Phosphorylation of Akt at Thr308 regulates p-eNOS Ser1177 during physiological conditions

Abstract

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays a crucial role in maintaining vascular homeostasis. As a hallmark of eNOS activation, phosphorylation of eNOS at Ser1177 induced by activated protein kinase B (PKB/Akt) is pivotal for NO production. The complete activation of Akt requires its phosphorylation of both Thr308 and Ser473. However, which site plays the main role in regulating phosphorylation of eNOS Ser1177 is still controversial. The purpose of the present study is to explore the specific regulatory mechanism of phosphorylated Akt in eNOS activation. Inhibition of Akt Thr308 phosphorylation by a specific inhibitor or by siRNA in vitro led to a decrease in eNOS phosphorylation at Ser1177 and to lower NO concentration in the cell culture medium of HUVECs. However, inhibiting p-Akt Ser473 had no effect on eNOS phosphorylation at Ser1177. Next, we administered mice with inhibitors to downregulate p-Akt Ser473 or Thr308 activity. Along with the inhibition of p-Akt Thr308, vascular p-eNOS Ser1177 protein was simultaneously downregulated in parallel with a decrease in plasma NO concentration. Additionally, we cultured HUVECs at various temperature conditions (37, 22, and 4 °C). The results showed that p-Akt Ser473 was gradually decreased in line with the reduction in temperature, accompanied by increased levels of p-Akt Thr308 and p-eNOS Ser1177. Taken together, our study indicates that the phosphorylation of Akt at Thr308, but not at Ser473, plays a more significant role in regulating p-eNOS Ser1177 levels under physiological conditions.

Keywords: Akt; endothelial nitric oxide synthase; nitric oxide; phosphorylation.

Fig. 1

Inhibiting phosphorylation of Akt at Thr308 accompanied by decreased p‐eNOS Ser1177 expression. HUVECs were treated with GSK2334470 at different concentrations (0.01, 0.1, 0.5, 1 μm) or for different durations (2, 5, 10 min) to explore the effects of GSK2334470 on p‐eNOS Ser1177 expression. (A) Western blot and quantitative analysis of the protein expressions of p‐Akt Thr308, p‐Akt Ser473, and total‐Akt in HUVECs treated with different concentrations of GSK2334470 for 30 min. (B) Western blot and quantitative analysis of the protein expressions of p‐Akt Thr308, p‐Akt Ser473, and total‐Akt in HUVECs treated with GSK2334470 (1 μm) for different durations. (C) Western blot and quantitative analysis of the protein expressions of p‐eNOS Ser1177, p‐eNOS Thr495, and total‐eNOS in HUVECs treated with GSK2334470 (1 μm) for different durations. The relative levels of p‐Akt Thr308, p‐Akt Ser473, p‐eNOS Ser1177, and p‐eNOS Thr495 were quantified as the ratios of p‐Akt Thr308/Akt, p‐Akt Ser473/Akt, p‐eNOS Ser1177/eNOS, and p‐eNOS Thr495/eNOS, respectively. Data were expressed as mean ± SE, n = 4. Data were analyzed with one‐way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 vs. Control group; ***P < 0.001 vs. Control group.

Fig. 2

p‐eNOS Ser1177 was independent of phosphorylation of Akt at Ser473. HUVECs were treated with PP242 at different concentrations (25, 50, 75, 100, and 200 nm) or for different durations (5, 10, and 15 min) to explore the effects of p‐Akt Ser473 on p‐eNOS Ser1177 expression. (A) Western blot and quantitative analysis of the protein expressions of p‐Akt Thr308, p‐Akt Ser473, and total‐Akt in HUVECs treated with different concentrations of PP242 for 30 min. (B) Western blot and quantitative analysis of the protein expressions of p‐Akt Thr308, p‐Akt Ser473, and total‐Akt in HUVECs treated with PP242 (100 nm) for different durations. (C) Western blot and quantitative analysis of the protein expressions of p‐eNOS Ser1177, p‐eNOS Thr495, and total‐eNOS in HUVECs treated with GSK2334470 (100 nm) for different durations. The relative levels of p‐Akt Thr308, p‐Akt Ser473, p‐eNOS Ser1177, and p‐eNOS Thr495 were quantified as the ratios of p‐Akt Thr308/Akt, p‐Akt Ser473/Akt, p‐eNOS Ser1177/eNOS, and p‐eNOS Thr495/eNOS, respectively. Data were expressed as mean ± SE, n = 4. Data were analyzed with one‐way ANOVA followed by Tukey's multiple comparisons test. **P < 0.01 vs. Control group; ***P < 0.001 vs. Control group.

