It is well accepted that extracellular ligands trigger nuclear signals through a cascade of protein-protein interactions. Many of these pathways have been carefully defined and provide an important framework by which we can understand and intervene in the processes they initiate. Recent data in the literature indicate that many extracellular ligands generate and/or require reactive free radicals or derived species to successfully transmit their signals to the nucleus. Thus, a novel signaling mechanism akin to one solely dependent on protein-protein interactions may exist. Here, we review this information, identify both the sources and targets of free radicals generated by various growth factors and cytokines, discuss how specificity can be achieved, and explore the pathophysiological implications.

Advanced glycation end products (AGEs) exert their cellular effects on cells by interacting with specific cellular receptors, the best characterized of which is the receptor for AGE (RAGE). The transductional processes by which RAGE ligation transmits signals to the nuclei of cells is unknown and was investigated. AGE-albumin, a prototypic ligand, activated p21 ras in rat pulmonary artery smooth muscle cells that express RAGE, whereas nonglycated albumin was without effect. MAP kinase activity was enhanced at concentrations of AGE-albumin, which activated p21 ras and NF-B. Depletion of intracellular glutathione rendered cells more sensitive to AGE-mediated activation of this signaling pathway. In contrast, signaling was blocked by preventing p21 ras from associating with the plasma membrane or mutating Cys 118 on p21 ras to Ser. Signaling was receptor-dependent, because it was prevented by blocking access to RAGE with either anti-RAGE IgG or by excess soluble RAGE. These data suggest that RAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving p21 ras and MAP kinase, culminating in transcription factor activation. The molecular mechanism that triggers this pathway likely involves oxidant modification and activation of p21 ras .In the presence of aldoses, proteins become nonenzymatically glycated and oxidized (1-3). This initially reversible glycation is followed by further irreversible rearrangements leading to a class of permanently modified proteins known as advanced glycation end products (AGEs).1 Although glycated proteins are found at low levels in normal individuals during aging, significantly higher levels are found in certain disease states such as diabetes and renal failure (4, 5). We have identified a cellular receptor for AGEs, termed RAGE, which exhibits a wide tissue distribution (6 -9). We have recently demonstrated the enhanced presence of RAGE in vascular smooth muscle of diabetic vasculature (renal arterial vessel) compared with a similar sized vessel from a nondiabetic age-matched control. These areas of enhanced RAGE immunoreactivity colocalize with enhanced immunostaining for AGE-reactive epitopes (10). Our previous data in endothelial cells and in vivo demonstrated that interaction of AGEs with RAGE results in triggering a range of cellular responses, including transcription factor activation and changes in gene expression (11)(12)(13)(14). However, the means by which a signal reflecting AGE engagement of RAGE is transmitted to the nucleus is not known. Given the enhanced expression of AGE and RAGE in diabetic vascular smooth muscle, we focused on elucidating the signaling pathways in smooth muscle cells that are triggered upon ligation of RAGE by AGE-albumin, a prototypical ligand.Recent evidence supports a role for reactive oxygen species in mediating signaling by several receptor systems (15-21). For example, platelet-derived growth factor has recently been shown to stimulate H 2 O 2 production in vascular smooth muscle cells (15). When pro...

The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.

Reactive free radicals have been implicated in mediating signal transduction by a variety of stimuli. We have investigated the role of p21 ras in mediating free radical signaling. Our studies revealed that signaling by oxidative agents which modulate cellular redox status, such as H 2 O 2 , hemin, Hg 2؉ , and nitric oxide was prevented in cells in which p21ras activity was blocked either through expression of a dominant negative mutant or by treating with a farnesyltransferase inhibitor, as assessed by NF-B binding activity. Furthermore, the NF-B response to these oxidative stress stimuli was found to be enhanced when cells from the human T cell line, Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis. We directly assayed p21 ras and mitogen-activated protein kinase activities in Jurkat cells and found both of these signaling molecules to be activated in cells treated with the redox modulating agents. Blocking glutathione synthesis made cells 10-to 100-fold more sensitive to these agents. Finally, using recombinant p21 ras in vitro, we found that redox modulators directly promoted guanine nucleotide exchange on p21 ras . This study suggests that direct activation of p21 ras may be a central mechanism by which a variety of redox stress stimuli transmit their signal to the nucleus.Free radicals have been shown to play important roles in carcinogenesis by directly damaging DNA and acting as tumor promoters (1-4). Free radicals and redox stress are now thought to participate in cellular signaling (5-9), and, thus, additional targets may exist. The transcription factor NF-B has been demonstrated to mediate signaling by reactive oxygen (5) and reactive nitrogen (10). The exact target of these species is unknown, although it has been postulated to be upstream of p21 ras and involve tyrosine phosphorylation (8, 9, 11). We have previously identified G proteins (12), and particularly p21 ras (13), as central targets by which nitric oxide transmits signals. Therefore, we explored whether p21 ras is a more general target for reactive free radicals and senses cellular redox status. EXPERIMENTAL PROCEDURESMaterials-The farnesyltransferase inhibitor, ␣-hydroxyfarnesylphosphonic acid, was obtained from Biomol (Plymouth Meeting, PA), and L-buthionine-(S,R)-sulfoximine, H 2 O 2 , phorbol 12-myristate 13-acetate, hemin, sodium nitroprusside, and HgCl 2 from Sigma. Purified, recombinant p21 ras was generously provided by Dr. Daniel Manor, Dept. of Pharmacology, Cornell University.Cell Culture and Treatment-The rat pheochromocytoma PC12 cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% horse serum. The p21 ras

