Differential effects of nitric oxide-mediated S-nitrosylation on p50 and c-jun DNA binding.

Abstract

Nitric oxide (NO) regulates a variety of cellular functions. One mechanism by which NO may exert its influence is through formation of S-nitrosothiols at critical thiol residues in protein-active sites, including those of nuclear protein transcription factors. NF-kappa B p50 and AP-1 c-jun were S-nitrosylated in the presence of acidic NaNO2. Wild-type protein and protein subjected to nitrosylating conditions in the absence of NaNO2 served as controls. Confirmatory evidence for S-nitrosothiol bond formation was obtained by ultraviolet-visible spectrophotometry with the absorption maximum for S-NO bonds at approximately 320 to 360 nm. With consensus oligonucleotide probes, gel-shift analysis was used to examine DNA binding characteristics. In the case of NF-kappa B p50, S-nitrosylation resulted in significantly decreased DNA binding. In contrast, S-nitrosylation did not alter c-jun DNA binding. The S-nitrosylating conditions themselves did not alter p50 or c-jun DNA binding. Quantitative analysis was performed according to the Scatchard plot technique to generate the respective dissociation constants. S-nitrosylated p50 was associated with a fourfold greater dissociation constant than that of the wild-type p50. S-nitrosylation of transcription factors may be one mechanism by which NO may selectively regulate gene transcription.