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Am J Respir Cell Mol Biol. 2009 Dec;41(6):671-9. doi: 10.1165/rcmb.2008-0365OC. Epub 2009 Mar 13.

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

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1
Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M300, Chicago, IL 60611, USA. e-lecuona@northwestern.edu

Abstract

The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na(+) transport and plays an important role in keeping the lungs free of edema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasma membrane of AEC in steady-state conditions. The half-life 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 microM) and infection with an adenovirus expressing dominant-negative PKCzeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha1-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 prevented its degradation. Interestingly, mutation of the four lysines described to be necessary for ubiquitination and endocytosis 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.

PMID:
19286978
PMCID:
PMC2784405
DOI:
10.1165/rcmb.2008-0365OC
[Indexed for MEDLINE]
Free PMC Article
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