Fig. 3

Inhibiting phosphorylation of Akt at Thr308 decreased NO production in HUVECs. HUVECs were treated with GSK2334470 (1 μm) or PP242 (100 nm) for 30 min, respectively. The NO concentration was measured. Data were expressed as mean ± SE, n = 4. Data were analyzed with two‐tailed unpaired Student’s t‐test. **P < 0.01 vs. Control group.

Fig. 4

Knockdown of PDK1 induced the downregulation of p‐eNOS Ser1177. HUVECs were transfected with siRNA targeting PDK1 or SIN1 and NC scrambled siRNA (100 nm) for 48 h, the transfection efficiency as well as the total and phosphorylated levels of Akt and eNOS were detected by western blot. (A) Western blot and quantitative analysis of the protein expressions of PDK1 and SIN1 in HUVECs transfected with different siRNA oligos. (B) Western blot and quantitative analysis of the protein expressions of p‐Akt Thr308, p‐Akt Ser473, and total‐Akt in HUVECs transfected with different siRNA oligos. (C) Western blot and quantitative analysis of the protein expressions of p‐eNOS Ser1177, p‐eNOS Thr495, and total‐eNOS in HUVECs transfected with different siRNA oligos. The relative levels of p‐Akt Thr308, p‐Akt Ser473, p‐eNOS Ser1177, and p‐eNOS Thr495 were quantified as the ratios of p‐Akt Thr308/Akt, p‐Akt Ser473/Akt, p‐eNOS Ser1177/eNOS, and p‐eNOS Thr495/eNOS, respectively. Data were expressed as mean ± SE, n = 4. Data were analyzed with one‐way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 vs. siRNA‐NC group; **P < 0.01 vs. siRNA‐NC group; ***P < 0.001 vs. siRNA‐NC group.

Fig. 5

Inhibition of p‐Akt Thr308 decreased p‐eNOS Ser1177 expression and NO production in mice. Mice were administrated by a single intraperitoneal injection of GSK2334470 (40 mg·kg⁻¹) or PP242 (5 mg·kg⁻¹) for 6 h, respectively. The mesenteric artery of mice was harvested at the indicated time points. (A) Western blot and quantitative analysis of the protein expressions of p‐eNOS Ser1177, p‐Akt Ser473, p‐Akt Thr308, total‐eNOS, and total‐Akt in the mesenteric artery of mice treated with GSK2334470. (B) Western blot and quantitative analysis of the protein expressions of p‐eNOS Ser1177, p‐Akt Ser473, p‐Akt Thr308, total‐eNOS, and total‐Akt in the mesenteric artery of mice treated with PP242. (C) Quantitative detection of plasma NO concentration of mice treated with GSK2334470 or PP242. The relative levels of p‐Akt Thr308, p‐Akt Ser473, and p‐eNOS Ser1177 were quantified as the ratios of p‐Akt Thr308/Akt, p‐Akt Ser473/Akt, and p‐eNOS Ser1177/eNOS, respectively. Data were expressed as mean ± SE, n = 6. Data were analyzed with two‐tailed unpaired Student’s t‐test. *P < 0.05 vs. Control group; **P < 0.01 vs. Control group; ***P < 0.001 vs. Control group.

Fig. 6

p‐Akt Thr308 regulated p‐eNOS Ser1177 expression in HUVECs exposed to different temperature. HUVECs were exposed to different temperatures (37, 22, and 4 °C) for 1 h; then the total and phosphorylated levels of Akt and eNOS were detected by western blot. (A) Western blot of the protein expressions of p‐Akt Ser473, p‐Akt Thr308, total‐Akt, p‐eNOS Ser1177, and total‐eNOS in HUVECs. (B) Quantitative analysis of the protein expressions of p‐Akt Ser473, p‐Akt Thr308, total‐Akt, p‐eNOS Ser1177, and total‐eNOS in HUVECs. The relative levels of p‐Akt Ser473, p‐Akt Thr308, p‐eNOS Ser1177 were quantified as the ratios of p‐Akt Ser473/Akt, p‐Akt Thr308/Akt, and p‐eNOS Ser1177/eNOS, respectively. Data were expressed as mean ± SE, n = 3. Data were analyzed with one‐way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 vs. 37 °C; **P < 0.01 vs. 37 °C; ***P < 0.001 vs. 37 °C.