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Ubiquitination Participates in the Lysosomal Degradation of Na,K-ATPase in Steady-State Conditions

¹ Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; and ² Center for Vascular and Tumor Biology, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel

Correspondence and requests for reprints should be addressed to Emilia Lecuona, Ph.D., Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M300, Chicago, IL 60611. E-mail: e-lecuona{at}northwestern.edu

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 µM) and infection with an adenovirus expressing dominant-negative PKC prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase 1-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.

Key Words: Na,K-ATPase • alveolar epithelial cells • ubiquitination • lysosome • degradation

CLINICAL RELEVANCE This is the first report studying the degradation in steady-state conditions of the plasma membrane Na,K-ATPase in alveolar epithelial cells. We found that ubiquitination was necessary for the plasma membrane Na,K-ATPase lysosomal degradation and that the mechanism differs from the one under pathologic conditions.

 

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