Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia.
As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.
Dada
Laura A
LA
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States.
Welch
Lynn C
LC
Zhou
Guofei
G
Ben-Saadon
Ronen
R
Ciechanover
Aaron
A
Sznajder
Jacob I
JI
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D021122:Protein Subunits; D025801:Ubiquitin; D010768:Phosphoserine; D000254:Sodium-Potassium-Exchanging ATPase; D008239:Lysine