Ubiquitination participates in the lysosomal degradation of Na,K-ATPase in steady-state conditions

Emilia Lecuona*, Haiying Sun, Christine Vohwinkel, Aaron Ciechanover, Jacob I. Sznajder

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na+ transport and plays an important role in keeping the lungs free ofedema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasmamembrane of AEC in steady-state conditions. The halflife of the plasma membrane Na,K-ATPase was about 4 hours, and the incorporation of new Na,K-ATPase to the plasma membrane was Brefeldin A sensitive. Both protein kinase C (PKC) inhibition with bisindolylmaleimide (10 μM) and infection with an adenovirus expressing dominant-negative PKCζ prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPaseα1-subunit lacking the PKC phosphorylation sites, the plasma membrane Na,K-ATPase had a moderate increase in half-life. We also found that the Na,K-ATPase was ubiquitinated in steady-state conditions and that proteasomal inhibitors preventedits degradation. Interestingly,mutation of the four lysines described to be necessary for ubiquitination an dendocytosis of the Na,K-ATPase in injurious conditions did not have an effect on its half-life in steady-state conditions. Lysosomal inhibitors prevented Na,K-ATPase degradation, and co-localization of Na,K-ATPase and lysosomes was found after labeling and chasing the plasma membrane Na,K-ATPase for 4 hours. Accordingly, we provide evidence suggesting that phosphorylation and ubiquitination are necessary for the steady-state degradation of the plasma membrane Na,K-ATPase in the lysosomes in alveolar epithelial cells.

Original languageEnglish (US)
Pages (from-to)671-679
Number of pages9
JournalAmerican journal of respiratory cell and molecular biology
Volume41
Issue number6
DOIs
StatePublished - Dec 1 2009

Keywords

  • Alveolar epithelial cells
  • Degradation
  • Lysosome
  • Na,K-ATPase
  • Ubiquitination

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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