AMP-activated protein kinase regulates the vacuolar H⁺-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

Abstract

(28 -30, 49). We have demonstrated that in kidney intercalated cells and in epididymal clear cells, which share a common developmental origin in the Wolffian duct, the V-ATPase redistributes from the apical membrane to the cytosol with stimulation of the metabolic sensor AMPK (16,18). In our previous studies we first identified the V-ATPase A subunit as an AMPK substrate using the unbiased "MudSeeK" proteomic screening method and subsequently showed that the V-ATPase A subunit is phosphorylated directly by AMPK in kidney cells (18,52). AMPK is an important cell energy-sensing kinase that has been shown to downregulate several kidney membrane transport proteins (17,39). We have shown that AMPK reduces apical V-ATPase accumulation acutely in kidney intercalated cells, and that AMPK activation antagonizes the ability of PKA to increase V-ATPase localization at the apical pole of type A intercalated cells (16).Phosphatidylinositol 3-kinase and PKA are additional kinases that regulate the function of the V-ATPase in the mammalian kidney (16,41,43). PKA agonists also regulate the V-ATPase in other animal models (42, 54). However, it was not until recently that work by our group linked the direct phosphorylation of the V-ATPase by PKA at Ser-175 to increases in V-ATPase plasma membrane activity in mammalian cells (2). Ser-175 A subunit phosphorylation is likely to occur downstream of acid-base-sensing pathways, which require the presence of active carbonic anhydrase, the soluble

Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

“…The diffuse intracellular localization induced by the AMPK activator AICAR was also associated with a qualitative decrease in V-ATPase-associated fluorescence in the S3 segment tubule cells, a finding that we had observed previously in collecting duct intercalated cells of rat kidney slices treated with AICAR (22). We then tested whether acute AMPK activation for 75 min affected the steady-state levels of the V-ATPase A subunit, a subunit that is directly phosphorylated by AMPK (1,23). Quantification of the anti-V-ATPase A subunit immunoblot signal (Fig.…”

“…In addition, the V 0 sector "a" subunit (ATP6V0A) senses endosomal acidification by recruiting ARNO in the proximal tubule (30). Our group also showed that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits V-ATPase apical accumulation in kidney intercalated cells and in epididymal clear cells (22,23) via phosphorylation at Ser-384 in the V-ATPase A subunit (1).…”

“…Although AMPK is abundantly expressed in kidney (20), an understanding of its role in kidney physiology and disease is less developed than in other organs (24,47). We have recently identified a key AMPK phosphorylation site in the V-ATPase A subunit that when mutated abrogates the inhibitory effects of AMPK on the V-ATPase in a cell line of intercalated cell origin (1). Of clinical significance, AMPK activation in the proximal tubule during ischemia protects the epithelial cells against apoptosis and loss of polarity (37,58).…”

Adaptation by the collecting duct to an exogenous acid load is blunted by deletion of the proton-sensing receptor GPR4

“…We did not observe such differences in the response of the apical transporters pendrin and H ϩ -ATPase. It seems unlikely to anticipate identification of a single or unique acid sensor in the kidney because current evidence suggests a complex network of kinases [e.g., Pyk2 adhesion kinase (34,39,58), ErbB1/2 tyrosine kinase (87), and AMPK (2)], enzymes [e.g., ammoniagenic enzymes (24,25,43) and soluble adenylyl cyclase (14,64)], G protein-coupled receptors like GPR4 (23,88) or OGR1 (63) or CaSR (28,73), the insulin receptor-related receptor (InsR-RR) (68), ion channels [e.g., TASK2 (101)], and transporters [e.g., vATPase (2,62)], which may all be involved in the response to a reduction in pH. Furthermore, multiple sensors may offer advantages based on different ranges of pH sensitivity, or cellular and subcellular localization.…”

Collecting Duct Intercalated Cell Function and Regulation

“…These findings have led to an intense search for the pHsensing mechanism within the intercalated cells. For example, the H 1 -ATPase in type A intercalated cells is acutely activated (within minutes to hours) by kinases such as protein kinase A (PKA), while it is acutely inhibited by the metabolic sensor AMP activated protein kinase (AMPK) ( Figure 5) (72)(73)(74). This downregulation of the proton pump by AMPK is preliminary evidence that intercalated cell function can be altered when the tubule is under metabolic stress, such as under conditions of ischemia.…”