{alpha}1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase C{zeta}

doi:10.1128/MCB.00054-09

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${alpha}$ 1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase C ${zeta}$

Galina A. Gusarova,¹^, Laura A. Dada,¹^, Aileen M. Kelly,¹ Chaya Brodie,² Lee A. Witters,³ Navdeep S. Chandel,¹ and Jacob I. Sznajder¹^*

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois,¹ Department of Neurosurgery and Hermelin Brain Tumor Center, Henry Ford Health System, Detroit, Michigan,² Departments of Medicine and Biochemistry, Dartmouth Medical School, and Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire³

Received 13 January 2009/ Returned for modification 13 February 2009/ Accepted 10 April 2009

Hypoxia promotes Na,K-ATPase endocytosis via protein kinaseC ${zeta}$ (PKC ${zeta}$ )-mediated phosphorylation of the Na,K-ATPase ${alpha}$ subunit.Here, we report that hypoxia leads to the phosphorylation of5'-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolarepithelial cells. The overexpression of a dominant-negativeAMPK ${alpha}$ subunit (AMPK-DN) construct prevented the hypoxia-inducedendocytosis of Na,K-ATPase. The overexpression of the reactiveoxygen species (ROS) scavenger catalase prevented hypoxia-inducedAMPK activation. Moreover, hypoxia failed to activate AMPK inmitochondrion-deficient ${rho}$ ⁰-A549 cells, suggesting that mitochondrialROS play an essential role in hypoxia-induced AMPK activation.Hypoxia-induced PKC ${zeta}$ translocation to the plasma membrane andphosphorylation at Thr410 were prevented by the pharmacologicalinhibition of AMPK or by the overexpression of the AMPK-DN construct.We found that AMPK ${alpha}$ phosphorylates PKC ${zeta}$ on residue Thr410 withinthe PKC ${zeta}$ activation loop. Importantly, the activation of AMPK ${alpha}$ was necessary for hypoxia-induced AMPK-PKC ${zeta}$ binding in alveolarepithelial cells. The overexpression of T410A mutant PKC ${zeta}$ preventedhypoxia-induced Na,K-ATPase endocytosis, confirming that PKC ${zeta}$ Thr410 phosphorylation is essential for this process. PKC ${zeta}$ activationby AMPK is isoform specific, as small interfering RNA targetingthe ${alpha}$ 1 but not the ${alpha}$ 2 catalytic subunit prevented PKC ${zeta}$ activation.Accordingly, we provide the first evidence that hypoxia-generatedmitochondrial ROS lead to the activation of the AMPK ${alpha}$ 1 isoform,which binds and directly phosphorylates PKC ${zeta}$ at Thr410, therebypromoting Na,K-ATPase endocytosis.

* Corresponding author. Mailing address: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M-300, Chicago, IL 60611. Phone: (312) 908-7737. Fax: (312) 908-4650. E-mail: j-sznajder{at}northwestern.edu

Published ahead of print on 20 April 2009.

These authors contributed equally to this paper.