Neph1 Signaling

描述（由申请人提供）： 与肾小球功能障碍相关的肾脏疾病导致进行性肾损伤和肾功能丧失，是发病率和死亡率的重要原因。家族性肾病综合征的研究和肾小球疾病动物模型的分析导致足细胞生物学领域的重大进展。已经鉴定了许多参与狭缝隔膜连接的结构完整性和组成的蛋白质。它们中的几种（Nephrin、Podocin、CD 2AP和Neph 1）在维持肾小球滤过屏障中的重要性已在小鼠模型和人类疾病中得到证实，其中这些基因的缺陷显示蛋白尿和足细胞消失。Neph 1是一种新发现的Nephrin相关蛋白复合物的组成部分，存在于足突之间的细胞间连接处，通过质膜平面中的顺式相互作用与Nephrin直接相互作用。Holzman实验室证明，Nephrin通过Src家族激酶（SFK）在酪氨酸残基上磷酸化，表明Nephrin相关复合物参与信号转导。由于Fyn被发现是Nephrin相关蛋白复合物的一个组成部分，我们研究了Neph 1是否也被酪氨酸磷酸化。在初步研究中，发现Neph 1在体内条件下以SFK依赖的方式被酪氨酸磷酸化。结合研究中，磷酸化的Neph 1被用作诱饵，从肾小球裂解物中拉下Neph 1相互作用的蛋白质，显示出Neph 1与SFK的相互作用，包括Fyn和Yes和衔接蛋白Grb 2（生长因子受体结合蛋白）。基于这些结果，我们假设Neph 1被SFK磷酸化，并且这种磷酸化是参与正常足细胞结构和滤器完整性的发展的重要信号事件。具体目标如下：1）研究Neph 1被Fyn和/或Yes酪氨酸磷酸化的假说，并定位Neph 1的磷酸化位点。2)探索Neph 1通过Grb 2向下游靶点发出信号的假设。3)探索Neph 1磷酸化的生理条件。4)在小鼠中研究由Fyn和/或其他SFK引起的Neph 1酪氨酸磷酸化的生理相关性。5)分离并鉴定足细胞或狭缝隔膜的细胞间连接的组分Neph 1相关蛋白。

DESCRIPTION (provided by applicant): Kidney diseases associated with glomerular dysfunctioning that result in the progressive kidney damage and loss of kidney function are important causes of morbidity and mortality. The study of familial nephrotic syndromes and the analysis of animal models of glomerular disease have resulted in major advancements in the field of podocyte biology. A number of proteins participating in the structural integrity and composition of the slit diaphragm junction have been identified. The importance of several of them (Nephrin, Podocin, CD2AP, and Neph1) in the maintenance of the glomerular filtration barrier has been demonstrated in mouse models and human diseases where defects in these genes showed proteinuria and podocyte effacement. Neph1 is a newly recognized component of the Nephrin-associated protein complex existent at the intercellular junction between foot processes that directly interacts with Nephrin via a cis-interaction in the plan of the plasma membrane. The Holzman lab demonstrated that Nephrin is phosphorylated on tyrosine residues by Src family kinases (SFK's) suggesting that the Nephrin associated complex is involved in signal transduction. Since Fyn was found to be a component of the Nephrin associated protein complex we investigated if Neph1 is also tyrosine phosphorylated. In preliminary studies, Neph1 was found to be tyrosine phosphorylated under in vivo conditions in a SFK-dependent fashion. Binding studies where phosphorylated Neph1 was used as a bait to pull down Neph1 interacting proteins from glomerular lysate showed interaction of Neph1 with SFK's including Fyn and Yes and adaptor protein Grb2 (growth factor receptor-bound protein). Based on these results, we hypothesize that Neph1 is phosphorylated by SFK's and that this phosphorylation is an important signaling event that is involved in the development of normal podocyte structure and filter integrity. The following specific aims are proposed: 1) Study the hypothesis that Neph1 is tyrosine phosphorylated by Fyn and/or Yes and map the phosphorylation sites in Neph1. 2) Explore the hypothesis that Neph1 signals to downstream targets via Grb2. 3) Explore the physiological conditions during which Neph1 is phosphorylated. 4) Investigate in mice the physiological relevance of Neph1 tyrosine phosphorylation by Fyn and/or other SFK's. 5) Isolate and identify Neph1 associated proteins that are components of the intercellular junction of podocytes or slit diaphragm.