Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel.
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 (Pf) of LLC-PK1 cells transfected with WT was 69.6 +/- 6.5 microm/s (24.8 +/- 2.2 microm/s for mock-transfected), and stimulation by 500 microM 8-(4-chlorophenylthio)-cAMP increased the Pf by 85 +/- 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the Pf 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 [32P]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.
Fushimi
K
K
Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. kfushimi.med2@med.tmd.ac.jp
Sasaki
S
S
Marumo
F
F
D000409:Alanine; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D005089:Exocytosis; D007473:Ion Channels / Q000096:biosynthesis / Q000378:metabolism*; D008027:Light; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012542:Scattering, Radiation; D012694:Serine; D013873:Thionucleotides / Q000494:pharmacology*; D014162:Transfection
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D007473:Ion Channels; D011994:Recombinant Proteins; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D012694:Serine; D000242:Cyclic AMP; D000409:Alanine