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Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides

机译:SH3结构域结合肽对肥大细胞激活中Lyn功能的抑制

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摘要

While Lyn tyrosine kinase has been shown to be necessary for IgE-receptor (FcepsilonRI)-mediated mast cell activation, the mechanism of Lyn activation is not yet understood. Using a micro-electroporation technique to quantitatively introduce peptides into the cytosol of tumor mast cells, we show that proline-rich peptides that preferentially bind Src family SH3 domains block receptor-induced repetitive calcium spikes in a concentration dependent manner. The Src family member Lyn was the likely target, since a series of phage displaying derived peptides with increased Lyn SH3 domain binding specificity inhibited FcepsilonRI-mediated calcium signaling at concentrations consistent with binding to Lyn rather than other Src-type kinases. Furthermore, SH3 binding peptides prevented the plasma membrane translocation of a fluorescently labeled Syk tandem SH2 domain, which binds to phosphorylated FcepsilonRI, suggesting that the peptides specifically block the Lyn-mediated step by which FcepsilonRI cross-linking leads to receptor phosphorylation. Our study suggests that the binding of proline-rich peptides, or corresponding cellular interaction partners, to Lyn SH3 domain suppresses the Lyn-mediated phosphorylatation of FcepsilonRI and calcium signaling.
机译:虽然已经证明Lyn酪氨酸激酶对于IgE受体(FcepsilonRI)介导的肥大细胞激活是必需的,但Lyn激活的机制尚不清楚。使用微电穿孔技术将肽定量引入肿瘤肥大细胞的细胞质中,我们显示优先结合Src家族SH3域的富含脯氨酸的肽以浓度依赖的方式阻断受体诱导的重复性钙峰值。 Src家族成员Lyn是可能的靶标,因为展示具有增加的Lyn SH3结构域结合特异性的衍生噬菌体的一系列噬菌体以与Lyn而不是其他Src型激酶结合的浓度抑制FcepsilonRI介导的钙信号传导。此外,SH3结合肽阻止了荧光标记的Syk串联SH2结构域的质膜移位,该区域与磷酸化的FcepsilonRI结合,表明这些肽特异性地阻断了Lyn介导的FcepsilonRI交联导致受体磷酸化的步骤。我们的研究表明,富含脯氨酸的肽或相应的细胞相互作用伴侣与Lyn SH3结构域的结合会抑制FcepsilonRI的Lyn介导的磷酸化和钙信号传导。

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