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

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Title: The Role of Putative Phosphorylation Sites in the Targeting and Shuttling of the Aquaporin-2 Water Channel
Authors: van Balkom, Bas; Savelkoul, Paul J.M.; Markovich, D.; Hofman, Erik; Nielsen, Soren; van der Sluijs, Peter; Deen, Peter M.T.
Publisher: Journal of Biological Chemistry
Date Published: November 01, 2002
Reference Number: 571
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Abstract • Article • Translation
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.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Aquaporin-2 (AQP2) is a protein in the kidney collecting duct cells. It resides in the cell interior but, when stimulated by a molecular sequence initiated by the hormone, vasopressin, AQP2 makes its way to a specific portion of the cell membrane (the apical membrane) by way of little sacs called vesicles. Once AQP2 is in the apical membrane, it acts as channels through which water may pass into the cell. When protein kinase C (PKC) is activated by certain hormones, it initiates a process which returns AQP2 from the apical membrane to the cell interior.

Scientists conduct research to get an ever clearer picture of all the compounds and biochemical processes that are involved in the transfer of AQP2 from the cell interior to the cell membrane and back again. They know that a process called phosphorylation, which introduces a phosphate group to the AQP2, must take place for the AQP2?s journey to the cell membrane to occur. They know Ser256, the 256th amino acid residue in the chain of amino acid residues that comprise the AQP2, is one of the phosphorylation sites on the AQP2. They know some of the specific amino acid residues in AQP2 that might be subject to phosphorylation by different classes of kinases, enzymes that catalyze the phosphorylation process. But until the current work of van Balkon, et al., it wasn?t clear if the AQP2 movement to the vesicle in which it traveled to the membrane and back could occur without phosphorylation of other amino acids than Ser256. Nor was it known if the phosphorylation of Ser256 was both necessary and sufficient for AQP2 to be expressed into the apical membrane.

To answer these questions, van Balkom, et al., tested four places on the AQP2 called kinase sites, i.e., places where specific kinase enzymes would transfer high?energy phosphate groups to the AQP2. The researchers altered these sites so they could study their relevance to the AQP2 transport process. They found:

  1. The AQP2 movement to transport vesicles does not require phosphorylation to occur;
  2. Phosphorylation is necessary and sufficient for AQP2 to be expressed in the apical membrane;
  3. AQP2?s return trip to the cell interior is independent of the phosphorylation state.