Neph1 Signaling

描述(由申请人提供)： 肾脏疾病与肾小球功能不全相关，导致进行性肾损害和肾功能丧失，是导致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在足细胞生物学领域取得了重大进展。一些参与缝隙横隔膜连接的结构完整性和组成的蛋白质已被鉴定。在小鼠模型和人类疾病模型中，已经证明了其中几种基因(Nephin、Podocin、CD2AP和Neph1)在维持肾小球滤过屏障方面的重要性，这些基因的缺陷表现为蛋白尿和足细胞消失。Neph1是一种新发现的Nephin相关蛋白复合体，存在于足突之间的细胞间连接处，通过质膜平面上的顺式相互作用与Nephin直接相互作用。Holzman实验室证明，NePhin在酪氨酸残基上被Src家族激酶(SFK)磷酸化，这表明NePhin相关复合体参与了信号转导。由于Fyn被发现是Nephin相关蛋白复合体的一个组成部分，我们研究了Neph1是否也被酪氨酸磷酸化。在初步研究中，发现Neph1在活体条件下以SFK依赖的方式被酪氨酸磷酸化。用磷酸化的Neph1作为诱饵从肾小球裂解液中拉下Neph1相互作用蛋白的结合研究表明，Neph1与SFK的相互作用，包括Fyn和Yes，以及接头蛋白Grb2(生长因子受体结合蛋白)。根据这些结果，我们假设Neph1被SFK磷酸化，这种磷酸化是一个重要的信号事件，参与了正常足细胞结构和过滤器完整性的发展。具体目标如下：1)研究Neph1是被Fyn和/或Yes磷酸化的假设，并绘制Neph1的磷酸化位点图。2)探索Neph1通过Grb2向下游靶标发出信号的假设。3)探讨Neph1被磷酸化的生理条件。4)研究Fyn和/或其他SFK对Neph1酪氨酸磷酸化的生理相关性。5)分离和鉴定足细胞或裂隙隔膜细胞间连接的Neph1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.