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 8

Nck binds directly with nephrin via an SH2 domain–dependent interaction.

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Nck binds directly with nephrin via an SH2 domain–dependent interaction....
(A) Purified recombinant GST-nephrinCD was prepared from BL21 cells or TKB1 cells and was incubated in vitro with GST-Nck or a Nck mutant in the SH2 domain. The specified mixtures of recombinant proteins were immunoprecipitated with anti-Nck. Immune complexes were resolved by SDS-PAGE and were immunoblotted with either nephrin or P-nephrin antibody. Coomassie blue–stained gel demonstrates GST-Nck proteins used in this experiment. (B) COS7 cells were transiently transfected as indicated with plasmids encoding nephrin and Fyn. Independently, COS7 cells were transfected with plasmid encoding Nck1 or Nck1ΔSH2. Lysate obtained from these independently transfected cells were mixed, then immunoprecipitated and/or immunoblotted as indicated. (C) COS7 cells were transfected with plasmid encoding nephrin or its tyrosine mutants, then treated with pervanadate for 15 minutes as indicated prior to lysis. Independently, COS7 cells were transfected with plasmid encoding Nck1. Lysate obtained from these independently transfected cells were mixed, then immunoprecipitated and/or immunoblotted as indicated. (D) Coimmunoprecipitation experiments from isolated wild-type mouse glomerular lysate using pan-Nck and nephrin antibodies demonstrating association of endogenous Nck and nephrin. (E) Similar experiments performed on isolated glomerular lysate obtained from wild-type and Fyn-null mice.