Cerebral Palsy

Hypoxia promotes Na,K-ATPase endocytosis via PKCzeta-mediated phosphorylation of its alpha subunit. Here, we describe that hypoxia leads to phosphorylation of AMPK at Thr172 in rat alveolar epithelial cells. Over-expression of a dominant-negative AMPKalpha construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. Overexpression of the reactive oxygen species (ROS) scavenger catalase prevented the hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondria-deficient A549-rho(0) cells, suggesting that mitochondrial ROS play an essential role in the hypoxia-induced AMPK activation. The hypoxia-induced PKCzeta translocation to the plasma membrane and phosphorylation at Thr410 was prevented by pharmacologically inhibiting AMPK or by over-expression of AMPK-DN construct. We found that AMPKalpha phosphorylates PKCzeta on residue Thr410 within the PKCzeta activation loop. Importantly, activation of AMPKalpha was necessary for hypoxia-induced AMPK-PKCzeta binding in alveolar epithelial cells. Overexpression of mutant PKCzeta-T410A prevented the hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCzeta Thr410 phosphorylation is essential for this process. PKCzeta activation by AMPK is isoform specific as siRNA against the alpha1 but not the alpha2 catalytic subunit prevented PKCzeta activation. Accordingly, we provide first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1 isoform, which binds and directly phosphorylates PKCzeta at Thr410, thereby promoting Na,K-ATPase endocytosis.