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Many studies have identified nitric oxide (NO) and related chemical species (NO x ) as having critical roles in neurotransmission, vasoregulation, and cellular signaling. Previous work in this laboratory has focused on elucidating the mechanism of NO x signaling in cells. We have demonstrated that NO x -induced activation of the guanine nucleotide-binding protein p21 ras leads to nuclear translocation of the transcription factor NFB. Here, we investigated whether intermediary signaling elements, namely the mitogen-activated protein (MAP) kinases, are involved in mediating NO x signaling. We found that NO x activates the extracellular signal-regulated kinase (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK) subgroups of MAP kinases in human Jurkat T cells. JNK was found to be 100-fold more sensitive to NO x stimulation than p38 and ERK. In addition, the activation of JNK and p38 by NO x was more rapid than ERK activation. Depletion of intracellular glutathione augmented the NO x -induced increase in kinase activity. Furthermore, endogenous NO, generated from NO synthase, activated ERK, and NO x -induced MAP kinase activation was effectively blocked by the farnesyl transferase inhibitor ␣-hydroxyfarnesylphosphonic acid. These data support the hypothesis that critical signaling kinases, such as ERK, p38, and JNK, are activated by NO-related species and thus participate in NO signal transduction. These findings establish a role for multiple MAP kinase signaling pathways in the cellular response to NO x .Our previous work focused on identifying the signaling cascade responsible for the positive regulatory effects of nitric oxide (NO) 1 and related chemical species (NO x ) on human peripheral blood mononuclear cells (1, 2). NO x was found to activate human lymphocytes, as evidenced by increased glucose uptake, tumor necrosis factor ␣ secretion, and nuclear translocation of the transcription factor NFB. Further studies demonstrated that NO x directly activates p21 ras

We have identified the site of molecular interaction between nitric oxide (NO) and p21 ras responsible for initiation of signal transduction. We found that p21 It is well known that signal transduction pathways initiated by extracellular ligands are dependent on protein-protein interactions for propagation and amplification of their signal. Many of these interactions lead to phosphorylation events. For example, receptor-tyrosine kinases require a series of protein interactions utilizing SH2 and SH3 domains of adaptor proteins to generate an activated form of p21 ras , a critical signaling enzyme (1-3).Recent studies have identified reactive free radicals as central participants in certain signaling events (4 -7). Enhancing free radical destruction, either enzymatically or chemically, prevented ligand-stimulated transcription factor (8) and mitogen-activated protein (MAP) 1 kinase (4) activation and also prevented smooth muscle cell mitogenesis and chemotaxis (4). Thus, a role is emerging for reactive free radicals in mediating signal transduction.Among the many recently discovered functions of NO, a role in signaling has surfaced (9). Although soluble guanylyl cyclase is an important target of NO in mediating some of its physiologic functions such as the regulation of blood pressure (10, 11), other signaling events, some culminating in transcriptional activation, may be cGMP-independent (12-15). Our studies have focused on how NO initiates cGMP-independent signaling within cells (16,17). We have identified p21ras as a critical target of NO and other redox modulators (17-19). Here, we sought an understanding of the structural basis of the NOp21 ras interaction in the hope of gaining insight into how redox signaling is achieved. MATERIALS AND METHODS Preparation of p21ras Proteins-p21 ras (1-166) was expressed and purified as described previously (20). p21 ras C118S(1-166) was expressed and purified similarly.Generation of p21 ras C118S cDNA Constructs-Codon 118 of truncated (codons 1-166) Ha-ras cDNA was mutated from TGT (cysteine) to TCT (serine) using the polymerase chain reaction. The generated cDNA fragment was then sequenced (Sequenase) and cloned into the pATras bacterial expression vector. To generate full-length p21 ras C118S an NcoI/BamHI fragment (encoding residues 111-166 of the ras(1-166) mutant) was exchanged for a 0.8-kilobase fragment encoding residues 111-189 plus 3Ј-noncoding region and the coding junction sequenced. A BglII/BamHI fragment encoding full-length Ras(C118S) was then subcloned into the BamHI site of the pCDNA3 mammalian expression plasmid and orientation confirmed by BstXI digestion.CNBr Digestion and ESI-MS Analysis of p21 ras -One small crystal of CNBr (Fluka) was added to 100 pmol of p21 ras in 20 l of 0.1 N HCl in a 0.5-ml polypropylene tube. Digestion was carried out at room temperature for 10 min prior to analysis by ESI-MS. After 10 min, samples were directly electrosprayed into a Finnigan-MAT TSQ-700 triple quadrupole instrument for analysis of S-nitrosylation exactly as we d...

Recent studies have demonstrated the biological importance of the interaction of nitric oxide (NO) with proteins. Protein-associated targets of NO include heme, Cys, and Tyr. Electrospray ionization-mass spectrometry was used to monitor the results of exposure of model peptides and an enzyme to NO under different conditions and thus addressed aspects of NO-protein interactions. The molecular mass of a decapeptide containing a single Cys residue increased by 29 Da upon treatment with NO under aerobic and acidic conditions, consistent with the substitution of one NO moiety. The mass of reduced somatostatin, a peptide containing two Cys residues, increased by 58 Da, consistent with the substitution of two NO moieties. These substitutions were prevented by pretreatment of the peptides with N-ethylmaleimide. The strength of the nitrosothiol bond was examined by varying the amount of energy applied to the peptide ions and indicated a labile species. Cys residues were very rapidly nitrosated, while other reactions were observed to occur at much slower rates. These include the further oxidation of nitrosothiol to sulfonic acid and nitration of Tyr. Peptides treated with NO at physiological pH were observed to undergo dimerization as well as nitrosation. These studies were extended to the enzyme p21ras, whose activity has been postulated to be modulated by nitrosothiol formation, and revealed the formation of a single nitrosothiol on p21ras upon NO treatment. These data suggest that electrospray ionization-mass spectrometry allows for quantitation and characterization of nitrosothiol bonds in peptides and proteins.