A Molecular Redox Switch on p21

Abstract

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...

Role of redox potential and reactive oxygen species in stress signaling

“…Mutant forms of p21 ras at cysteine 118 no longer mediate guanine nucleotide exchange and further downstream signaling (i.e. activation of MAPK cascade) by NO (Lander et al, 1997). ROS were also reported to mediate recruitment of PI3K to p21 ras , an association that contributed to activation of PKB/Akt and MAPK, which are downstream targets for these kinases, respectively (Deora et al, 1998).…”

Identification of S-nitrosylation motifs by site-specific mapping of the S -nitrosocysteine proteome in human vascular smooth muscle cells

“…From the 16 proteins identified as potential targets of S-nitrosylation, 14-3-3 protein , 14-3-3 protein , annexin A2, elongation factor 2, and elongation factor 1 A-1 had been identified by the biotin switch method in various other systems (17)(18)(19)24). In addition, Cys-137 of RAB3B has been proposed as susceptible to S-nitrosylation based on a conserved NKCD motif (25). Because these experiments identified S-nitrosocysteine at residue 184, further work will be necessary to examine the site-specificity of S-nitrosylation in RAB3B.…”

Nitric Oxide and Cancer Development

“…The Ras activation pathway primarily involves the following activation sequence Ras/Raf/Mek/ Erk 172,173 , however Ras activation can also influence other pathways, including Akt 171,173 , which is also important in cancer development. It has been known for many years that NO activates Ras to enhance guanine nucleotide exchange [174][175][176][177][178][179] . The first experiments demonstrating that NO enhances guanine nucleotide exchange was done by exposing jurkat cells to NO gas 174 .…”