Hypoxia promotes Na,K-
ATPase endocytosis via
protein kinase C zeta (PKC zeta)-mediated phosphorylation of the Na,K-
ATPase alpha subunit. Here, we report that
hypoxia leads to the phosphorylation of
5'-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK alpha subunit (AMPK-DN) construct prevented the
hypoxia-induced endocytosis of Na,K-
ATPase. The overexpression of the
reactive oxygen species (ROS) scavenger
catalase prevented
hypoxia-induced AMPK activation. Moreover,
hypoxia failed to activate AMPK in mitochondrion-deficient rho(0)-A549 cells, suggesting that mitochondrial ROS play an essential role in
hypoxia-induced AMPK activation.
Hypoxia-induced PKC zeta translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK alpha phosphorylates PKC zeta on residue Thr410 within the PKC zeta activation loop. Importantly, the activation of AMPK alpha was necessary for
hypoxia-induced AMPK-PKC zeta binding in alveolar epithelial cells. The overexpression of T410A mutant PKC zeta prevented
hypoxia-induced Na,K-
ATPase endocytosis, confirming that PKC zeta Thr410 phosphorylation is essential for this process. PKC zeta activation by AMPK is
isoform specific, as
small interfering RNA targeting the alpha1 but not the alpha2 catalytic subunit prevented PKC zeta activation. Accordingly, we provide the first evidence that
hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1
isoform, which binds and directly phosphorylates PKC zeta at Thr410, thereby promoting Na,K-
ATPase endocytosis.