Structural and biochemical studies of p21^RasS-nitrosylation and nitric oxide-mediated guanine nucleotide exchange

Abstract

Ras is a guanine nucleotide-binding protein that cycles between inactive GDP-bound and active GTP-bound states to regulate a diverse array of cellular processes, including cell growth, apoptosis, and differentiation. The guanine nucleotide-bound state of Ras is tightly maintained by regulatory factors to promote regulated growth control. A class of regulatory molecules that lead to Ras activation are guanine nucleotide exchange factors (GEFs). Ras GEFs bind to Ras and facilitate GDP release, followed by GTP incorporation and Ras activation. Nitric oxide (NO) has also been shown to promote guanine nucleotide exchange (GNE) on Ras and increase cellular Ras-GTP levels, but the process by which NOmediated GNE occurs is not clear. We initiated NMR structural and biochemical studies to elucidate how nitrosylation of Ras might lead to enhanced GNE. Surprisingly, our studies show that stable S-nitrosylation of Ras at Cys-118, does not affect the structure of Ras, its association with the Ras-binding domain of Raf (a downstream effector of Ras), or GNE rates relative to non-nitrosylated Ras. We have found, however, that the actual chemical process of nitrosylation, rather than the end-product of Ras S-nitrosylation, accounts for the enhanced GNE that we have observed and that has been previously observed by others.guanosine triphosphatase ͉ redox ͉ cancer ͉ reactive oxygen species ͉ reactive nitrogen species N early 20 years ago, nitric oxide (NO) was identified as a vaso-relaxation factor and was found to mediate its effects on smooth muscle by coordinating the heme moiety of guanylate cyclase, leading to enhanced production of cGMP (1). It was later shown that increased levels of cGMP modulate the function of other physiological targets, such as protein kinases, phosphodiesterases, and ion channels, causing vaso-relaxation (as reviewed in refs. 1-3). NO has also been shown to modulate cGMP-independent pathways by modifying a multitude of cellular biomolecules containing reactive metal centers, as well as cysteine residues (4-6). Although NO is able to modulate the activity of tyrosine kinases, serine͞threonine kinases, guanosine triphosphatases (GTPases), and phosphatases, the molecular basis of NO modification and the subsequent effect on enzyme activity are not completely understood. NO is generally believed to exert its effects on biomolecules by direct modification of a residue important for protein structure or enzymatic catalysis.The Ras GTPase is a protein product of the most commonly mutated oncogene in all of human cancer (ras), and has been identified as a target for S-nitrosylation by NO (7). Ras is a molecular switch that cycles between an inactive GDP-bound state and an active GTP-bound state, to regulate a number of cellular processes, including cell growth, differentiation and apoptosis (for reviews, see refs. 8-10). In its oncogenic form, Ras is populated in a GTP-bound state and is constitutively active. The chronic activation of Ras leads to persistent activation of downstream signaling cascades to pr...

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

“…Furthermore, endogenous NO synthesized by neuronal NOS in rat neurons in culture also activates Ras signaling (44). NO may directly activate Ras in part by promoting exchange of the guanine nucleotide GDP for GTP (45). In contrast, our data suggest that NO inhibits Ras signaling, not by modulating Ras activity but by decreasing Ras expression.…”

New Insights into Protein S-Nitrosylation

“…Ras have raised the possibility that the S-nitrosylated anion radical may be stabilized by protein conformation (56,61). The oxidized RS-NO product could then form in the presence of an electron acceptor (21), and in sarcosine dehydrogenase, the bound FAD may fulfill this requirement.…”

“…In this pathway, NO could first react with a thiolate to form a radical anion intermediate [RS-NO] . (60,61). Structural studies of Hb and p21…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Those proteins are included here as the same critical cysteine(s) can be modified by either ROS or RNS and very likely by HNE as well when the cysteine is in the thiolate form. Among the signaling proteins (and their critical cysteines) that have been well characterized are the PTPs, PTP1B (cys 215 ) (26,27,(34)(35)(36), low molecular weight PTP (cys12,17) (37-39) and Srchomology 2 domain-containing PTP (SHP-2) (40), the small G protein, Ras (cys 118 ) (41)(42)(43)(44), the large G proteins, G i (cys 267 ) and G o (cys 326 ) (45) and the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, as mentioned above, in its reduced form Trx binds and inhibits ASK1 and possibly other signaling proteins as well (46)(47)(48)(49)(50)(51)(52)(53).…”