Chronic insulin treatment phosphorylates the renal Na-K-ATPase α1-subunit at serine 16/23 and reduces its activity involving PI3-kinase-dependent PKC activation

Abstract

The regulation of Na-K-ATPase in various tissues is under the control of a number of hormones and peptides that exert both short- and long-term control over its activity. The present study was performed to investigate the effect of chronic insulin treatment on Na-K-ATPase in renal proximal tubular cells. Incubation of opossum kidney (OK) cells, transfected with the rat Na-K-ATPase α₁-subunit, with 1 nmol/l insulin for 48 h decreased Na-K-ATPase activity. Insulin decreased α₁-protein content and increased α₁-serine phosphorylation and α₁-adaptor protein 2 (AP2) interaction. Removal of the 26 NH₂-terminal (-NT) amino acid from the α₁-subunit containing serine/threonine sites abolished the insulin-mediated serine phosphorylation and inhibition of Na-K-ATPase. Substitution of serine 16 and 23 with alanine showed a comparable effect on -NT. Insulin increased the activity of protein kinase C (PKC), which was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. Both PI3K and PKC inhibitors abolished the insulin-mediated inhibition of Na-K-ATPase. Insulin increased the expression of PKC-β₁, -δ, -ξ, and-λ; however, only PKC-ξ/λ-specific inhibitors blocked insulin-induced phosphorylation and inhibition of Na-K-ATPase. Our data demonstrate that insulin activates the atypical PKC isoforms-ξ/λ via the PI3K pathway. PKC-ξ/λ-induced phosphorylation of the α₁-subunit at serine 16 and 23 leads to AP2 recruitment, degradation, and a decrease in Na-K-ATPase activity.

Keywords: adaptor protein 2; kinase; serine kinase; serine phosphorylation; sodium transporter; tyrosine; tyrosine phosphorylation.

Fig. 1.

RNA interference downregulates Na-K-ATPase α₁-subunit expression. A: α₁-subunit expression was determined by ELISA in opossum kidney (OK) cells incubated with media alone (basal), transfection reagent only (reagent), sequence 1 siRNA (siRNA1), and sequence 2 siRNA (siRNA2). B: cell samples of varying protein concentration were assayed for α₁-subunit expression by ELISA. OD, optical density. *P < 0.05 vs. basal.

Fig. 2.

Time- and concentration-dependent insulin-mediated Na-K-ATPase stimulation. A: cells were incubated with 1 nmol/l insulin or DMEM-ouabain alone (basal) for indicated time periods at 37°C. B: cells were incubated with DMEM-ouabain alone (basal) or increasing insulin concentration for 20 min. *P < 0.05 vs. basal.

Fig. 3.

Insulin-induced Na-K-ATPase stimulation and α₁-subunit tyrosine phosphorylation in OK cells. A: cells were incubated with 1 nmol/l insulin for 20 min in the absence and presence of genistein (Gen), HNPA-(AH)₃ (HNM), GF109203X (GFX), and H89. B: insulin-mediated membrane α₁-subunit tyrosine phosphorylation (Tyr-P). Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal and #P < 0.05 vs. insulin.

Fig. 4.

Basal Na-K-ATPase expression and activity with α₁-subunit tyrosine and serine mutations or NH₂-terminal truncation. OK cells were transfected with rat α₁-subunit wild-type (WT), NH₂-terminal truncated (-NT), tyrosine 10 mutated to alanine (Y10A), or glycine (Y10E) and serine 16 and 23 mutated to alanine (S16A/S23A).

Fig. 5.

Effect of α₁-subunit tyrosine and serine mutations on insulin-induced acute tyrosine phosphorylation (A) and Na-K-ATPase stimulation (B). OK cells transfected with rat α₁-subunit wild-type (WT), NH₂-terminal truncated (-NT), tyrosine 10 mutated to alanine (Y10A), or glycine (Y10E) and serine 16 and 23 mutated to alanine (S16A/S23A) were incubated with 1 nmol/l insulin or DMEM-ouabain alone (basal) for 20 min at 37°C. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 6.

