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 language | English (US) |
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Pages (from-to) | 671-679 |
Number of pages | 9 |
Journal | American journal of respiratory cell and molecular biology |
Volume | 41 |
Issue number | 6 |
DOIs | |
State | Published - 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