title: Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-{gamma}1.
abstract: A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
authors.affiliation: Division of Cellular Proteomics (BML) and Department of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Harita
authors.forename: Hidetake
authors.initials: H
authors.lastname: Kurihara
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kosako
authors.forename: Tohru
authors.initials: T
authors.lastname: Tezuka
authors.forename: Takashi
authors.initials: T
authors.lastname: Sekine
authors.forename: Takashi
authors.initials: T
authors.lastname: Igarashi
authors.forename: Ikuroh
authors.initials: I
authors.lastname: Ohsawa
authors.forename: Shigeo
authors.initials: S
authors.lastname: Ohta
authors.forename: Seisuke
authors.initials: S
authors.lastname: Hattori
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007152:Immunologic Capping / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000235:genetics / Q000378:metabolism; D007365:Intercellular Junctions / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D050199:Podocytes / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D016827:CD8 Antigens; D008565:Membrane Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; C113267:nephrin; D015544:Inositol 1,4,5-Trisphosphate; C494957:FYN protein, human; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; C522157:PLCG1 protein, human; D051966:Phospholipase C gamma; C117399:Plcg1 protein, mouse