PMC

Stimulation of Na⁺,K⁺-ATPase activity by insulin is linearly correlated and cosaturates with the tyrosine phosphorylation level of its α-subunit. The ouabain-sensitive ⁸⁶Rb uptake was plotted as a function of the fractional tyrosine phosphorylation of the α-subunit of Na⁺,K⁺-ATPase. Values were from the experiments depicted in Figure .

Phosphoamino acid analysis of the Na⁺, K⁺-ATPase α-subunit. Kidney proximal tubules were labeled with 1 mCi/ml [³²P]orthophosphoric acid for 2 h at 30°C in the absence (A) or presence of 10⁻³ M orthovanadate (B). ³²P-Labeled Na⁺, K⁺-ATPase was immunoprecipitated with anti-NK1 antibody, and the α-subunit was subjected to phosphoamino acid analysis. The dashed circles represent the positions of cold phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr). This experiment was performed twice with the same results.

Estimation of the percentage of Na⁺, K⁺-ATPase α-subunits immunoprecipitated with antiphosphotyrosine antibodies under control or stimulated conditions. Proximal tubules were preincubated for 30 min at 37°C without or with stimulators. Extracts were prepared and aliquots were subjected to immunoblotting with the McK1 antibody before (b) or after (a) immunoprecipitation with the PY20 antibody. (A) Tyrosine phosphorylation of the α-subunit from control or insulin-treated (10⁻⁸ M) tubules. Upper panels, immunoblots. Lower panel, quantification of data shown in upper panels and expressed as percent of the total α-subunit population immunoprecipitated with the PY20 antibody. Results are means ± SEM from four independent experiments. (B) Tyrosine phosphorylation of the α-subunit from control or orthovanadate-treated (10⁻³ M) tubules. Upper panels, immunoblots. Lower panel, quantification of data shown in upper panels and expressed as percent of α-subunits of the total α-subunit population immunoprecipitated with the PY20 antibody (*, p < 0.05 vs. control).

Substitution of Tyr-10 by either Ala (Y10A) or Glu (Y10E) abolishes the stimulation of Na⁺,K⁺-ATPase by insulin in transfected OK cells. ⁸⁶Rb (K) uptake was measured in the presence of 5 × 10⁻⁶ M ouabain under initial rates of influx in OK cells expressing the WT rat α1-subunit, its tyrosine phosphorylation site mutants (Y10A and Y10E). Cells were preincubated for 30 min in the absence (C) or presence of 10⁻⁸ M insulin (I). Results are expressed as a percentage of control and are means ± SEM from seven independent experiments (*, p < 0.05 vs. control). In each cell line studied, the ouabain-insensitive ⁸⁶Rb (K) uptake was not altered by insulin.

Substitution of Tyr-10 by either Ala (Y10A) or Glu (Y10E) does not alter the stimulation of Na⁺,K⁺-ATPase by PMA in transfected OK cells. ⁸⁶Rb (K) uptake was measured in the presence of 5 × 10⁻⁶ M ouabain under initial rates of influx in OK cells expressing the WT rat α1-subunit and its tyrosine phosphorylation site mutants (Y10A and Y10E). Cells were preincubated for 30 min in the absence (C) or presence of 10⁻⁶ M PMA (P). Results are expressed as a percentage of control and are means ± SEM from six independent experiments (* p < 0.05 vs. control). In each cell line studied, the ouabain-insensitive ⁸⁶Rb (K) uptake was not altered by PMA.

The effect of insulin is prevented by genistein and is not additive to the effect of orthovanadate. (A) Kidney proximal tubules were incubated for 30 min at 37°C in the absence (C) or presence of 10⁻⁸ M insulin (I) and/or 10⁻⁴ M genistein (G). Upper panel, immunoblot of proximal tubule extracts with McK1 antibody showing the effects of insulin and genistein on the amount of Na⁺, K⁺-ATPase α-subunit immunoprecipitated by the PY20 antibody. Lower panel, densitometric quantification of data shown in the upper panel. Results are expressed as percent of control optical density and are means ± SEM from seven independent experiments. (B) Proximal tubules were incubated for 30 min at 37°C in the absence (C) or presence of 10⁻⁸ M insulin (I) and/or 10⁻³ M orthovanadate (V). Upper panel, immunoblot of proximal tubule extracts with McK1 antibody showing the effects of insulin and orthovanadate on the amount of Na⁺, K⁺-ATPase α-subunit immunoprecipitated with the PY20 antibody. Lower panel, densitometric quantification of data shown in the upper panel. Results are expressed as percent of control optical density and are means ± SEM from six independent experiments (*, p < 0.05 vs. control).

Orthovanadate increases the phosphorylation level of the Na⁺, K⁺-ATPase α-subunit and its phosphotyrosine immunoreactivity in rat kidney proximal tubules. Kidney proximal tubules were incubated for 30 min at 37°C in the absence (C) or presence of 10⁻³ M orthovanadate (V). (A) Immunoblots of tubular extracts with the McK1 antibody before (left panel) and after (right panel) immunoprecipitation with the PY20 antibody. (B) Densitometric quantification of data shown in A (right panel). Results are expressed as percent of the control optical density and are means ± SEM from six independent experiments. (C) Lysates from ³²P-labeled proximal tubules were prepared, and immunoprecipitation was performed with the anti-NK1 antibody. Samples were subjected to immunoblotting with the McK1 antibody (left panel) or directly to SDS-PAGE and autoradiography (right panel). (D) Densitometric quantification of data shown in C (right panel). Results are expressed as percent of the control optical density and are means ± SEM from six independent experiments. (E) Immunoblots of tubular extracts with McK1 antibody (left panel) and 4G10 antibody (right panel) after immunoprecipitation with the anti-NK1 antibody. (F) Densitometric quantification of data shown in E (right panel). Results are expressed as percent of the control optical density and are means ± SEM from four independent experiments (**, p < 0.01; *, p < 0.05 vs. control).

