Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals

Abstract

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Protection of Human Endothelial Cells From Oxidative Stress

“…17 We have found recently that the effects of Ha-Ras and Ki-Ras on ROS levels are complementary; Ha-Ras activates, and Ki-Ras reduces, intracellular ROS. 18,19 In the present study, we show that Ha-Ras protein increases the number of cells undergoing apoptosis after oxidative stress; conversely, the Ki-Ras isoform has a protective effect, encoded by a polybasic stretch of lysine residues at the COOH terminus of the molecule, not present in the Ha-Ras sequence.…”

“…We have recently found that Ki-Ras but not Ha-Ras stimulates the mitochondrial enzyme manganese superoxide dismutase (MnSOD) via ERK1/2 signaling and that the polybasic region of Ki-Ras is essential for this induction. 19 Taken together, the data suggest that Ki-Ras reduces H 2 O 2 -induced apoptosis by buffering the production of endogenous ROS via MnSOD-ERK1/2 signaling ( Figure 6). The difference between ERK1/2 activated by Ha-Ras or Ki-Ras is probably accounted for either by a different membrane compartmentalization 27,28 or by a discrete organelle localization encoded by the polybasic lysine stretch.…”

“…[1][2][3][4][5] Recent insights into the p21 Ras ERK1/2 signaling cascade suggest that Ras is directly involved in the regulation of the intracellular redox state. 8,18,19 HUVECs, exposed to 2 mmol/L H 2 O 2 for 2 hours, undergo apoptosis 6 to 24 hours later. This is accompanied by activation of stress-related kinases that culminates in the apoptotic death of stressed cells.…”

“…8,18 Ras (specifically Ha-Ras) stimulates intracellular ROS levels by activating NADPH oxidase, further amplifying the cascade initiated by H 2 O 2 . 18,19 Ki-Ras and Ha-Ras regulate H 2 O 2 -induced apoptosis in a complementary fashion; Ki-Ras increases the tolerance, whereas Ha-Ras decreases it and promotes apoptosis induced by H 2 O 2 . Both Ki-Ras and Ha-Ras effects are dependent on the Ras signaling cascade because the transdominant negative Ras variant Leu61-Ser186 completely suppresses both the Ki-and the Haeffects (Figure 2).…”

“…ERK1/2-dependent transcription was not affected in Lys Ϫ mutants. 19 Determination of ROS in Ki-Ras-transfected cells indicates that low ROS levels parallel resistance to H 2 O 2 -induced apoptosis. Moreover, mutation of lysines at the COOH terminus of Ki-Ras significantly increased ROS levels, although Ki-Ras Cys Ϫ also showed a partial effect (Figure 4b).…”