Src-Family Kinase Fyn Phosphorylates the Cytoplasmic Domain of Nephrin and Modulates Its Interaction with Podocin

Abstract

Abstract. Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Srcfamily kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was cotransfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.

Analysis of the Cell Adhesion Molecule Sticks-and-Stones Reveals Multiple Redundant Functional Domains, Protein-Interaction Motifs and Phosphorylated Tyrosines That Direct Myoblast Fusion in Drosophila melanogaster

“…Phosphotyrosines can also be critical intermediates in signal transduction pathways and mediate interactions with proteins containing SH2, WW, or phosphotyrosine-binding (PTB) domains. Tyrosine phosphorylation is a key modification in the SNS ortholog, nephrin (Li et al 2004;Jones et al 2006), and regulates signaling to two downstream pathways: one that activates Akt by phosphoinositide 3-kinase (PI3K) and another that directs actin rearrangements via Nck binding. Both the Akt signaling and Nck pathways are critical for nephrin function (Huber et al 2003a;Jones et al 2006).…”

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture

“…Nephrin was phosphorylated on Y1217 in nephrin WT/WT animals, whereas we could not detect any signal above background in nephrin Y3F/Y3F animals ( Figure 2C). Given that nephrin phosphorylation can affect nephrin-podocin interactions, 17,23 we performed dual-immunofluorescence staining for nephrin and podocin (Figure 2C) and found that both proteins were similarly expressed in a continuous pattern in the glomeruli of nephrin WT/WT and nephrin Y3F/Y3F animals. In accordance with this staining pattern, transmission electron microscopy (EM) revealed the presence of slit diaphragm structures in nephrin Y3F/Y3F animals ( Figure 2D).…”

“…Nephrin is tyrosine phosphorylated during glomerulogenesis and throughout life, 8,16,17 and decreases in nephrin phosphorylation on Y1217 are seen coincident with foot process effacement in human kidney diseases, including minimal change disease (MCD) 18 and membranous nephropathy. 19 Similarly, reduced phosphorylation of the downstream survival factor Akt on serine (S) 473 can be observed in MCD, 20,21 and inactivation of the actin binding protein cofilin is detected in patients with MCD, membranous nephropathy, and FSGS.…”

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.…”

“…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. (2) Elle stabilise le complexe fonctionnel néphrine-podocine [10]. En effet, la déphos-peuvent être à l'origine d'une protéinu-rie massive quel que soit le mécanisme lésionnel initial.…”