Functional expression of WT or mutant rat α1-subunits in stably transfected OK cells. ⁸⁶Rb (K) uptake was measured under initial rate in OK cells preincubated in the absence or presence of increasing concentrations of ouabain. The Na⁺,K⁺-ATPase-mediated ⁸⁶Rb uptake was obtained by subtraction of ouabain-insensitive ⁸⁶Rb (K) uptake measured in the presence of 5 × 10⁻³ M ouabain. Results from three or four independent experiments are expressed as percent of control (absence of ouabain) ± SEM. OK cells expressing the WT (WT) rat α1-subunit or its tyrosine phosphorylation site mutants (Y10A and Y10E) exhibited a bimodal ouabain inhibition pattern. The IC₅₀ values of Na⁺,K⁺-ATPase with high ouabain sensitivity were WT, 2.2 ± 0.9 × 10⁻⁸ M; Y10A, 3.8 ± 1.5 × 10⁻⁸ M; and Y10E, 3.8 ± 2.5 × 10⁻⁸ M. The IC₅₀ values of Na⁺,K⁺-ATPase with low ouabain sensitivity were WT, 4.3 ± 0.9 × 10⁻⁵ M; Y10A, 8.7 ± 2.7 × 10⁻⁵ M; and Y10E, 3.0 ± 1.6 × 10⁻⁵ M. The percentages of ⁸⁶Rb (K) uptake mediated by the ouabain-resistant Na⁺,K⁺-ATPase were WT, 57.1 ± 2.6%; Y10A, 47.6 ± 3.0%; and Y10E, 45.8 ± 5.7%.

Insulin increases the phosphorylation level of the Na⁺, K⁺-ATPase α-subunit and its phosphotyrosine immunoreactivity in rat kidney proximal tubules. Kidney proximal tubules were incubated for 30 min at 37°C in the absence (C) or presence of 10⁻⁸ M insulin (I). (A) Immunoblots of tubular extracts with the McK1 antibody before (left panel) and after (right panel) immunoprecipitation with the PY20 antibody. (B) Densitometric quantification of data shown in A (right panel). Results are expressed as percent of the control optical density and are means ± SEM from 12 independent experiments. (C) Lysates from ³²P-labeled proximal tubules were prepared, and immunoprecipitation was performed with the anti-NK1 antibody. Samples were subjected to immunoblotting with the McK1 antibody (left panel) or directly to SDS-PAGE and autoradiography (right panel). (D) Densitometric quantification of data shown in C (right panel). Results are expressed as percent of the control optical density and are means ± SEM from six independent experiments. (E) Immunoblots of tubular extracts with McK1 antibody (left panel) and 4G10 antibody (right panel) after immunoprecipitation with the anti-NK1 antibody. (F) Densitometric quantification of data shown in E (right panel). Results are expressed as percent of the control optical density and are means ± SEM from four independent experiments (**, p < 0.01; *, p < 0.05 vs. control).

The time course and the dose dependence of insulin-induced tyrosine phosphorylation of the Na⁺,K⁺-ATPase α-subunit and stimulation of the ouabain-sensitive ⁸⁶Rb uptake are similar in rat PCT. (A) The tyrosine phosphorylation of the α-subunit of Na⁺,K⁺-ATPase from kidney proximal tubules (closed squares) and ouabain-sensitive ⁸⁶Rb uptake by isolated PCTs (open circles) was determined after 30 min preincubation at 37°C. Prewarmed insulin solution (10⁻⁸ M final) was added or not for various times (5–30 min). Upper panel, typical immunoblot with McK1 antibody showing the amount of α-subunit immunoprecipitated with the PY20 antibody. Lower panel, averaged results expressed as fractional tyrosine phosphorylation of the α-subunit (assuming that 10% of α-subunits are phosphorylated under baseline) and as pmol Rb × mm⁻¹ × min⁻¹ were plotted on the same graph and are means ± SEM from five or six independent experiments. (B) The tyrosine phosphorylation of the α-subunit of Na⁺,K⁺-ATPase from proximal tubules (closed squares) and ouabain-sensitive ⁸⁶Rb uptake by isolated PCTs (open circles) was determined after preincubation for 30 min in the absence (C) or presence of various concentrations of insulin. Upper panel, typical immunoblot with McK1 antibody showing the amount of α-subunit immunoprecipitated with the PY20 antibody. Lower panel, averaged results expressed as in A were plotted on the same graph and are means ± SEM from five or six independent experiments.

The Na⁺, K⁺-ATPase α-subunit is phosphorylated at tyrosine 10. (A) Proximal tubules were preincubated for 30 min at 37°C with 10⁻⁸ M insulin, and 200 μg protein were incubated for 5 min at 4°C without (C) or with trypsin (T) at a trypsin:protein ratio (wt/wt) of 0.005–0.01. Immunoblots of total protein with McK1 antibody (Anti NH₂-α; left panel) and with an antibody raised against the extreme COOH-terminal ETYY motif (Anti COOH-α; middle panel) show that tryptic cleavage of the α-subunit has occurred exclusively at the extreme NH₂ terminus. This limited trypsinolysis removed the tyrosine phosphorylation site recognized by PY20 antibody (right panel). Results are representative of four independent experiments. (B) OK cells were stably transfected with the WT rat α1-subunit (WT) or its Y10A or Y10E mutants. Immunoblot of total protein (left panel) shows that transfected cell lines express similar amounts of α-subunit, but substitution of Tyr-10 by Ala or Glu greatly reduces the amount of α-subunit immunoprecipitated by PY20 antibody (right panel).