title: Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases.
abstract: BACKGROUND
  Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of nephrin with antibodies on cell surface mimics the situation where the interaction between nephrin and its extracellular ligand(s) is altered.
  
  
  METHODS
  Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antinephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in nephrin phosphorylation.
  
  
  RESULTS
  Clustering of nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce nephrin phosphorylation when cotransfected to HEK293 cells with nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with nephrin. Interestingly, clustering of nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with nephrin.
  
  
  CONCLUSIONS
  Nephrin is a signaling protein phosphorylated by Src family kinases.
authors.affiliation: Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
authors.forename: Juhani
authors.initials: J
authors.lastname: Lahdenperä
authors.forename: Pekka
authors.initials: P
authors.lastname: Kilpeläinen
authors.forename: Xiao Li
authors.initials: XL
authors.lastname: Liu
authors.forename: Timo
authors.initials: T
authors.lastname: Pikkarainen
authors.forename: Paula
authors.initials: P
authors.lastname: Reponen
authors.forename: Vesa
authors.initials: V
authors.lastname: Ruotsalainen
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
mesh_terms: D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011940:Receptor Aggregation / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011506:Proteins; C113267:nephrin; D014443:Tyrosine; D019061:src-Family Kinases