title: Phospholipase C-gamma1 is involved in signaling the activation by high NaCl of the osmoprotective transcription factor TonEBP/OREBP.
abstract: High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-gamma1 (PLC-gamma1), which has a predicted binding site at TonEBP/OREBP-phospho-Y143. We find the following. (i) Activation of TonEBP/OREBP transcriptional activity by high NaCl is reduced in PLC-gamma1 null cells and in HEK293 cells in which PLC-gamma1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of TonEBP/OREBP at Y143. (iii) Wild-type PLC-gamma1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-gamma1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-gamma1 or overexpression of a PLC-gamma1-SH3 deletion mutant reduces high NaCl-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-gamma1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-gamma1, if PLC-gamma1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-gamma1 restores nuclear localization of wild-type TonEBP/OREBP in PLC-gamma1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-gamma1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-gamma1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization.
authors.affiliation: Department of Health and Human Services, Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1603, USA.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Irarrazabal
authors.forename: Morgan
authors.initials: M
authors.lastname: Gallazzini
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schnetz
authors.forename: Margarita
authors.initials: M
authors.lastname: Kunin
authors.forename: Brigitte L
authors.initials: BL
authors.lastname: Simons
authors.forename: Chester K
authors.initials: CK
authors.lastname: Williams
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology*; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C118125:NFAT5 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D012965:Sodium Chloride; D051966:Phospholipase C gamma