Phosphorylation of Serine 256 Is Required for cAMP-dependent Regulatory Exocytosis of the Aquaporin-2 Water Channel*
- From the Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan
Abstract
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 μm 8-(4-chlorophenylthio)-cAMP increased theP f by 85 ± 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in theP 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[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.
Footnotes
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↵* This work was supported in part by Grants-in-aid for Scientific Research A and C from the Ministry of Education, Science, Sports, and Culture and grants-in-aid from the Japan Medical Association and the Tokyo Hypertension Conference.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence and reprint requests should be addressed: Second Dept. of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-5803-5216; Fax: 81-3-5803-0132; E-mail: kfushimi.med2{at}med.tmd.ac.jp.
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↵1 The abbreviations used are: AQP2, aquaporin-2; WT, wild type; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; cpt-cAMP, 8-(4-chlorophenylthio)-cAMP;P f, osmotic water permeability; PAGE, polyacrylamide gel electrophoresis.
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- Received March 24, 1997.
