Multivalent nephrin–Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis

Abstract

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier

“…The non-catalytic region of the tyrosine kinase (Nck) family of adaptors including Nck1 and Nck2, plays key roles in the remodeling of the cytoskeleton ( 91 ). Phosphorylation on the tyrosine residues of nephrin promotes the nephrin-Nck (Nck1/Nck2) complex formation which binds via the SH3 domain of Nck and results in actin assembly ( 92 ). Nck also interacts with GTPase dynamin, lipid phosphatase synaptojanin1, and actin-polymerizing protein N-WASP to regulate the actin assembly and endocytosis ( 91 , 93 , 94 ).…”

“…Although the role of the actin cytoskeleton in podocytes has not been fully understood, the actin filament accumulating around the endocytic pits provides the constriction force required for pulling the invaginated vesicles inwards ( 92 , 100 ). Its importance in counteracting the membrane tension caused by the significant stretch and shear strain due to the cell adhesion to the glomerular basement membrane has been solidified.…”

“…Phosphorylation (activation) and dephosphorylation (deactivation) of nephrin have been identified as an essential posttranslational event that maintains proper podocyte structure and function. Nephrin tyrosine residue phosphorylation by Src family kinases Fyn and Yes, promotes Nck adaptor protein binding mediating actin polymerization and facilitating the endocytic process ( 92 , 113 , 114 ). Nck's SH3 domains also play a role in coupling dynamin to nephrin, as it allows dynamin to bind through its proline-rich domain.…”

“…Increased nephrin tyrosine phosphorylation upon podocyte injury correlates with a decreased membrane surface nephrin. Interestingly, mice lacking-Nck binding sites on nephrin are resistant to podocyte injury, which suggests that nephrin-Nck interaction regulates the nephrin endocytosis at the slit diaphragm ( 92 ). Nephrin has also been shown to undergo caveolin-dependent endocytosis ( 20 ) and CIE (raft-mediated endocytosis) ( 115 ).…”

Murine Epsins Play an Integral Role in Podocyte Function

“…When these cells are injured, foot process effacement and detachment from the glomerular basement membrane (GBM) ensue, resulting in proteinuria and glomerulosclerosis. 1 Recently, continuing evidence supports the importance of podocyte endocytic trafficking, not only in murine models, [2][3][4][5] but also in humans. 6 It has been shown previously that proteins within the clathrinmediated endocytic pathway, such as dynamin, synaptojanin1, and endophilin, are critical to normal kidney function, because mice lacking these genes developed massive albuminuria.…”