The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel.
In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.
van Balkom
Bas W M
BW
Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, The Netherlands.
Savelkoul
Paul J M
PJ
Markovich
Daniel
D
Hofman
Erik
E
Nielsen
Soren
S
van der Sluijs
Peter
P
Deen
Peter M T
PM
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004285:Dogs; D004705:Endocytosis; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D021381:Protein Transport; D012694:Serine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D005576:Colforsin; D012694:Serine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate