title: High NaCl-induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization.
abstract: When activated by high NaCl, tonicity-responsive enhancer-binding protein/osmotic response element-binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.
authors.forename: Morgan
authors.initials: M
authors.lastname: Gallazzini
authors.forename: Gary E
authors.initials: GE
authors.lastname: Heussler
authors.forename: Margarita
authors.initials: M
authors.lastname: Kunin
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Izumi
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007679:Kidney Medulla / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: C118125:NFAT5 protein, human; C492199:Nfat5 protein, rat; D014157:Transcription Factors; D010769:Phosphothreonine; D012965:Sodium Chloride; C426236:ARG tyrosine kinase; D011505:Protein-Tyrosine Kinases; D051076:Proto-Oncogene Proteins c-fyn; D051360:Cyclin-Dependent Kinase 5; D000409:Alanine