title: AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney.
abstract: The vacuolar H(+)-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) inhibited V-ATPase-dependent H(+) secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3(-)-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.
authors.affiliation: Renal-Electrolyte Div., Dept. of Medicine, S976.1 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. hallows@pitt.edu.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Alzamora
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Al-Bataineh
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Fan
authors.initials: F
authors.lastname: Gong
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Ramon F
authors.initials: RF
authors.lastname: Thali
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Joho-Auchli
authors.forename: René A
authors.initials: RA
authors.lastname: Brunisholz
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Satlin
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
authors.forename: Núria M
authors.initials: NM
authors.lastname: Pastor-Soler
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D000136:Acid-Base Equilibrium; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D013058:Mass Spectrometry; D010766:Phosphorylation; D011817:Rabbits; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D055372:AMP-Activated Protein Kinases; D025262:Vacuolar Proton-Translocating ATPases
keywords: AMPK; Intercalated cells; V-ATPase; kidney; mass spectrometry