Neph1, a component of the kidney slit diaphragm, is tyrosine-phosphorylated by the Src family tyrosine kinase and modulates intracellular signaling by binding to Grb2
Neph1, a component of the kidney slit diaphragm, is tyrosine-phosphorylated by the Src family tyrosine kinase and modulates intracellular signaling by binding to Grb2
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DOI：
10.1074/jbc.m707247200
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发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Hattori, Seisuke
中科院分区：
文献类型：
--
作者：
Harita, Yutaka;Kurihara, Hidetake;Hattori, Seisuke
有多项证据表明，足细胞裂隙隔膜 (SD) 作为肾小球滤过屏障的结构框架，也作为信号平台发挥着重要作用。已知包括去氧肾上腺素和 TRPC6 在内的几种 SD 成分可被 Src 家族酪氨酸激酶 (SFK) Fyn 磷酸化。在这里，我们将另一种 SD 成分 Neph1 表征为 SFK 的新型底物。在体外和完整细胞中，Fyn 与 Neph1 的细胞质结构域相互作用并使其磷酸化。肽质量指纹分析和定点诱变鉴定了几个酪氨酸磷酸化位点。在使用大鼠肾小球裂解物的 Pull-down 测定中，Neph1（而非去氧肾上腺素）以磷酸化依赖性方式特异性结合接头蛋白 Grb2 和酪氨酸激酶 Csk。 Neph1 的酪氨酸 637 和 638 对于 Neph1-Grb2 结合至关重要。在足细胞损伤的体内模型中，酪氨酸 637 的磷酸化显着上调。此外，Neph1 减弱 Fyn 引起的 ERK 激活，这种抑制作用需要 Grb2 SH2 结构域的完整结合基序。我们的结果表明 Neph1 是 SFK 的一种新型体内底物，并表明 Neph1 通过与 Grb2 的磷酸化依赖性相互作用来调节 ERK 信号传导。因此，SFK 通过酪氨酸磷酸化在 SD 上协调广泛的蛋白质-蛋白质相互作用和细胞内信号网络。
There are several lines of evidence that the podocyte slit diaphragm ( SD), which serves as a structural framework for the filtration barrier in kidney glomerulus, also plays an essential role as a signaling platform. Several SD components including nephrin and TRPC6 are known to be phosphorylated by a Src family tyrosine kinase ( SFK), Fyn. Here we have characterized Neph1, another SD component, as a novel substrate of SFK. Fyn interacts with and phosphorylates the cytoplasmic domain of Neph1 in vitro and in intact cells. Peptide mass fingerprinting and site-directed mutagenesis identified several tyrosine phosphorylation sites. In pull-down assays using rat glomerular lysates, Neph1 but not nephrin specifically binds to adaptor protein Grb2 and tyrosine kinase Csk in a phosphorylation-dependent manner. Both tyrosine 637 and 638 of Neph1 are crucial for Neph1-Grb2 binding. Phosphorylation of tyrosine 637 is significantly up-regulated in in vivo models of podocyte injury. Furthermore, Neph1 attenuates ERK activation elicited by Fyn, and this inhibitory effect requires the intact binding motif for the Grb2 SH2 domain. Our results shown here demonstrate that Neph1 is a novel in vivo substrate of SFK and suggest that Neph1 modulates ERK signaling through phosphorylation-dependent interaction with Grb2. Thus, SFK orchestrates a wide spectrum of protein-protein interactions and intracellular signaling networks at SD through tyrosine phosphorylation.