PKA Regulates Vacuolar H⁺-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells
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Date
2010-08-06
Publication Type
Journal Article
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yes
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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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published
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Volume
285 (32)
Pages / Article No.
24676 - 24685
Publisher
American Society for Biochemistry and Molecular Biology
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Subject
ATPases; H⁺-ATPase; Kidney; Protein Kinase A (PKA); Vacuolar ATPase; ATP6V1A; Clone C; Bicarbonate; Proximal Tubule
Organisational unit
02207 - Functional Genomics Center Zurich / Functional Genomics Center Zurich