Effects of ERK1/2 S-nitrosylation on ERK1/2 phosphorylation and cell survival in glioma cells

Abstract

Aberrant activation of extracellular signal‑regulated kinase 1/2 (ERK1/2) by phosphorylation modification can trigger tumor cell development in glioma. S‑nitrosylation, which refers to the covalent addition of a nitric oxide (NO) group to a cysteine (Cys) thiol, is an important post‑translational modification that occurs on numerous cancer‑associated proteins. Protein S‑nitrosylation can increase or decrease protein activity and stability, and subsequent signal transduction and cellular processes. However, the association between ERK1/2 S‑nitrosylation and ERK1/2 phosphorylation, and the effects of ERK1 S‑nitrosylation on glioma cell survival are currently unknown. U251 glioma cells were treated with NO donors sodium nitroprusside (SNP) or S‑nitrosoglutathione (GSNO). CCK8 assay was used to assess the cell viability. NO levels in the medium were detected by Griess assay. Western blot analysis and biotin switch assay were employed to detect the ERK1/2 phosphorylation and S-nitrosylation. ERK1 wild-type and mutant plasmids were constructed, and used to transfect the U251 cells. Caspase-3 western blot analysis and flow cytometry were employed to assess cell apoptosis. The present study demonstrated that treatment with the NO donors SNP or GSNO led to an increase in ERK1/2 S‑nitrosylation, and a reduction in ERK1/2 phosphorylation, which was accompanied by growth inhibition of U251 glioma cells. Mutational analysis demonstrated that Cys183 was vital for S‑nitrosylation of ERK1, and that preventing ERK1 S‑nitrosylation by replacing Cys183 with alanine partially reversed GSNO‑induced cell apoptosis, and reductions in cell viability and ERK1/2 phosphorylation. In addition, increased ERK1/2 phosphorylation was associated with decreased ERK1/2 S‑nitrosylation in human glioma tissues. These findings identified the relationship between ERK1/2 S‑nitrosylation and phosphorylation in vitro and in vivo, and revealed a novel mechanism of ERK1/2 underlying tumor cell development and apoptotic resistance of glioma.

Figure 1

NO donor treatment inhibits the growth of U251 glioma cells. (A and B) Results of a CCK-8 assay indicated a concentration-dependent decrease in the viability of U251 glioma cells following exposure to 0–2 mM SNP or 0–500 µM GSNO for 36 h. (C and D) CCK-8 assay exhibited a reduction in cell viability at different time-points following SNP (2 mM) or GSNO (500 µM) treatment. Relative absorbance was normalized to the untreated group (0). ^*P<0.05 and ^***P<0.001 compared with the untreated group (0). CCK-8, Cell Counting kit-8; GSNO, S-nitrosoglutathione; NO, nitric oxide; SNP, sodium nitroprusside.

Figure 2

NO donors SNP and GSNO can release NO into the medium of cultured glioma cells. The Griess method was used to quantify NO levels in the culture supernatant of U251 cells following (A) 1 and 2 mM SNP or (B) 250 and 500 µM GSNO treatment. ^**P<0.01 and ^***P<0.001 compared with the untreated group (0). GSNO, S-nitrosoglutathione; NO, nitric oxide; SNP, sodium nitroprusside.

Figure 3

NO donor treatment attenuates ERK1/2 phosphorylation in U251 glioma cells. (A and B) Alterations in p-ERK1/2 expression at the indicated time-points following SNP (2 mM) or GSNO (500 µM) treatment were detected by western blotting and were semi-quantified. (C and D) Alterations in p-ERK1/2 expression following treatment with the indicated concentrations of SNP or GSNO were examined by western blotting and were semi-quantified. p-ERK1/2 expression was normalized to total ERK1/2. ^*P<0.05 and ^**P<0.01 compared with the control group (0). ERK1/2, extracellular signal-regulated kinase 1/2 GSNO, S-nitrosoglutathione; NO, nitric oxide; p-ERK1/2, phosphorylated-ERK1/2; SNP, sodium nitroprusside.

Figure 4

NO donor treatment promotes S-nitrosylation of ERK1/2 in U251 glioma cells. (A) Cells were treated with 500 µM GSNO for the indicated time and ERK1/2-SNO was detected by biotin switch assay followed by western blotting. (B) Cells were treated with 250 and 500 µM GSNO for 24 h and the level of ERK1/2-SNO was detected as aforementioned. Total ERK1/2 was used as an endogenous control. ERK1/2, extracellular signal-regulated kinase1/2; GSNO, S-nitrosoglutathione; NO, nitric oxide; SNO, S-nitrosothiol.

Figure 5

Point mutation at Cys¹⁸³ partially prevents S-nitrosylation of ERK1 in glioma cells. (A) Location of Cys and the adjacent amino acids in the ERK1 protein sequence. The red 'C' in the middle of each box is cysteine residue. (B) Transfection efficiency and S-nitrosylation of ERK1^WT and ERK1^C183A were determined by biotin switch assay, followed by western blotting. (C) Semi-quantitative analysis of ERK1-SNO levels. β-actin was used as a loading control. ^*P<0.05 compared with the ERK1^WT group. Cys, cystein; ERK, extracellular signal-regulated kinase; SNO, S-nitrosothiol; IB, immunoblotting; WT, wild-type.

Figure 6

Preventing S-nitrosylation of ERK1 promotes ERK phosphorylation and cell survival, and suppresses apoptosis. Following transfection of U251 glioma cells with ERK1-Flag or ERK1 mutant form (ERK^C183A), cells were treated with 500 µM GSNO for 24 h. (A and B) p-ERK1/2 was detected by western blotting and was semi-quantified. p-ERK1/2 levels were compared with β-actin levels, and results were normalized to vector group. (C) Cell Counting kit-8 assay was performed to examine the viability of U251 glioma cells. Cell survival percentage was normalized to the vector group. (D and E) Caspase-3 protein expression was detected by western blotting and semi-quantified. Cleaved caspase-3 levels were compared with β-actin levels, and results were normalized to the vector group. The lower panel of cleaved caspase-3 blot in part D was obtained after a longer exposure time compared with the upper panel. (F) Flow cytometric detection of apoptosis of U251 glioma cells. The percentage of apoptotic cells was quantified and compared. ^*P<0.05, ^**P<0.01 and ^***P<0.001. IB, immunoblotting; ERK, extracellular signal-regulated kinase; fITC, fluorescein isothiocyanate; GSNO, S-nitrosoglutathione; p-ERK1/2, phosphorylated ERK1/2; PI, propidium iodide; WT, wild-type.

Figure 7

Alterations in the levels of ERK1/2 phosphorylation and S-nitrosylation in noncancerous and glioma tissues. In noncancerous brain samples (n=9) and various grades of glioma (n=11 for each grade), western blotting was used to detect the expression levels of p-ERK1/2 and total ERK1/2 levels, and biotin switch assay followed by western blotting was employed to detect ERK1/2-SNO. (A) Representative blot images are presented. Semi-quantification for the ratio of (B) p-ERK1/2/total ERK1/2 and (C) ERK1/2-SNO/total ERK1/2. ^*P<0.05 compared with the noncancerous group. ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated-ERK1/2; SNO, S-nitrosothiol.