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

作者：Lecuona Emilia; Sun Haiying; Vohwinkel Christine; Ciechanover Aaron; Sznajder Jacob I

来源：American Journal of Respiratory Cell and Molecular Biology, 2009, 41(6): 671-679.

DOI：10.1165/rcmb.2008-0365OC

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

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 mu M) and infection with an adenovirus expressing dominant-negative PKC zeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha 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.

• 出版日期2009-12
• 单位西北大学