RRC ID 72947
Author Fujii M, Ohtsubo M, Ogawa T, Kamata H, Hirata H, Yagisawa H.
Title Real-time visualization of PH domain-dependent translocation of phospholipase C-delta1 in renal epithelial cells (MDCK): response to hypo-osmotic stress.
Journal Biochem Biophys Res Commun
Abstract Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.
Volume 254(2)
Pages 284-91
Published 1999-1-19
DOI 10.1006/bbrc.1998.9936
PII S0006-291X(98)99936-3
PMID 9918830
MeSH Animals Calcium / metabolism Catalytic Domain Cell Line Cell Membrane / enzymology* Cytosol / enzymology Dogs Enzyme Inhibitors / pharmacology Epithelial Cells Estrenes / pharmacology Green Fluorescent Proteins Hypotonic Solutions Inositol 1,4,5-Trisphosphate / metabolism Isoenzymes / chemistry* Isoenzymes / metabolism* Kidney Kinetics Luminescent Proteins / metabolism Phospholipase C delta Pyrrolidinones / pharmacology Recombinant Fusion Proteins / metabolism Substrate Specificity Time Factors Transfection Type C Phospholipases / chemistry* Type C Phospholipases / metabolism* src Homology Domains
IF 2.985
Human and Animal Cells MDCK(RCB0995)