title: Alpha1-AMP-activated protein kinase regulates hypoxia-induced Na,K-ATPase endocytosis via direct phosphorylation of protein kinase C zeta.
abstract: 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.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M-300, Chicago, IL 60611, USA.
authors.forename: Galina A
authors.initials: GA
authors.lastname: Gusarova
authors.forename: Laura A
authors.initials: LA
authors.lastname: Dada
authors.forename: Aileen M
authors.initials: AM
authors.lastname: Kelly
authors.forename: Chaya
authors.initials: C
authors.lastname: Brodie
authors.forename: Lee A
authors.initials: LA
authors.lastname: Witters
authors.forename: Navdeep S
authors.initials: NS
authors.lastname: Chandel
authors.forename: Jacob I
authors.initials: JI
authors.lastname: Sznajder
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000166:cytology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D000099221:Protein Kinase C zeta
chemical_list: D055372:AMP-Activated Protein Kinases; D007527:Isoenzymes; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000254:Sodium-Potassium-Exchanging ATPase; D000099221:Protein Kinase C zeta