S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF 4 NO), a NO؉ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Snitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function. Nitric oxide (NO)1 is an important biological messenger that plays a role in physiological and pathophysiological conditions such as endothelium-dependent vasorelaxation, inflammation, and septic shock (1, 2). These multiple effects are based on its redox chemistry. NO can react with oxygen species and transition metals to form NO x , peroxynitrite (ONOO Ϫ ), and metal-NO adducts, respectively (3, 4). The interactions of NO with sulfhydryl-containing molecules and enzymes has gained considerable importance (5, 6). In many biological systems, nitrosation reactions transferring NO ϩ from a NO donor to a protein S Ϫ group affect protein function. Targets for this type of modification, among others, are bovine serum albumin (7), tissuetype plasminogen activator (8), gyceraldehyde-3-phosphate dehydrogenase (GAPDH) (9, 10), the N-methyl-D-aspartate receptor (11), oncogenic p21 ras (12), and transcriptional activators (13).The S-nitrosothiol of glutathione (GSNO) may be the most relevant biological molecule to carry out nitrosation reactions under physiological conditions (14 -16). It has been reestablished that the actions of the endothelium-derived relaxing factor more closely resemble a low molecular weight nitrosothiol rather than the NO ⅐ radical itself (17). However, S-nitrosothiols can decompose to form NO ⅐ and thiyl radicals (18), and the thiyl radical can lead to the production of protein-mixed disulfides also known as protein S-glutathionylation.In this study, we investigated the influence of NO donors on the glycolytic enzyme GAPDH which catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate by NAD ϩ and inorganic phosphate. GAPDH is comprised of four identical 37-kDa subunits. Each subunit contains four cysteines; two of them (Cys-149 and Cys-153) are located in the catalytic site of each GAPDH subunit. The catalytically active cysteine 149 interacts with a histidine to form a highly reactive thiolate group (cys-S Ϫ ) which is required for GAPDH...

Rap1 proteins belong to the Ras superfamily of small molecular weight GTP-binding proteins. Although Rap1 and Ras share approximately 50% overall amino acid sequence identity, the effector domains of the two proteins are identical, suggesting either similar or antagonistic signaling roles. Several pathways leading to Ras activation have been defined, including those initiated by agonist binding to tyrosine kinase or Gi-coupled receptors. Nothing is known about such events for Rap1 proteins. The cAMP-mediated inhibition of Ras-dependent MAP kinase activation is well documented and resembles that caused by expression of GTPase-deficient Rap1. We have developed a system whereby signals leading to Rap1b activation, i.e. an increase in Rap1b-bound GTP/GDP ratio, can be measured. We report here that treatment of cells with agents that elevate intracellular cAMP levels result in Rap1b activation. These results demonstrate for the first time agonist-dependent activation of Rap1 proteins.

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