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.
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.
Kamsteeg
Erik-Jan
EJ
Department of Physiology, Radboud University Nijmegen Medical Center, Research Tower, 7th floor; UMC St Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Savelkoul
Paul J M
PJ
Hendriks
Giel
G
Konings
Irene B M
IB
Nivillac
Nicole M I
NM
Lagendijk
Anne Karine
AK
van der Sluijs
Peter
P
Deen
Peter M T
PM
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
D051399:Aquaporin 2; D014452:Ubiquitins; D011493:Protein Kinase C