Mechanisms of Kinase-dependent Regulation of the V-ATPase in Kidney

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

DESCRIPTION (provided by applicant): V-ATPase function in kidney is important for excretion of non-volatile acids generated by metabolism. Individuals with defective V-ATPase activity in kidney intercalated cells (ICs) may suffer from severe chronic metabolic acidosis with potential severe sequelae in bone, kidney, and other organs. V-ATPase activity is also critical to the function of epithelial proton-secreting epididymal clear cells, which contribute to the acidic environment where spermatozoa mature. Therefore, understanding V-ATPase regulation may provide insights involving the physiology and pathophysiology of proton secretion in the urogenital tract and lead to better treatments for severe metabolic acidosis and male infertility. V-ATPase activity is likely orchestrated by a number of regulatory mechanisms;direct V-ATPase phosphorylation in mammalian proton-secreting epithelial cells, however, has been addressed by only a few studies. We and others have shown that in clear cells, V-ATPase translocation to the apical membrane responds to alkaline luminal pH, increased luminal HCO3 - concentration, soluble adenylyl cyclase (sAC), and protein kinase A (PKA) activity. Our preliminary studies demonstrate that PKA is also required for apical V-ATPase in ICs and that sAC regulates V-ATPase-mediated intracellular pH (pHi) recovery in isolated perfused outer medullary collecting ducts (OMCDs). In addition, activation of the metabolic sensor AMP-activated protein kinase (AMPK) prevents V-ATPase translocation in clear cells and ICs, and that both PKA and AMPK phosphorylate V-ATPase A and C2b subunits in vitro and in vivo. Based on these findings, we hypothesize that subcellular localization and V-ATPase activity in ICs depend on the direct phosphorylation of its subunits by PKA and AMPK, and that V-ATPase activity may be coupled to the sensing of extracellular acid-base status via PKA and metabolic status via AMPK. The Aims of this proposal are to: 1) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by PKA in ICs;and 2) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by AMPK in ICs. Initially, we will focus our efforts toward mapping PKA and AMPK candidate phosphorylation sites in the V1 sector A subunit. To identify relevant phosphorylation sites, we will use site-directed mutagenesis followed by in vitro and in vivo phosphorylation assays, as well as mass spectrometry. Additionally, we will examine the roles of relevant PKA and AMPK phosphorylation sites in the A subunit on V-ATPase subcellular localization and activity using ICs in cell culture. We will determine the effects of PKA or AMPK activation on V-ATPase-dependent pHi changes in OMCDs. Finally, we will examine the effects of AMPK activation on PKA-mediated phosphorylation of the V-ATPase A subunit in vitro and in vivo. The successful completion of the Aims of this application will contribute to new strategies to treat disorders of acidification in the kidney and in the urogenital tract. PUBLIC HEALTH RELEVANCE: The vacuolar ATPase (V-ATPase) is an important protein complex that functions in the kidney to help excrete acid generated by the body's metabolism. Patients with defective V-ATPase activity in kidney intercalated cells may suffer from severe chronic metabolic acidosis with potential serious sequelae in bone, kidney, and other organs. We hypothesize that V-ATPase function depends on direct phosphorylation of its subunits by enzymes that are coupled to the sensing of acid/base and metabolic status. The successful completion of the Specific Aims detailed in this application will, we believe, contribute to new strategies to treat disorders of acidification in the kidney and in the urogenital tract.