反馈发生的分子成分和机制。我们建议首先确定在Nik-IKK1反馈复合体中发挥作用的额外分子成分，然后确定这些成分如何在刺激诱导的NIK降解复合体中组装，最后确定这种反馈机制在调节典型和非典型NF-：B信号中的作用。总之，我们相信这些研究将显著提高我们对非规范的NF-：B调控的理解。鉴于该通路过度活跃可能导致自身免疫性疾病和癌症，进一步了解该通路中负反馈的分子因素、生化关系和功能作用将有助于进一步尝试药物干预该通路的活性。 与公共卫生相关：现在人们认识到，非规范的NF-：B通路的过度活跃可以导致多种自身免疫性疾病，如系统性红斑狼疮和癌症，包括多发性骨髓瘤和弥漫性B细胞淋巴瘤。这项R21基金的目标是揭开在非规范的NF-kB信号中负责一种新的反馈控制途径的分子成分和机制，这是我们根据最近令人兴奋的初步结果确定的。我们相信，进一步了解非典型性核因子-B通路中负反馈的分子因素、生化关系和功能作用，将有助于未来药物干预治疗自身免疫性疾病和癌症的尝试。

DESCRIPTION (provided by applicant): Dimeric NF-:B transcription factors play critical roles in a wide variety of immune processes. Cytosolically sequestered in resting cells, NF-:B dimers are released to initiate gene transcription through the action of two basic signaling pathways known as the canonical and non-canonical NF-:B signaling pathways. These two pathways lead to release of NF-:B dimers on vastly different time scales and are regulated distinctly. While canonical NF-:B signaling is activated within minutes by a large number of receptors on a wide variety of cell types, non-canonical NF-:B signaling is activated over hours by a select group of receptors such as BAFF, LT2R, CD40 and RANK on a limited number of cell types such as B-cells, fibroblasts, and macrophages. However, both of these pathways play critical, non-redundant roles in the generation and survival of B-cells, osteoclasts, and secondary lymphoid tissues. It is now appreciated that hyperactivity of both canonical and non-canonical NF-:B signaling can lead to a variety autoimmune and proliferative diseases including Systemic Lupus Erythematosus, Multiple Myeloma, and Diffuse B-cell Lymphoma. While multiple negative feedback mechanisms have been described in the case of canonical NF-:B signaling which serve to terminate signaling and prevent pathological hyperactivation, no negative feedback mechanisms have been described for the non- canonical NF-:B signaling pathway. Our preliminary studies have demonstrated that IKK1, activated by NIK, can induce not only p100 phosphorylation and processing but also phosphorylation and destabilization of NIK. We have further mapped the IKK1 phosphorylation sites in NIK and shown that disruption of IKK1-dependent NIK phosphorylation can significantly increase NIK levels after receptor activation. Based on these preliminary results, we hypothesize that while previously reported TRAF-cIAP complex is responsible for NIK degradation in unstimulated cells, this novel IKK1-dependent NIK phosphorylation and destabilization mechanism plays an important negative feedback role in regulating non-canonical NF-:B activity after receptor activation. The goal of this R21 grant is to unravel the molecular components and mechanisms by which this feedback occurs. We propose to first identify the additional molecular components functioning within NIK-IKK1 feedback complex, then determine how these components assemble within the stimulus-induced NIK degradative complex, and finally determine the role of this feedback mechanism in regulation of both canonical and non-canonical NF-:B signaling. Together, we believe these studies will significantly enhance our understanding of non-canonical NF- :B regulation. Given the pathological potential of this pathway's hyperactivity in causing autoimmune diseases and cancers, further understanding of the molecular factors, biochemical relationships, and functional roles of negative feedback within the pathway will assist in future attempts to pharmacologically intervene in the pathway's activity. PUBLIC HEALTH RELEVANCE: It is now appreciated that hyperactivity of the non-canonical NF-:B pathway can lead to a variety autoimmune diseases such as Systemic Lupus Erythematosus and cancers including Multiple Myeloma and Diffuse B-cell Lymphoma. The goal of this R21 grant is to unravel the molecular components and mechanisms responsible for a novel feedback control pathway within non-canonical NF-kB signaling which we have identified based on our recent exciting preliminary results. We believe further understanding of the molecular factors, biochemical relationships, and functional roles of negative feedback within the non-canonical NF- :B pathway will assist in future attempts to pharmacologically intervene in treating autoimmune diseases and cancers.