Nephrin and CD2AP Associate with Phosphoinositide 3-OH Kinase and Stimulate AKT-Dependent Signaling

Abstract

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/ AKT-dependent signal transduction in glomerular podocytes.The renal glomerulus is the site of a number of disorders that lead to proteinuria and chronic renal failure (19,25). Recent studies of the hereditary nephrotic syndrome have emphasized the role of the glomerular podocytes in generating a size-selective filtration barrier and have provided a new understanding of the mechanisms leading to proteinuria, in both inherited and acquired diseases. The hereditary nephrotic syndrome is a heterogeneous disease characterized by heavy proteinuria and renal failure. Mutations of NPHS1 (20) or NPHS2 (4, 13), the genes encoding the glomerular podocyte proteins nephrin and podocin, as well as the targeted disruption of the CD2-associated protein (CD2AP) in mice (43), lead to a severe nephrotic syndrome, suggesting that these proteins are indispensable for an intact glomerular filter. All three proteins localize at opposing sites of the secondary foot processes formed by podocytes, specialized epithelial cells that ensure size-and charge-selective ultrafiltration (14,15,39,41,42). This specialized cell-cell contact between opposing secondary foot processes is called the slit diaphragm (38). Nephrin is a member of the immunoglobulin superfamily and appears to form a zipper-like filter structure within the ϳ40-nm-wide slit between two foot processes (reviewed in reference 48). It was recently shown that nephrin is a signaling protein that interacts with podocin, a podocyte protein of the stomatin family, facilitating nephrin signaling (18) and regulating its recruitment to lipid raft microdomains (41,45). CD2AP is an adaptor molecule that was first ide...

MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin

“…Moreover, ZO-1, podocin, and CD2AP are proposed to be involved in the signalings of cell adhesion molecules. 21,45,46 The significance of the interactions among slit diaphragm proteins and slit diaphragm-associated proteins is underscored by the fact that some molecular interactions are lost and the components redistribute in podocyte foot processes under the proteinuric state. In this study, we have demonstrated a novel interaction between nephrin and MAGI-1.…”

“…45,56 Nephrin also associates with p85 regulatory subunit of PI3K and stimulates PI3K-dependent AKT signaling in podocytes. 46 In polarized epithelial cells, MAGI-1 interacts with several signaling molecules including RapGEP, b-catenin and mNET1. [57][58][59] MAGI-1 may modulate nephrinmediated signaling in podocytes, although the roles of these signaling molecules at the podocytes are not known.…”

Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin

“…The pathways by which nephrin influences cell behavior have just begun to be investigated. Nephrin, together with podocin, stimulates the AP-1 transcription factor via activation of mitogen-activated protein kinases, 57 associates with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K), stimulates PI3K-dependent AKT signaling in podocytes, 58 and is phosphorylated by Fyn, a Src family kinase. 36 The events that trigger these effects and the functional consequences remain to be worked out.…”

Podocyte Mitosis – A Catastrophe

“…The cytoplasmic tail of nephrin, like other transmembrane Ig-SF members, is anchored to the actin cytoskeleton of podocyte by means of CD2-associated protein, CD2AP , a cytoplasmic adaptor protein which interacts with actin [16] and the actin-binding proteins cortactin [17], capping protein Z (CapZ) [18], and the actin polymerization complex Arp2/3 [17]. CD2AP and nephrin interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K), recruit PI3K to the plasma membrane, and stimulate PI3K-dependent AKT signaling in podocytes [19], thus participating in a common signaling pathway necessary to maintain crucial podocyte functions. The external, highly glycosylated, Ig-like domain of nephrin forms dimers across the slit diaphragm, producing pores with a predicted diameter slightly smaller than the radius of albumin [20] and regulates glomerular permeability [15].…”