title: Missorting of the Aquaporin-2 mutant E258K to multivesicular bodies/lysosomes in dominant NDI is associated with its monoubiquitination and increased phosphorylation by PKC but is due to the loss of E258.
abstract: To stimulate renal water reabsorption, vasopressin induces phosphorylation of Aquaporin-2 (AQP2) water channels at S256 and their redistribution from vesicles to the apical membrane, whereas vasopressin removal results in AQP2 ubiquitination at K270 and its internalization to multivesicular bodies (MVB). AQP2-E258K causes dominant nephrogenic diabetes insipidus (NDI), but its subcellular location is unclear, and the molecular reason for its involvement in dominant NDI is unknown. To unravel these, AQP2-E258K was studied in transfected polarized Madin-Darby canine kidney (MDCK) cells. In MDCK cells, AQP2-E258K mainly localized to MVB/lysosomes (Lys). Upon coexpression, wild-type (wt) AQP2 and AQP2-E258K formed multimers, which also localized to MVB/Lys, independent of forskolin stimulation. Orthophosphate labeling revealed that forskolin increased phosphorylation of wt-AQP2 and AQP2-E258K but not AQP2-S256A, indicating that the E258K mutation does not interfere with the AQP2 phosphorylation at S256. In contrast to wt-AQP2 but consistent with the introduced protein kinase C (PKC) consensus site, AQP2-E258K was phosphorylated by phorbol esters. Besides the 29-kDa band, however, an additional band of about 35 kDa was observed for AQP2-E258K only, which represented AQP2-E258K uniquely monoubiquitinated at K228 only. Analysis of several mutants interfering with AQP2-E258K phosphorylation, and/or ubiquitination, however, revealed that the MVB/lysosomal sorting of AQP2-E258K occurred independent of its monoubiquitination or phosphorylation by PKC. Instead, our data reveal that the loss of the E258 in AQP2-E258K is fundamental to its missorting to MVB/Lys and indicate that this amino acid has an important role in the proper structure formation of the C-terminal tail of AQP2.
authors.affiliation: Department of Physiology, Radboud University Nijmegen Medical Center, Research Tower, 7th floor; UMC St Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Erik-Jan
authors.initials: EJ
authors.lastname: Kamsteeg
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Savelkoul
authors.forename: Giel
authors.initials: G
authors.lastname: Hendriks
authors.forename: Irene B M
authors.initials: IB
authors.lastname: Konings
authors.forename: Nicole M I
authors.initials: NM
authors.lastname: Nivillac
authors.forename: Anne Karine
authors.initials: AK
authors.lastname: Lagendijk
authors.forename: Peter
authors.initials: P
authors.lastname: van der Sluijs
authors.forename: Peter M T
authors.initials: PM
authors.lastname: Deen
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism*; D003720:Densitometry; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006207:Half-Life; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D051399:Aquaporin 2; D014452:Ubiquitins; D011493:Protein Kinase C