Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance
Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance
复制标题
DOI：
10.1074/jbc.m606781200
复制
发表时间：
2007-06-01
影响因子：
4.8
通讯作者：
Vogel, Stefanie N.
中科院分区：
文献类型：
--
作者：
Medvedev, Andrei E.;Piao, Wenji;Vogel, Stefanie N.
在这项研究中，我们研究了Toll样受体(TLR)4的Toll-IL-1抗性(TIR)区域的酪氨酸磷酸化是否是信号传递所必需的，并在内毒素耐受中被阻断。将导致C3H/HeJ小鼠脂多糖(LPS)无反应性的P712H突变引入到具有固有活性的小鼠Delta TLR4的TIR结构域中，以及人CD4-TLR4中同源P714的突变使它们失去信号功能并阻止TLR4酪氨酸磷酸化。酪氨酸残基Y674A和Y680A在CD4-TLR4的TIR结构域上的突变削弱了其诱导p38和JNK丝裂原激活蛋白激酶的磷酸化、I kappa B-α降解以及NF-kappa B和RANTES报告蛋白的激活的能力。同样，表达Y674A或Y680A突变的全长人类TLR4显示出介导内毒素诱导的细胞激活的能力受到抑制。Y674A和Y680A TLR4的信号缺陷与MyD88-TLR4相互作用的改变、与短IRAK-1亚型的关联增加以及与TLR4的复合体中激活的IRAK-1的数量减少有关。人胚胎肾(HEK)293/TLR4/MD-2细胞经蛋白酪氨酸激酶或Src激酶抑制剂处理后，可抑制脂多糖诱导的TLR4酪氨酸磷酸化、p38和核因子-kappaB的活化。TLR2和TLR4激动剂诱导高表达CD14、MD-2和TLR4或TLR2的HEK293细胞TLR酪氨酸磷酸化。HEK293/TLR4/MD-2细胞和人单核细胞对内毒素耐受的诱导可显著抑制脂多糖介导的TLR4酪氨酸磷酸化和Lyn激酶向TLR4的募集，但不影响TLR4-MD-2的相互作用。因此，我们的数据表明，TLR4酪氨酸磷酸化在内毒素耐受细胞中是重要的信号转导和受损的，并提示Lyn激酶参与了这些过程。
In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse Delta TLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, I kappa B-alpha degradation, and activation of NF-kappa B and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappa B activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.