PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells |
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Authors: | Rodrigo Alzamora Ramon F. Thali Fan Gong Christy Smolak Hui Li Catherine J. Baty Carol A. Bertrand Yolanda Auchli René A. Brunisholz Dietbert Neumann Kenneth R. Hallows Núria M. Pastor-Soler |
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Affiliation: | From the Renal-Electrolyte Division, ‡Departments of Medicine and ;¶Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.;the §Department of Biology, Institute of Cell Biology, ETH Zurich, 8093 Zurich, Switzerland, and ;the ‖Functional Genomics Center Zurich (FGCZ), University of Zurich, 8057 Zurich, Switzerland |
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Abstract: | The vacuolar H+-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V1 sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO3−-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H+ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO3−-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175. |
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Keywords: | ATPases H+-ATPase Kidney Protein Kinase A (PKA) Vacuolar ATPase ATP6V1A Clone C Bicarbonate Proximal Tubule |
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