title: Clustered PI(4,5)P₂ accumulation and ezrin phosphorylation in response to CLIC5A.
abstract: CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] reporter RFP-PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA-PIP5Kα colocalized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Kα abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5Kα, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2-generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, AL T6G 2V2, Canada.
authors.forename: Abass
authors.initials: A
authors.lastname: Al-Momany
authors.affiliation: Department of Medicine (Nephrology), University of Alberta, Edmonton, AL T6G 2V2, Canada.
authors.forename: Laiji
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AL T6G 2V2, Canada.
authors.forename: R Todd
authors.initials: RT
authors.lastname: Alexander
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, AL T6G 2V2, Canada Department of Medicine (Nephrology), University of Alberta, Edmonton, AL T6G 2V2, Canada barbara.ballermann@ualberta.ca.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Ballermann
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D020868:Gene Silencing / Q000187:drug effects; D006016:Glycolates / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007678:Kidney Glomerulus / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D058105:Polymerization / Q000187:drug effects; D012795:Sialoglycoproteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D000099149:Ezrin; D000096926:Benzenesulfonamides
chemical_list: D000199:Actins; D018118:Chloride Channels; D003598:Cytoskeletal Proteins; D006016:Glycolates; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D012795:Sialoglycoproteins; D013449:Sulfonamides; D010738:Type C Phospholipases; D000099149:Ezrin; C473783:2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide; D000096926:Benzenesulfonamides; C411466:CLIC5 protein, human; C514190:CLIC5 protein, mouse; C001643:podocalyxin; C071058:moesin; C071776:radixin; C015765:MK 473; C020077:1-phosphatidylinositol-4-phosphate 5-kinase
keywords: CLIC; Chloride intracellular channel; ERM protein; Glomerular; Phosphatidylinositol 4,5-bisphosphate; Podocyte; ezrin