The ubiquitin conjugation system is necessary for Na,K-ATPase endocytosis during hypoxia. A, A549 cells were exposed to 21 or 1.5% O₂ for 60 min at 37° C. Na,K-ATPase α₁ subunit abundance in basolateral (BLM), and late endosomes (LE) was determined. Equal amounts of protein (5 μg) were loaded in each lane. A representative Western blot is shown, n=3. B, A549 cells were incubated in the presence or absence MG132 (20 μM) and then exposed to 1.5% O₂ for 60 min. Na,K-ATPase endocytosis was determined by biotin labeling of surface proteins followed by streptavidin pull-down and Western blot analysis using a specific anti α₁ subunit antibody. Top, quantitative densitometric scan of three experiments (mean ± SEM), **p<0.01, bottom representative Western blot of the α₁ subunit abundance. C and D, E36 or CHO-ts20 cells were incubated at 21% O₂ or 1.5% O₂ for 60 min at either 30°C or 43°C. Na,K-ATPase endocytosis was studied as describe for B. Top, quantitative densitometric scan of three experiments (mean ± SEM), **p<0.01, bottom representative Western blot of the α₁ subunit abundance.

Hypoxia increases Na,K-ATPase (NKA)-ubiquitin conjugates at the plasma membrane. GFP-α₁-A549 cells were incubated at 21% O₂ in the presence or absence MG132 (20 μM) and then exposed to 1.5% O₂ for 60 min. At the end of the incubation, BLMs were isolated; membrane proteins were solubilized with 1% Triton-X100. Equal amount of proteins (20−30 μg) were immunoprecipitated with an anti-GFP antibody. Immunoprecipitates were immunoblotted against ubiquitin. The membranes were then stripped and reblotted against GFP as a loading control. A representative Western blot is shown, n=3.

Hypoxia induction of Na,K-ATPase-ubiquitin conjugates is dependent on Ser-18 phosphorylation. WT or S18A GFP-α₁-A549 were incubated at 21% O₂ in the presence of MG132 (20 μM) and then exposed to 1.5% O₂ for 60 min Cells were processed as described for Fig. 2. A representative western blot is shown, n=3.

Lysines in the KKSKK have a redundant function. WT or Lys 16, Lys 17, Lys 19, Lys 20 α₁-A549 cells were exposed to 1.5 % O₂ for 60 min. At the end of the incubation, Na,K-ATPase endocytosis was studied as described above. Top, quantitative densitometric scan of three experiments (mean ± SEM), **p<0.01, bottom representative Western blot of the α₁-subunit abundance.

Mutations of ubiquitination sites prevent Na,K-ATPase endocytosis during hypoxia. A, The lysines in the KKSKK sequence are required for Na,K-ATPase endocytosis during hypoxia. WT or K4R-α₁-A549 cells were exposed to 1.5 %O₂ for 60 min. Na,K-ATPase endocytosis was determined as described above. Top, quantitative densitometric scan of three experiments (mean ± SEM), **p<0.01, bottom representative Western blot of the α subunit abundance. B, WT or K4R-GFP-α₁-A549 were incubated with MG132 (20 μM) and then exposed to 21 or 1.5% O₂ for 60 min. At the end of the incubation cell lysates were isolated and Na,K-ATPase-ubiquitin conjugates were immunoprecipitated with an antibody against GFP. Immunoprecipitates were immunoblotted against ubiquitin. The membranes were then stripped and reblotted against GFP as a loading control. A representative western blot is shown, n=3. C, GFP-α₁-A549 cells were exposed to 1.5% O₂ for 20 min, and Na,K-ATPase GFP α₁ subunit was immunoprecipitated using a polyclonal GFP antibody. An in vitro phosphorylation reaction with the immunoprecipitated Na,K-ATPase, purified PKC, and [γ³²P]ATP was conducted. A representative autoradiogram of the α₁ subunit phosphorylation is shown with the corresponding immunoblot performed with a monoclonal anti-GFP antibody (n=3).