Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization
Rakesh Verma, … , Kevin Patrie, Lawrence B. Holzman
Rakesh Verma, … , Kevin Patrie, Lawrence B. Holzman
Published May 1, 2006
Citation Information: J Clin Invest. 2006;116(5):1346-1359. https://doi.org/10.1172/JCI27414.
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Research Article Nephrology

Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization

Abstract

A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2–SH3 domain–containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics.

Authors

Rakesh Verma, Iulia Kovari, Abdul Soofi, Deepak Nihalani, Kevin Patrie, Lawrence B. Holzman

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Figure 1

Identification of Fyn-dependent tyrosine phosphorylation sites on nephrin.

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Identification of Fyn-dependent tyrosine phosphorylation sites on nephri...
For in vivo metabolic labeling of nephrin (A), DRM obtained from isolated mouse glomeruli was pelleted, resuspended in kinase buffer, and incubated in the presence of [32P-γ]ATP. These DRM were re-extracted in RIPA buffer, and nephrin was obtained thereafter by immunoprecipitation. For in vitro phosphorylation of nephrin by recombinant Fyn (B–I), purified recombinant GST-nephrinCD or various GST-nephrinCD point mutants were phosphorylated in vitro by recombinant Fyn. Phosphorylated bands representing nephrin protein or its mutants were separated by SDS-PAGE, identified by autoradiography, and processed for 2D tryptic peptide mapping. Representative tryptic peptide maps for radiophosphate-labeled wild type nephrin obtained by both in vivo (A) and in vitro (B) phosphorylation methods are shown juxtaposed for comparison, and corresponding peptides are labeled as defined in J (along with a summary of peptide characteristics). Mutagenesis of individual tyrosine residues was performed to map Fyn-dependent phosphorylation sites. Where single peptides contained multiple tyrosine residues, single, double, and triple combination tyrosine mutants were created. Shown are representative experiments. Note that for peptide 2, mutation of Y1154 had no effect, Y1153F mutation resulted in a shift in mobility (arrow denotes the shift from 2 to 2*), and mutation of both Y1153 and Y1154 to phenylalanine resulted in disappearance of peptide 2. O, origin; rNephrin, recombinant nephrin.