Phosphorylation of NLRC4 is critical for inflammasome activation
Phosphorylation of NLRC4 is critical for inflammasome activation
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DOI：
10.1038/nature11429
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发表时间：
2012-10-25
期刊：
影响因子：
64.8
通讯作者：
Dixit, Vishva M.
中科院分区：
文献类型：
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作者：
Qu, Yan;Misaghi, Shahram;Dixit, Vishva M.
NLRC4 是 NOD 样受体家族的胞质成员，在先天免疫细胞中表达。它间接感知细菌鞭毛蛋白和 III 型分泌系统，并通过组装促进 caspase-1 激活和细胞焦亡的炎性体复合物做出反应 (1-6)。在这里，我们使用表达具有羧基末端 3xFlag 标签的 NLRC4 的敲入小鼠来鉴定在用鼠伤寒沙门氏菌（也称为鼠伤寒沙门氏菌）感染巨噬细胞后，NLRC4 在单个进化保守残基 Ser 533 上的磷酸化。使用 NLRC4 磷酸化 Ser 533 抗体进行的蛋白质印迹证实，这种翻译后修饰仅在存在已知与 NLRC4 结合的刺激物的情况下发生，而不是在相关蛋白 NLRP3 或 AIM2 结合的情况下发生。与用野生型NLRC4重建的NLRC4(-/-)巨噬细胞不同，用NLRC4突变体S533A重建的Nlrc4(-/-)巨噬细胞不会响应鼠伤寒沙门氏菌而激活caspase-1和焦亡，表明S533磷酸化对于NLRC4炎症小体功能至关重要。相反，磷酸模拟物NLRC4 S533D在没有感染的情况下引起巨噬细胞快速焦亡。 NLRC4 磷酸化活性的生化纯化和激酶抑制剂筛选将 PRKCD (PKC delta) 确定为候选 NLRC4 激酶。重组 PKC δ 体外磷酸化 NLRC4 S533，巨噬细胞裂解物中的 PKC δ 免疫耗竭可阻断 NLRC4 S533 体外磷酸化，Prkcd(-/-) 巨噬细胞表现出大大减弱的 caspase-1 激活和 IL-1 β 分泌，特别是针对鼠伤寒沙门氏菌。磷酸化缺陷的 NLRC4 S533A 在鼠伤寒沙门氏菌感染期间无法募集 procaspase-1，并且不会组装炎性小体斑点 (7)，因此 NLRC4 S533 的磷酸化可能会驱动 NLRC4 炎性小体活性和宿主先天免疫所需的构象变化。
NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis(1-6). Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3xFlag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKC delta) as a candidate NLRC4 kinase. Recombinant PKC delta phosphorylated NLRC4 S533 in vitro, immunodepletion of PKC delta from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1 beta secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks(7) during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.