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

Abstract

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.The urinary side of the capillary loop in kidney glomerulus is covered by highly branched glomerular visceral epithelial cells, called podocytes (1). Podocytes form primary processes that further extend numerous foot processes. Foot processes from neighboring podocytes interdigitate with each other and surround the entire surface of capillary loops. These foot processes are bridged by a unique cell adhesion structure, the slit diaphragm (SD).

Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray Scattering (SWAXS)

“…Importantly, a comparative DLS analysis of these purified proteins stored in the same buffer at 4°C showed that although aggregation occurred in the tagless and GST-tagged proteins (at concentrations of ϳ1 mg/ml), His-Neph1-CD protein was remarkably more stable for longer durations. An earlier report also indicated that Neph1-CD protein was unstable in the solution and prone to aggregation (15). Overall, our results indicate that the His tag provides increased stability to Neph1-CD; however, the mechanism for this stability is uncertain.…”

“…Recent studies indicate that the interactions mediated by the PDZbinding domain of Neph1 are critical for Neph1 function (12). Currently, ZO-1 and Par proteins have been shown to interact with this domain, and our recent study demonstrates that Neph1 interacts with ZO-1 in a dynamic fashion that is regulated by Neph1 tyrosine phosphorylation (15,16). Further, Fyn-mediated tyrosine phosphorylation of Neph1 has been shown as a key signaling event that induces actin polymerization through recruitment of the adapter protein Grb2 (10).…”

Slit Diaphragm Protein Neph1 and Its Signaling

“…In the rat model, PAN treatment has been shown to induce podocyte effacement, a characteristic of actin reorganization, and loss of podocyte function associated with proteinuria (12,18). More importantly, these changes in cultured podocytes and rat PAN model were associated with rapid tyrosine phosphorylation of Neph1 (10). Apart from PAN, the tyrosine phosphorylation of Neph1 was also noted in the ischemia reperfusion model of podocyte injury (12).…”

“…This phosphorylation event that is primarily mediated by the phosphorylation of residues 637 and 638 in Neph1 was shown to recruit several proteins, including Grb2, Csk tyrosine kinase, and ZO-1 (10,12). Although the recruitment of Grb2 in cultured podocytes induced actin cytoskeleton reorganization at the cytoplasmic tail of Neph1 (6), the phosphorylation-dependent increased association between Neph1 and ZO-1 played a key role in defining the tight junction formation in podocytes (12).…”

“…Studies in recent years have highlighted the significance of tyrosine phosphorylation in these proteins that plays a central role in regulating the function of these proteins particularly in the actin remodeling of podocytes that may occur during development and in response to glomerular injury (5)(6)(7)(8)(9)(10)(11). The cytoplasmic domains of Neph1 and nephrin are tyrosine-phosphorylated by Src family tyrosine kinases, namely Fyn (11), leading to the recruitment of adapter proteins Grb2 and Nck that are required for actin cytoskeleton rearrangement (5,7,10,11).…”