PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

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

doi:10.1074/jbc.m110.106278

Rodrigo Alzamora, Ramon F Thali + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m110.106278

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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 V(1) 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 HCO(3)(-)-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 HCO(3)(-)-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.

Highlights

Somes, lysosomes, and the Golgi complex [1]
We and others have shown that regulation of the V-ATPase at the apical membrane of intercalated and clear cells is tightly linked to alkaline luminal pH, HCO3Ϫ, carbonic anhydrase activity, activation of the soluble adenylyl cyclase,2 cAMP, and protein kinase A (PKA) [19, 31,32,33]
It has been shown that V-ATPase subcellular localization and/or V-ATPase-dependent proton secretion are regulated by a variety of stimuli, including changes in intracellular and extracellular pH, extracellular [CO2], intracellular [Ca2ϩ], and [HCO3Ϫ] in protonsecreting cells derived embryologically from the Wolffian duct

Summary

Introduction

Somes, lysosomes, and the Golgi complex [1]. The V-ATPase has 14 subunits distributed into two domains. PKA-dependent in Vivo Phosphorylation of the V-ATPase A Subunit in HEK-293T Cells Occurs at Ser-175—To determine whether Ser-175 is a target for PKA-dependent phosphorylation in intact HEK-293T cells, we compared [32P]orthophosphate labeling of the FLAG-A-WT and FLAG-A-S175A mutant subunits under control conditions or following treatment with PKA activators (100 ␮M 6-MB-cAMP ϩ 500 ␮M IBMX) or a specific PKA inhibitor (10 ␮M mPKI) (Fig. 3A).

Results

Conclusion