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. IntroductionGlomerular visceral epithelial cells play a central role in maintaining the selective filtration barrier of the renal glomerulus. These cells are also termed podocytes to describe the foot-like appearance of numerous interdigitating foot processes that arise from their cell bodies and surround glomerular capillary walls. Glomerular filtrate passes across the specialized intercellular junction -also termed the slit diaphragm -formed at the interface of these interdigitating foot processes.In response to injury, podocytes undergo a dramatic change in morphology termed foot process effacement resulting from alteration in cytoskeletal and intercellular junctional architecture. By electron microscopy, effacement is manifested by retraction and spreading of podocyte processes. Effacement is a fluid and reversible process that directly correlates with the development of proteinuria both in human disease and in experimental models.The cellular and molecular mechanisms that integrate podocyte morphology and filter integrity are incompletely defined. Recent investigations have focused on identifying and characterizing the interrelationships and functions of the molecular components of the foot process intercellular junction because several of these components are necessary for development of normal podocyte structure and filter integrity (reviewed in refs. 1, 2).

Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain

“…This finding may be relevant to address the effect of rostafuroxin in the nephrin-signaling network, since evidence has indicated that SH2-domain interactions are involved in the complex signaling cascade originating from the slit diaphragm (Verma et al, 2006). The tyrosine kinases on which we evaluated the effect of rostafuroxin were selected on the basis of a score matrix-assisted ligand identification analysis performed on nephrin and b-adducin protein sequences.…”

“…Recently, we produced data indicating that mutant b-adducin and EO/ouabain affect the expression of nephrin (Ferrandi et al, 2010b;Bignami et al, 2013), a selective podocyte marker protein (Verma et al, 2006), both in animal models and in patients.…”

Mécanismes moléculaires du syndrome néphrotique idiopathique à rechutes

“…La liaison de la néphrine à son ligand entraîne un regroupement des complexes dans des microdomaines lipidiques (lipid rafts). Cet événement va activer la protéine Fyn, un membre de la famille Src kinase, qui phosphoryle plusieurs résidus tyrosine situés à l'extré-mité carboxy-terminale de la néphrine ; à ce niveau, des protéines kinases et des protéines adaptatrices sont recrutées qui se lient ensuite à la néphrine via leurs domaines SH2 [7][8][9][10]. La phosphorylation de la néphrine par Fyn a deux conséquences essentielles ( Figure 3) : (1) elle active la PI3K via l'interaction du domaine SH2 de la sous-unité régulatrice p85 de cette dernière avec le domaine cytoplasmique de la néphrine tandis que la sous-unité catalytique p110 génère des phosphoinositides capables de recruter et d'activer la sérine thréonine kinase Akt, qui joue un rôle clé dans les processus de survie du podocyte.…”

“…Cette fente est fermée par une mince membrane, le diaphragme de fente, qui joue un rôle essentiel dans la perméabilité aux protéines de la barrière glomérulaire » (repris de l'encadré de Raymond Ardaillou, dans [20] phorylation de la néphrine provoque le détachement de la podocine et la liaison de la -arrestine-2 à la néphrine qui subit alors une endocytose suivie d'une dégradation, interrompant ainsi la voie de signalisation [11]. Outre son rôle sur l'activation de la néphrine, Fyn interagit avec N-WASP (Wiskott Aldrich syndrome protein) qu'elle phosphoryle, entraînant le recrutement et l'ancrage de NCK aux microdomaines lipidiques [7,8]. La famille des protéines adaptatrices Nck comprend Nck1 (Nck) et Nck2 (Nck ou GRB4).…”

“…Chacune possède trois domaines SH3 et un domaine SH2 carboxy-terminal [12]. Nck se lie, via ses domaines SH3, à différentes protéines impliquées dans la régulation de l'actine tels que WASP et le complexe ARP2/3 [8] ; via ses domaines SH2, Nck se lie aux phosphotyrosines de la néphrine, ce qui permet une connexion étroite entre la plate-forme de signalisation et l'organisation du cytosquelette d'actine.…”