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

描述（由申请人提供）：二聚体NF-:B转录因子在多种免疫过程中发挥关键作用。NF-:B二聚体被隔离在静息细胞中，通过两种基本的信号通路，即典型和非典型NF-:B信号通路，释放并启动基因转录。这两种途径导致NF-:B二聚体在不同的时间尺度上释放，并且受到不同的调节。典型的NF-:B信号可以在几分钟内被多种细胞类型上的大量受体激活，而非典型的NF-:B信号可以在数小时内被少量细胞类型（如B细胞、成纤维细胞和巨噬细胞）上的BAFF、LT2R、CD40和RANK等特定受体激活。然而，这两种途径在b细胞、破骨细胞和次级淋巴组织的产生和存活中都起着关键的、非冗余的作用。现在人们认识到，典型和非典型NF-:B信号的过度活跃可导致多种自身免疫性和增生性疾病，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。虽然在典型的NF-:B信号通路中描述了多种负反馈机制，这些机制有助于终止信号传导并防止病理性过度激活，但对于非典型的NF-:B信号通路，没有描述负反馈机制。我们的初步研究表明，NIK激活的IKK1不仅可以诱导p100的磷酸化和加工，还可以诱导NIK的磷酸化和不稳定。我们进一步绘制了NIK中IKK1磷酸化位点，并表明IKK1依赖性NIK磷酸化的破坏可以在受体激活后显著增加NIK水平。基于这些初步结果，我们假设，尽管之前报道的TRAF-cIAP复合物在未受刺激的细胞中负责NIK降解，但这种新的ikk1依赖性NIK磷酸化和不稳定机制在受体激活后调节非规范NF-:B活性方面发挥重要的负反馈作用。这项R21资助的目标是揭示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反馈复合体中起作用的其他分子成分，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，最后确定该反馈机制在规范和非规范NF-:B信号传导调节中的作用。总之，我们相信这些研究将显著增强我们对非规范NF-:B调控的理解。鉴于该通路在引起自身免疫性疾病和癌症方面的过度活跃的病理潜力，进一步了解该通路中的分子因素、生化关系和负反馈的功能作用将有助于未来尝试从药理学上干预该通路的活性。

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.