S-Nitrosylation Decreases the Adsorption of H-Ras in Lipid Bilayer and Changes Intrinsic Catalytic Activity

Abstract

Structural, chemical, and mutational studies have shown that C-terminal cysteine residues on H-Ras could potentially be oxidized by nitrosylation. For investigating the effect of nitrosylation of Ras molecule on the adsorption of farnesylated H-Ras into lipid layer, experiments with optical waveguide lightmode spectroscopy were used. The analysis of association/dissociation kinetics to planar phospholipids under controlled hydrodynamic conditions has shown that preliminary treatment of protein by S-nitroso-cysteine decreased the adsorption of farnesylated H-Ras. The authors have found that compared with nitrosylated forms, farnesylated H-Ras has more compact configuration, because of the smaller area occupied by protein upon absorption at the membrane. The association rate coefficient for unmodified H-Ras was lower than similar parameter for farnesylated and nitrosylated forms. However, the desorbability, i.e., parameter, which reflects the rate of dissociation of protein from lipids is higher for farnesylated H-Ras. In addition, it was have found that farnesylation of cytoplasmic H-Ras, in contrast to membrane-derived forms, inhibits intrinsic GTPase activity of protein, and preliminary treatment of H-Ras by S-nitroso-cysteine restores the activity to the control level. These data suggest that nitrosylation of H-Ras rearranges the adsorptive potential and intrinsic GTPase activity of H-Ras through modification of C-terminal cysteines of molecule.

Cysteine-based regulation of redox-sensitive Ras small GTPases

“…Although the Cys118 residue (present in the NKCD region, located in the invariant N -terminal domain up to amino acid 166) is considered the primary target of ROS and RNS [[12], [13], [14], [15], [16], [17]], the involvement of this residue in the redox-based modulation is controversial. Conflicting results likely reflect the use of different methodological approaches, redox inducers, and cell lines (Table 1).…”

“…Cys-nitrosylation of H-Ras leads to the stimulation of the intrinsic GTPase activity that shortens the lifetime of the active GTP-bound state of the protein. Only the membrane-bound H-Ras is nitrosylated at Cys118 and this modification decreases the rate of GTP hydrolysis [13]. Last, mass spectrometry analysis provided evidence that the endogenous Cys118-nitrosylation of the K-Ras4B isoform occurs in colon cancer cells and primary tissues [15].…”

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability

“…Farnesylation also regulates mouse cpx 3/4 [ 13 ] and Drosophila cpx function [ 14 – 17 ]. The Cys within CAAX motifs can also undergo S-nitrosylation, which interferes with the farnesylation signaling [ 18 ]; however, direct evidence in a physiological environment is lacking. Cpx function has been studied in many different systems and there is controversy regarding its fusion-clamp activity.…”

Regulation of Ras Signaling by S-Nitrosylation

“…S-Nitrosylation of Ras has been extensively studied and is one of the best examples of how a PTM regulates the signaling of a protein ( Figure 2 ) [ 6 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ].…”

“…In contrast, S-nitrosylation of the membrane-anchored H-Ras diminishes the rate of GTP hydrolysis ( Figure 3 A). In this case, S-nitrosylation occurred only at the Cys118 residue, in the GTP-binding pocket [ 44 ].…”