The Role of Putative Phosphorylation Sites in the Targeting and Shuttling of the Aquaporin-2 Water Channel

Bas W.M Van Balkom,Paul J.M Savelkoul,Daniel Markovich,Erik Hofman,Soren Nielsen,Peter Van Der Sluijs,Peter M.T Deen

doi:10.1074/jbc.m207525200

Bas W.M Van Balkom, Paul J.M Savelkoul + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m207525200

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Abstract

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.

Highlights

In humans, the kidney is the prime organ for regulation of body fluid osmolarity, which is maintained within strict boundaries
Other hormones, such as epidermal growth factor, prostaglandin E2, and agonists of muscarinic receptors did not interfere with the arginine vasopressin (AVP)-mediated acetate; CKII, casein kinase II; MDCK, Madin-Darby canine kidney; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; CLSM, confocal laser scanning microscope; PKA, protein kinase A
Because the inhibitory effect of these latter hormones was absent upon co-treatment with protein kinase C (PKC) inhibitors [10, 12] and phorbol 12-myristate 13acetate (PMA), which is a specific activator of several PKCs [14], inhibits AVP-induced water permeability [13], these hormones were suggested to activate PKC isotypes that interfere with shuttling of AQP2 to the apical membrane

Summary

Introduction

The kidney is the prime organ for regulation of body fluid osmolarity, which is maintained within strict boundaries. 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.

Results

Conclusion