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.
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