The anti-phospho-Na/K ATPase α
1 antibody we used in the present study recognizes the Na,K-ATPase α
1-subunit only when phosphorylated at Ser18. This phosphorylation triggers endocytosis of the Na,K-ATPase α
1-subunit and results in inhibition of the Na,K-ATPase activity.
48,49 The phospho-Na,K-ATPase α
1-subunit (Ser18) could be regarded as an inactive state of the Na,K-ATPase α
1-subunit. Ser18 itself may be phosphorylated directly by PKC.
54–56 In our study, although insulin increased PKC activity, insulin decreased the ratio of phospho-Na,K-ATPase α
1-subunit expression to total Na,K-ATPase α
1-subunit expression. As we previously reported, PKC exerts bidirectional (stimulatory and inhibitory) regulation of Na,K-ATPase activity in mouse corneal endothelial cells, and PKC stimulates Na,K-ATPase activity by activating PP1, PP2A, or both, which dephosphorylates the Na,K-ATPase α
1-subunit in corneal endothelial cells.
57 We also reported that PKC has an inhibitory effect on Na,K-ATPase activity,
57 and this effect may be attributed to Ser18 direct phosphorylation by PKC. In the present study, PDBu phosphorylated the Na,K-ATPase α
1-subunit at 30 minutes; phosphorylation was decreased at 6 hours. The time-response curve of Na,K-ATPase activity by insulin (
Fig. 1A) seemed to rise at 2 hours, and the effect became significant at 6 hours and 12 hours. There appears to be a time lag between PKC activation and Na,K-ATPase activation. Some time may be required for subsequent dephosphorylation and cell surface expression of Na,K-ATPase, and it may support our idea that PP1 or PP2A is subsequently activated by insulin-induced PKC. In addition, PP1- and PP2A-induced dephosphorylation of Na,K-ATPase may overcome direct phosphorylation by PKC in corneal endothelial cells. Previous reports also have shown that insulin activates phosphatidylinositol 3-kinase (PI-3 kinase) by insulin/IGF-I receptor, and that PI-3 kinase, presumably acting through PKC, subsequently activates PP1, PP2A, or both in porcine endometrial epithelial cells,
29 rat skeletal muscle cells,
58–60 and frog skin.
61 PP1 or PP2A subsequently dephosphorylates the α-subunit of Na,K-ATPase and stimulates its enzymatic activity.
29,58–60 Ser18 is one of the phosphorylation sites of Na,K-ATPase. Other phosphorylation mechanisms, such as Ser11 dephosphorylation and Tyr10 phosphorylation, may also play roles in Na,K-ATPase activation.
62–64 We selected Ser18 dephosphorylation to prove that dephosphorylation by protein phosphatase 1 or 2A affects Na,K-ATPase activity. Although we did not examine the effect of protein phosphatases on Tyr10 phosphorylation, activation phosphatases should be synergistic and may not prevent the increase in activity by insulin. In the immunocytochemistry phase, insulin increased cell surface expression of the Na,K-ATPase α
1-subunit, and the presence of inhibitors such as staurosporine, okadaic acid, and GF109203X decreased its expression. These results support our conclusions.