α1-AMP-activated protein kinase regulates hypoxia-induced Na,K-ATPase endocytosis via direct phosphorylation of protein kinase Cζ

Galina A. Gusarova, Laura A. Dada, Aileen M. Kelly, Chaya Brodie, Lee A. Witters, Navdeep S. Chandel, Jacob I. Sznajder

Research output: Contribution to journalArticle

77 Scopus citations

Abstract

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMPactivated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α 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 ρ0-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ 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 α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK ζ was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,KATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.

Original languageEnglish (US)
Pages (from-to)3455-3464
Number of pages10
JournalMolecular and cellular biology
Volume29
Issue number13
DOIs
StatePublished - Jul 2009

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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