title: Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²⁺-activated non-selective cation channel TRPM4.
abstract: Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.
authors.affiliation: Programa de Biología Celular y Molecular, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.|Centro de Estudios Moleculares de la Célula (CEMC), Facultad de Medicina, Universidad de Chile, Santiago, Chile.|Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis CA 95616, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Cerda
authors.affiliation: Programa de Biología Celular y Molecular, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.|Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis CA 95616, USA.
authors.forename: Mónica
authors.initials: M
authors.lastname: Cáceres
authors.affiliation: Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis CA 95616, USA.|Department of Physiology, Kyung Hee University School of Medicine, Seoul, South Korea 130-701.
authors.forename: Kang-Sik
authors.initials: KS
authors.lastname: Park
authors.affiliation: Centro de Estudios Moleculares de la Célula (CEMC), Facultad de Medicina, Universidad de Chile, Santiago, Chile.|Programa de Fisiopatología, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Elías
authors.initials: E
authors.lastname: Leiva-Salcedo
authors.affiliation: Programa de Biología Celular y Molecular, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Aníbal
authors.initials: A
authors.lastname: Romero
authors.affiliation: Centro de Estudios Moleculares de la Célula (CEMC), Facultad de Medicina, Universidad de Chile, Santiago, Chile.|Programa de Fisiopatología, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Diego
authors.initials: D
authors.lastname: Varela
authors.affiliation: Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis CA 95616, USA.|Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis CA 95616, USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Trimmer
authors.affiliation: Centro de Estudios Moleculares de la Célula (CEMC), Facultad de Medicina, Universidad de Chile, Santiago, Chile.|Programa de Fisiopatología, Facultad de Medicina, ICBM, Universidad de Chile, Santiago, Chile.
authors.forename: Andrés
authors.initials: A
authors.lastname: Stutzin
mesh_terms: D000595:Amino Acid Sequence; D047389:Casein Kinase I / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D012694:Serine; D050053:TRPM Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; D012694:Serine; D047389:Casein Kinase I