Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases
Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases
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DOI：
10.1046/j.1523-1755.2003.00097.x
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发表时间：
2003-08-01
影响因子：
19.6
通讯作者：
Tryggvason, K
中科院分区：
文献类型：
--
作者：
Lahdenperä, J;Kilpeläinen, P;Tryggvason, K
背景。肾素是最近发现的免疫球蛋白（IG）超家族的蛋白质。在肾脏中，它位于狭缝隔膜，形成了肾小球超滤屏障的决定性尺寸选择性滤波器，并位于足细胞的互相脚步之间。肾素在芬兰类型（NPHS1）的先天性肾脏中突变，已被证明是缝隙膜片的重要组成部分。基于其结构域的结构，肾素可能是可能具有信号传导功能的细胞细胞或细胞 - 矩阵粘附蛋白。在这项研究中，我们假设肾蛋白在细胞表面上的抗体聚类模仿了肾素与其细胞外配体之间相互作用的情况。通过单克隆抗肾素抗体和多克隆二抗体，将肾素聚集在稳定转染的HEK293细胞的表面上。通过免疫荧光显微镜观察簇。通过免疫沉淀和蛋白质印迹分析，研究了蛋白质磷酸化的变化。使用特定的抑制剂和共转染实验来研究SRC家族激酶在肾磷酸化中的作用。肾素的聚类诱导其自身的酪氨酸磷酸化。 SRC家族激酶的抑制剂PP2抑制了这种磷酸化。当用肾素转染HEK293细胞时，SRC家族激酶的几个成员能够诱导肾素磷酸化。此外，始终发现SRC家族激酶FYN与肾素共免疫沉淀。有趣的是，肾素的聚类也诱导的46 kD蛋白的酪氨酸磷酸化也被发现与肾蛋白共免疫沉淀。肾素是一种由SRC家族激酶磷酸化的信号蛋白。
Background. Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of nephrin with antibodies on cell surface mimics the situation where the interaction between nephrin and its extracellular ligand(s) is altered.Methods. Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antinephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in nephrin phosphorylation.Results. Clustering of nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce nephrin phosphorylation when cotransfected to HEK293 cells with nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with nephrin. Interestingly, clustering of nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with nephrin.Conclusion. Nephrin is a signaling protein phosphorylated by Src family kinases.