Ubiquitination participates in the lysosomal degradation of Na,K-ATPase in steady-state conditions.
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.
Lecuona
Emilia
E
Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M300, Chicago, IL 60611, USA. e-lecuona@northwestern.edu
Sun
Haiying
H
Vohwinkel
Christine
C
Ciechanover
Aaron
A
Sznajder
Jacob I
JI
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004705:Endocytosis; D004847:Epithelial Cells / Q000201:enzymology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000201:enzymology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000099221:Protein Kinase C zeta
D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D000254:Sodium-Potassium-Exchanging ATPase; D000099221:Protein Kinase C zeta