Extracellular proton-secreting transport systems that contribute to extracellular pH include the Vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis.
We previously showed that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay as compared to cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.
American journal of physiology. Renal physiology. 2016 Feb 24 [Epub ahead of print]
Mohammad M Al-Bataineh, Rodrigo Alzamora, Kazuhiro Ohmi, Pei-Yin Ho, Allison L Marciszyn, Fan Gong, Hui Li, Kenneth R Hallows, Núria M Pastor-Soler
University of Pittsburgh., University of Pittsburgh., University of Southern California., University of Southern California., University of Pittsburgh., University of Pittsburgh., University of Southern California., University of Southern California Keck School of Medicine of USC.