title: Phosphorylation of podocalyxin (Ser415) Prevents RhoA and ezrin activation and disrupts its interaction with the actin cytoskeleton.
abstract: Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.
authors.affiliation: Department of Medicine, Hamamatsu University School of Medicine, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Fukasawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Obayashi
authors.forename: Sandra
authors.initials: S
authors.lastname: Schmieder
authors.forename: Jaesung
authors.initials: J
authors.lastname: Lee
authors.forename: Pradipta
authors.initials: P
authors.lastname: Ghosh
authors.forename: Marilyn G
authors.initials: MG
authors.lastname: Farquhar
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors*; D007678:Kidney Glomerulus / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009401:Nephrosis / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors*; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D012795:Sialoglycoproteins; D020742:rhoA GTP-Binding Protein; D000099149:Ezrin; C001643:podocalyxin