Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling.
Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling.
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DOI：
10.1091/mbc.e16-09-0632
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发表时间：
2017-04-15
影响因子：
3.3
通讯作者：
Kwiatkowska K
中科院分区：
文献类型：
--
作者：
Borzęcka-Solarz K;Dembińska J;Hromada-Judycka A;Traczyk G;Ciesielska A;Ziemlińska E;Świątkowska A;Kwiatkowska K
细菌脂多糖激活Toll样受体4（TLR 4）并触发巨噬细胞的促炎反应。TLR 4信号传导受林恩酪氨酸激酶负调控，前提是该激酶由于棕榈酰化、催化活性以及SH 2和SH 3介导的分子间相互作用而在膜筏中积累。脂多糖（LPS）是革兰氏阴性菌的组分，其激活Toll样受体4（TLR 4）以触发促炎反应。我们研究了林恩酪氨酸激酶参与巨噬细胞TLR 4信号转导，区分其催化活性和分子间相互作用。为此，在RAW 264巨噬细胞样细胞或鼠腹膜巨噬细胞中过表达一系列在林恩的特定结构域中具有点突变的Lyn-GFP构建体，并分析它们对LPS诱导的应答的影响。过量表达野生型或组成型活性林恩可抑制RAW 264细胞中TNF-α和CCL 5/RANTES细胞因子的产生，并下调NFκB和IRF 3转录因子的活性。林恩催化结构域或Src同源2（SH 2）或SH 3结构域或经历LPS诱导的棕榈酰化的半胱氨酸残基的点突变消除了林恩的负面影响。取决于细胞类型，这些林恩突变形式的过量产生甚至可以上调LPS诱导的反应，并且这种效应通过内源性林恩表达的沉默来再现。同时，林恩突变阻断了LPS诱导的RAW 264细胞筏组分中Lyn的积累。这些数据表明，棕榈酰化，SH 2和SH 3介导的分子间相互作用，和催化活性的林恩是必需的，其在筏积累，从而确定TLR 4信号的负调控。
Bacterial lipopolysaccharide activates Toll-like receptor 4 (TLR4) and triggers proinflammatory reactions of macrophages. TLR4 signaling is negatively regulated by Lyn tyrosine kinase, provided the kinase accumulates in membrane rafts as a result of palmitoylation, the catalytic activity, and SH2- and SH3-mediated intermolecular interactions. Lipopolysaccharide (LPS) is the component of Gram-negative bacteria that activates Toll-like receptor 4 (TLR4) to trigger proinflammatory responses. We examined the involvement of Lyn tyrosine kinase in TLR4 signaling of macrophages, distinguishing its catalytic activity and intermolecular interactions. For this, a series of Lyn-GFP constructs bearing point mutations in particular domains of Lyn were overexpressed in RAW264 macrophage-like cells or murine peritoneal macrophages, and their influence on LPS-induced responses was analyzed. Overproduction of wild-type or constitutively active Lyn inhibited production of TNF-α and CCL5/RANTES cytokines and down-regulated the activity of NFκB and IRF3 transcription factors in RAW264 cells. The negative influence of Lyn was nullified by point mutations of Lyn catalytic domain or Src homology 2 (SH2) or SH3 domains or of the cysteine residue that undergoes LPS-induced palmitoylation. Depending on the cell type, overproduction of those mutant forms of Lyn could even up-regulate LPS-induced responses, and this effect was reproduced by silencing of endogenous Lyn expression. Simultaneously, the Lyn mutations blocked its LPS-induced accumulation in the raft fraction of RAW264 cells. These data indicate that palmitoylation, SH2- and SH3-mediated intermolecular interactions, and the catalytic activity of Lyn are required for its accumulation in rafts, thereby determining the negative regulation of TLR4 signaling.