Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-γ1
Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-γ1
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DOI：
10.1074/jbc.m806851200
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发表时间：
2009-03-27
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
足细胞之间的特化细胞间连接，称为狭缝隔膜（SD），形成肾脏中肾小球过滤的基本结构框架。此外，越来越多的证据表明，SD作为生理和病理状态的信号平台也起着至关重要的作用。Nephrin是SD的主要成分，在发育或损伤的足细胞中被Src家族酪氨酸激酶Fyn酪氨酸磷酸化，通过其Src同源2结构域将Nck募集到Nephrin以调节动态肌动蛋白重塑。失调的Ca 2+稳态也与足细胞损伤有关，但足细胞对损伤的反应机制在很大程度上尚不清楚。在这里，我们已经确定了磷脂酶C-γ 1（PLC-γ 1）作为一种新的磷酸化肾蛋白结合蛋白。当用抗CD 8和抗小鼠抗体处理表达由CD 8和Nephrin胞质结构域（CD）组成的嵌合蛋白的HEK 293 T细胞时，诱导了Nephrin的聚集和Nephrin-CD的磷酸化。在该聚集后，PLC-γ 1与磷酸化的Nephrin Tyr-1204结合，其诱导PLC-γ 1从细胞质易位至质膜上的CD 8/Nephrin簇。通过PLC-γ 1 Tyr-783的磷酸化和1，4，5-三磷酸肌醇水平的增加检测到PLC-γ 1向Nephrin的募集激活了PLC-γ 1。我们还发现，Nephrin Tyr-1204磷酸化以PLC-γ 1依赖性方式触发Ca 2+反应。此外，PLC-γ 1在体内受损足细胞中显著磷酸化。考虑到PLC-γ在不同细胞功能中的深远影响，通过Nephrin调节Ca 2+信号传导在调节肾小球滤过屏障功能中可能是重要的。
A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural framework for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca2+ homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma 1 (PLC-gamma 1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma 1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma 1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma 1 to Nephrin activated PLC-gamma 1, as detected by phosphorylation of PLC-gamma 1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca2+ response in a PLC-gamma 1-dependent fashion. Furthermore, PLC-gamma 1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca2+ signaling by Nephrin may be important in modulating the glomerular filtration barrier function.