title: Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase.
abstract: Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.
authors.affiliation: Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal.
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Mendes
authors.forename: Paulo
authors.initials: P
authors.lastname: Matos
authors.forename: Sónia
authors.initials: S
authors.lastname: Moniz
authors.forename: Simão
authors.initials: S
authors.lastname: Luz
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Farinha
authors.forename: Peter
authors.initials: P
authors.lastname: Jordan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D017346:Protein Serine-Threonine Kinases; C433806:WNK4 protein, human