Chronic insulin treatment attenuates Na-K-ATPase activity and expression. A: incubation of cells with increasing insulin concentration for 48 h. B: Na-K-ATPase activity in OK cells incubated with 1 nmol/l insulin or DMEM-ouabain alone (basal) for 24 h in the absence and presence of genistein (Gen), HNPA-(AH)₃ (HNM), wortmannin (Wot), GF109203X (GFX), and H89. C: Na-K-ATPase α₁-subunit expression in cells incubated with 1nmol/l insulin for 24 h in the absence and presence of genistein (Gen), HNPA-(AH)₃ (HNM), wortmannin (Wot), GF109203X (GFX), and H89. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 7.

Effect of chronic insulin treatment on Na-K-ATPase α₁-subunit serine phosphorylation. Cell were incubated with 1 nmol/l insulin or DMEM-ouabain alone (basal) for 24 h and tyrosine (Tyr-P) and serine (Ser-P) phosphorylation was determined in membrane α₁-subunits (A and B). α₁-Subunit serine phosphorylation (Ser-P) in cells transfected with rat α₁-subunit wild-type (WT), NH₂-terminal truncated (-NT), serine 16 and 23 mutated to alanine (S16A/S23A), and tyrosine 10 mutated to alanine (Y10A) and incubated with 1 nmol/l insulin or DMEM-ouabain alone (basal) for 24 h (C). Insulin (24 h treatment)-induced membrane α₁-subunit serine phosphorylation (Ser-P) in the absence (basal) and presence of genistein (Gen), HNPA-(AH)₃ (HNM), wortmannin (Wot), GF109203X (GFX), and H89. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 8.

Effect of α₁-subunit tyrosine and serine mutations on insulin-mediated Na-K-ATPase activity and expression. OK cells transfected with rat α1-subunit wild-type (WT), NH₂-terminal truncated (-NT), tyrosine 10 mutated to alanine (Y10A) or glycine (Y10E), and serine 16 and 23 mutated to alanine (S16A/S23A) were incubated with vehicle 1 nM insulin or DMEM-ouabain alone (basal) for 24 h. Insulin-mediated inhibition of Na-K-ATPase activity (A) and insulin-induced decrease in Na-K-ATPase α₁-subunit expression (B). Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 9.

Insulin-mediated protein kinase C (PKC) activation in OK cells. Cells were incubated with DMEM-ouabain alone (basal) or 1 nmol/l insulin (Ins) for 24 h in the absence and presence of genistein (Gen), HNPA-(AH)₃ (HNM), wortmannin (Wot), GF109203X (GFX), and H89. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 10.

Insulin-induced increase in PKC expression in OK cells. Cells were incubated with vehicle (Veh) or 1 nM insulin for 24 h and PKC isoforms α, β₁, and γ (A); δ, θ, and ε (B); and ζ and λ (C) immunoblotted in cell homogenates. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. basal.

Fig. 11.

Effect of PKC inhibitors on insulin-induced Na-K-ATPase activity, expression and phosphorylation. A: Na-K-ATPase activity in cells incubated with vehicle (Veh) or 1 nM insulin for 24 h without and with inhibitors (Inh) of PKCα, -β, and -ζλ. B: Na-K-ATPase α₁-subunit expression in cells incubated with insulin and PKCζλ inhibitor. C: Na-K-ATPase α₁-subunit serine phosphorylation (Ser-P) in cells incubated with insulin and PKCζλ inhibitor. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. Veh and #P < 0.05 vs. Ins + ζλ-Inh.

Fig. 12.

Insulin-induced membrane adaptor protein (AP)-2 and α₁-subunit colocalization. A: cells were incubated with vehicle (Veh) and insulin (Ins) for 24 h and immunoblotted for AP2 protein expression in whole cell lysate. B: cell membranes were immunoprecipitated with α₁-antibodies and immunoblotted for AP2 protein. Bars represent mean ± SE from 5–6 experiments performed in triplicate. *P < 0.05 vs. Basal and #P < 0.05 vs. Insulin.