Nephrin Regulates Lamellipodia Formation by Assembling a Protein Complex That Includes Ship2, Filamin and Lamellipodin

Abstract

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5′ inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells

“…This suggests that the presence of the SHIP2-Myo1c complex might be rather specific for N1 cells. Interestingly, in a different cell type, nephrin regulates lamellipodia formation by assembling a protein complex that includes SHIP2, filamin and lamellipodin (Venkatareddy et al, 2011). SHIP2 is part of the adhesome, and it has been proposed that, on average, each adhesome protein has approximately nine different potential partners (Zaidel-Bar and Geiger, 2010).…”

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

“…50 The previously described tyrosin phosphorylation site on nephrin was not detected, which may either be the result of the not optimal access of trypsin to this site or due to a low baseline phosphorylation because several studies have shown that nephrin tyrosine phosphorylation is inducible upon ligation of the extracellular domain. [51][52][53] We focused on studying phosphorylation of the PHB domain protein podocin. Mutations in the podocin gene (NPHS2) are a major cause of steroid-resistant nephrotic syndrome in children.…”

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

“…Nephrin's ability to regulate actin dynamics in a phosphorylation-dependent manner has been demonstrated by us and other investigators (2)(3)(4)(5)(6). A critical role for nephrin is suggested by the lack of normal foot process development in mice lacking nephrin or humans born with nephrin mutations (7,8).…”

“…In vitro studies have shown that engagement of the nephrin extracellular domain results in Src family kinase Fyn-dependent tyrosine phosphorylation of the nephrin cytoplasmic domain (6,9,10). Phosphorylated nephrin then recruits the Src homology 2 (sh2) domain-containing proteins Nck1/Nck2, the p85 subunit of phosphatidylinositol 3-kinase (PI3K), and Crk (3)(4)(5)(6)11) and other components of the actin polymerization complex (2,3,5,12,13). Mice with the Src family kinase Fyn deleted develop proteinuria and foot process defects that are evident at 7 weeks of age (10,14).…”