Effect of protein S-nitrosylation on cAMP response element-binding protein activity in RSC-364 cells

AIM: To investigate the effect of protein S-nitrosylation on cAMP response element-binding protein(CREB) activity in RSC-364 cells. METHODS: ① RSC-364 cells cytoplasmic extracts were incubated with 100 μmol/L GSH (control chemical), 100 μmol/L GSNO (a donor of NO) in the presence or absence of 10 mmol/L DTT (inhibitor of S-nitrosylation) for 15 min at room temperature. The S-nitrosylated proteins were determined by biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. ② RSC-364 cell nuclear extracts were subjected to S-nitrosylation and electrophoretic mobility shift assay (EMSA) to analyze the CREB DNA binding activity after 1 h stimulation of rIL-1β (10 μg/L). RESULTS: GSNO obviously increased the expression of nitrosylated proteins and inhibited the CREB activity, which was reversed by DTT. CONCLUSION: S-nitrosylation may inhibit the CREB activity in RSC-364 cells.