Phosphorylation of Serine 256 Is Required for cAMP-dependent Regulatory Exocytosis of the Aquaporin-2 Water Channel

• Published in: The Journal of biological chemistry Vol. 272; no. 23; pp. 14800 - 14804
• Main Authors: Fushimi, K, Sasaki, S, Marumo, F
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 06.06.1997
• Subjects: Alanine; Animals; Aquaporin 2; Aquaporin 6; Aquaporins; Cell Line; Cyclic AMP - analogs & derivatives; Cyclic AMP - metabolism; Cyclic AMP - pharmacology; Exocytosis; Ion Channels - biosynthesis; Ion Channels - metabolism; Light; Mutagenesis, Site-Directed; Phosphorylation; Point Mutation; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Scattering, Radiation; Serine; Thionucleotides - pharmacology; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.23.14800

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability ( P f ) of LLC-PK1 cells transfected with WT was 69.6 ± 6.5 μm/s (24.8 ± 2.2 μm/s for mock-transfected), and stimulation by 500 μ m 8-(4-chlorophenylthio)-cAMP increased the P f by 85 ± 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the P f was only 8 ± 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 ± 10%, significantly less than that for WT (88 ± 5%). In addition, an in vivo [ 32 P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.