F-actin-dependent activation of NADPH oxidase 5 by the vacuolar-type ATPase.

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme that produces superoxide in response to increases in intracellular calcium. NOX5 has been shown to play essential roles in processes such as specialized cell differentiation, proliferation, and cell contraction, while its overexpression and hyperactivation are associated with human diseases, including cardiovascular disorders, cancer, and diabetic kidney disease. Despite its importance in human health and disease, the regulatory factors and mechanistic signaling details of NOX5 remain incompletely resolved. We previously reported that chemical stabilization of F-actin by jasplakinolide induces a superoxide burst by NOX5 independent of changes in intracellular calcium. Here, we show that this F-actin-dependent activation of NOX5 superoxide production relies on the vacuolar-type ATPase (v-ATPase). In cells, jasplakinolide treatment stabilizes a complex comprising actin, actin-binding proteins, the v-ATPase, and NOX5. Chemical inhibition of the v-ATPase or v-ATPase subunit knockdown decreases NOX5 superoxide production in response to jasplakinolide while only minimally affecting NOX5 superoxide production in response to increases in intracellular calcium or NOX5 phosphorylation. Finally, in untreated cells, the v-ATPase and NOX5 localize to the cell leading edge and contribute to cell migration. Our findings represent a novel mechanism of NOX5-mediated superoxide production by the v-ATPase and provide a new foundation for developing strategies to disrupt protein interactions and activities that drive aberrant cell migration.