Hydrogen sulfide (H 2 S), a messenger molecule generated by cystathionine γ-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H 2 S signals have been elusive. We now show that H 2 S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.

The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1͞FRAP͞mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. Here, we show that RAFT1 directly phosphorylates p70 S6k , 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70 S6k and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70 S6k on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70 S6k much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70 S6k is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1.Increases in the translation of certain mRNAs are an important response to mitogen stimulation (1-3), but little is known about the signaling pathways that link growth stimuli to the activation of the protein synthesis machinery. Studies with rapamycin (4), a potent immunosuppressant, have uncovered a signaling pathway that modulates protein synthesis in yeast (5) and animal cells (6, 7). In vivo treatment with rapamycin affects the phosphorylation of several regulators of translation, including the ribosomal S6 protein and its specific kinase, p70 S6k (8, 9); the eIF-4E binding proteins, 4E-BP1 (6, 7) and 4E-BP2 (10); and elongation factor 2 (11).Ribosomal S6 and 4E-BP1 regulate the initiation of translation of distinct classes of mRNAs. Phosphorylation by p70 S6k of the S6 protein of the small ribosomal subunit permits, through unknown mechanisms, efficient translation of mRNAs containing oligopyrimidine tracts in their 5Ј untranslated regions (12, 13). Phosphorylation of 4E-BP1 controls capdependent translation of mRNAs with extensive secondary structure (3). Initiation factor 4F complexes with these mRNAs through the interaction of its eIF-4G subunit with eIF-4E, the cap-binding protein that recognizes the N 7 -methyl-GpppN structure of the 5Ј end of all nonorganellar mRNAs. In quiescent cells, 4E-BP1 competes with eIF-4G for binding to eIF-4E and represses translation by displacing the initiation factor 4F from the mRNA. Growth stimuli activate phosphorylation of 4E-BP1, which decreases its affinity for eIF-4E and releases the block on cap-dependent translation (3). By preventing the phosphorylation of specific residues on p70 S6k and 4E-BP1, rapamycin inhib...

Rationale Nitric oxide, the classic endothelial derived relaxing factor (EDRF), acts via cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelial derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H2S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. Objective The purpose of this study is to determine if H2S is a major physiologic EDHF. Methods and Results We now show that H2S is a major EDHF, as in blood vessels of CSE deleted mice hyperpolarization is virtually abolished. H2S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H2S-elicited hyperpolarization. The endothelial intermediate conductance (IKCa) and small conductance (SKCa) potassium channels mediate in part the effects of H2S, as selective IKCa and SKCa channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide insensitive H2S induced vasorelaxation. Conclusions H2S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. As EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H2S biosynthesis offer therapeutic potential.