FIG. 6.

AMPK phosphorylation of PKCζ at the Thr410 residue is required for hypoxia-induced downregulation of Na,K-ATPase. (A) ATII cells were infected with Ad-null (20 PFU/cell) or Ad-HA-DN AMPK-α (DN-AMPK α; 20 PFU/cell) or pretreated with compound C (2 μM for 30 min) and exposed to 21 or 1.5% O₂ for 10 min. PKCζ was immunoprecipitated from the cell lysates as described in Materials and Methods. PKCζ phosphorylated at Thr410 [pPKCζ (T410)] and the total amount of PKCζ were measured by Western blot analysis. Representative Western blots for pPKCζ (T410), PKCζ, and HA-AMPK in the cell lysates are shown (n = 4). (B) ATII cells were treated with AICAR (2 mM) for 15 min or Ad-CA AMPK (CA-AMPK; 20 PFU/cell). PKCζ was immunoprecipitated from the cell lysate as described in Materials and Methods. pPKCζ (T410), pACC as a control for AMPK activation, and the total amount of PKCζ were measured by Western blot analysis (n = 4). Representative Western blots are shown. V, vehicle. (C) COS-7 cells were transiently transfected with full-length FLAG-WT PKCζ, FLAG-T410A mutant PKCζ, or empty vector (EV); 48 h later, PKCζ was immunoprecipitated with 1 μl of FLAG antibody as described in Materials and Methods and incubated with AMPK α1-GST fusion protein (200 ng) and [γ-³²P]ATP. A representative autoradiograph of the phosphorylated PKCζ and a Western blot (WB) analyzing FLAG-PKCζ are shown (n = 3). (D) COS-7 cells were transiently transfected with a vector expressing full-length FLAG-WT PKCζ or FLAG-T410A mutant PKCζ or an empty vector; 48 h later, cells were exposed to 21 or 1.5% O₂ for 60 min. Na,K-ATPase α1 subunit abundance at the plasma membrane was determined from cell surface biotinylation, followed by streptavidin pulldown and Western blot analysis using specific antibodies. Representative Western blots analyzing the Na,K-ATPase α1 subunit at the plasma membrane (PM) and the Na,K-ATPase α1 subunit (as a loading control) and PKCζ in the cell lysates (TCL) are shown. Values are expressed as means ± SEM (n = 4). **, P < 0.01.