Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen.
Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen.
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DOI：
10.4049/jimmunol.158.4.1902
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发表时间：
1997-02
影响因子：
4.4
通讯作者：
S. R. Yan;M. Huang;G. Berton
中科院分区：
文献类型：
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作者：
S. R. Yan;M. Huang;G. Berton
Src 家族激酶参与多形核中性粒细胞 (PMN) 中整合素的信号传导。为了鉴定 Src 家族激酶复合物中存在的蛋白质，我们对在纤维蛋白原包被表面上孵育的 PMN Triton X-100 裂解物中的 p58(c-fgr) 或 p53/56(lyn) 免疫沉淀物进行体外激酶测定。对来自 PMN 的 Triton 裂解物的 p58(c-fgr) 或 p53/56(lyn) 免疫沉淀物进行的分析表明，几种蛋白质与 Src 家族激酶形成复合物，并且可以在体外被磷酸化。免疫印迹分析表明 p72(syk) 酪氨酸激酶是这些蛋白质之一。含有 Src 家族激酶和 p72(syk) 的蛋白质复合物的形成需要 PMN 扩散，因为在悬浮液中用 TNF-α 刺激的 PMN 的 p58(c-fgr) 或 p53/56(lyn) 免疫沉淀物中未检测到 p72(syk)。此外，在没有 TNF-α 的情况下，用 Mn2+ 诱导 PMN 扩散促进了含有 Src 家族激酶和 p72(syk) 的蛋白质复合物的形成。我们还发现 p72(syk)-自磷酸化激酶活性增强，并且 p72(syk) 的一部分易位到 PMN 中的 Triton 不溶性细胞骨架部分，通过 TNF-α 或 Mn2+ 诱导其在纤维蛋白原上扩散。对来自传播的 PMN 的 Triton 裂解物的 CD18（β2 整联蛋白亚基）免疫沉淀物进行的体外激酶测定表明，几种蛋白质与 CD18 形成复合物，并且可以在体外磷酸化。 CD18 免疫沉淀物的免疫印迹分析使我们能够将 p72(syk) 鉴定为这些蛋白质之一。这些发现表明，PMN 扩散伴随着 p72(syk) 的激活和 Src 家族激酶和 p72(syk) 共定位的多分子复合物的形成。
Src family kinases are implicated in signaling by integrins in polymorphonuclear neutrophils (PMN). To identify proteins present in complexes with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton X-100 lysates of PMN incubated on fibrinogen-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to spread over fibrinogen in response to TNF-alpha showed that several proteins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing Src family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition, induction of PMN spreading with Mn2+ in the absence of TNF-alpha promoted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta 2 integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipitates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(syk) and formation of multimolecular complexes in which Src family kinases and p72(syk) colocalize.