TY - JOUR
T1 - Ubiquitination participates in the lysosomal degradation of Na,K-ATPase in steady-state conditions
AU - Lecuona, Emilia
AU - Sun, Haiying
AU - Vohwinkel, Christine
AU - Ciechanover, Aaron
AU - Sznajder, Jacob I.
N1 - Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2009/12/1
Y1 - 2009/12/1
N2 - 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.
AB - 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.
KW - Alveolar epithelial cells
KW - Degradation
KW - Lysosome
KW - Na,K-ATPase
KW - Ubiquitination
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U2 - 10.1165/rcmb.2008-0365OC
DO - 10.1165/rcmb.2008-0365OC
M3 - Article
C2 - 19286978
AN - SCOPUS:70949097231
VL - 41
SP - 671
EP - 679
JO - American Journal of Respiratory Cell and Molecular Biology
JF - American Journal of Respiratory Cell and Molecular Biology
SN - 1044-1549
IS - 6
ER -