ShcA Adaptor Protein Promotes Nephrin Endocytosis and Is Upregulated in Proteinuric Nephropathies

Abstract

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.

Characterization of a novel disease-associated mutation within NPHS1 and its effects on nephrin phosphorylation and signaling

“…Lastly, we monitored activation of the AP-1 transcription factor by the nephrin mutants, as this is a well-established downstream target of nephrin signaling [ 12 , 16 ]. HEK293T cells were transfected with WT, A419T and/or C623F or vector alone in addition to plasmids encoding podocin, AP-1 luciferase and renilla luciferase (as a transfection control).…”

“…These include the Nck family of actin adaptors, which associate with tyrosine residues 1176, 1193 and 1217 on the cytoplasmic tail of nephrin [ 11 , 31 , 32 ] and further promote nephrin tyrosine phosphorylation [ 33 ]. Several endocytic adaptor proteins are also recruited to these tyrosines, and they differentially modulate nephrin turnover on the cell surface [ 16 , 34 , 35 ]. Mutations in the cytoplasmic region of nephrin have been reported with varying clinical severity.…”

“…These phenotypes are paralleled in mouse studies where total nephrin knockout results in neonatal lethality with massive proteinuria [ 37 ], while site-specific disruption of nephrin Y1176, Y1193 and Y1217 (nephrin-Y3F mice) induces juvenile onset proteinuria with foot process effacement [ 13 ]. Nephrin-Y3F mice further display a reduced capacity to repair injured podocytes [ 13 ], and changes in phosphorylation of these tyrosines are associated with mislocalization of nephrin and proteinuria [ 16 ]. In our study, both A419T and C623F are not properly trafficked to the cell surface, which likely explains their reduced phosphorylation, though both of these variants induce a dominant negative effect on WT nephrin.…”

“…Point mutations were introduced into Myc-tagged human nephrin cDNA in pcDNA3.1 [ 11 ] using site-directed mutagenesis with Pfx Platinum polymerase (Invitrogen) and confirmed by DNA sequencing. Plasmids encoding podocin, pGL3-AP-1 firefly luciferase and pRL-SV40-renilla luciferase were described previously [ 16 ]. Plasmids encoding myristoylated (plasma membrane targeted) GFP (pEGFP-N3 Myr-GFP) and pEYFP-ER were provided by Dr. George Harauz (University of Guelph).…”

Nephrin Signaling in the Podocyte: An Updated View of Signal Regulation at the Slit Diaphragm and Beyond

“…Beyond its role as a physical barrier, nephrin acts a central signaling platform within the podocyte, facilitated by an extensive network of cytoplasmic binding partners which have been identified over the past two decades (Figure 1 B) ( 18 , 29 , 45 – 76 ). These proteins represent key components of diverse signaling cascades that affect podocyte polarity, cell survival, calcium mechano-signaling, membrane trafficking, and actin organization.…”