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Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-γ1*

By Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Tohru Tezuka, Takashi Sekine, Takashi Igarashi, Ikuroh Ohsawa, Shigeo Ohta and Seisuke Hattori

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 Ca2+ 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-γ1 (PLC-γ1) 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-γ1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-γ1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-γ1 to Nephrin activated PLC-γ1, as detected by phosphorylation of PLC-γ1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca2+ response in a PLC-γ1-dependent fashion. Furthermore, PLC-γ1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-γ in diverse cellular functions, regulation of the Ca2+ signaling by Nephrin may be important in modulating the glomerular filtration barrier function

Topics: Mechanisms of Signal Transduction
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2659252
Provided by: PubMed Central
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