The nitric oxide-sensitive p21Ras–ERK pathway mediates S-nitrosoglutathione-induced apoptosis Maristela Tsujita a,b , Wagner L. Batista a , Fernando T. Ogata a , Arnold Stern c , Hugo P. Monteiro a, * , Roberto J. Arai a, * a Departamento de Bioquímica/Biologia Molecular, Centro Interdisciplinar de Terapia Gênica CINTERGEN, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil b Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, SP, Brazil c Department of Pharmacology, New York School of Medicine, New York University, New York, USA article info Article history: Received 14 February 2008 Available online 4 March 2008 Keywords: p21Ras ERK SNOG Apoptosis abstract p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras–ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras C118S ) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly atten- uated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras–ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG. Ó 2008 Elsevier Inc. All rights reserved. Signaling pathways that control cell viability are associated with the regulation of the cell cycle and apoptosis. A critical cross- road in signaling pathways that regulate either cell proliferation or cell cycle arrest involves Ras proteins [1–3]. The distinct actions of Ras proteins (21 kDa proteins: H-Ras, N-Ras, K4a-Ras, K4b-Ras) [4] are due to its interactions with a number of downstream pathways (for review see: [5]). p21Ras activity is stimulated by guanine exchange factors (GEF) that promote the exchange of GDP to GTP and its GTPase activity is accelerated by GTPase-activating proteins (GAP) which result in the formation of the inactive GDP-bound form of p21Ras [6]. In normal tissues, p21Ras activity is tightly controlled by such proteins. In several cancer tissues p21Ras pre- vails in a GTP-bound state and is constitutively active leading to persistent activation of downstream signaling cascades that may promote cellular transformation [7]. p21Ras proteins are activated by several stimuli including growth factors, cytokines or neurotransmitters through cell surface receptors [8]. Nitric oxide (NO) can activate p21Ras directly by reversible post-translational modifications. Studies have shown that NO is capable of mediating S-nitrosation or S-gluthatiolation of the critical cysteine residue 118 nearby the guanine nucleotide binding site [9,10]. This mode of control has the same effect as the binding of GEF to p21Ras and can lead to stimulation of down- stream signaling pathways. Among major downstream pathways, MAP kinases are critical in regulating the cell cycle and cell viability. The extracellular signal regulating kinases 1 and 2 (ERK1/2 MAP kinases) are mainly associated with proliferation and differentia- tion, while the c-jun N-terminal kinases 1 and 2 (JNK1/2 MAP ki- nase) and p38 MAP kinase regulate cell responses to several stressors that may induce either apoptosis or cell resistance [11– 15]. The combined actions of MAP kinases and their temporal and spatial activation are crucial in defining the specificity of the cellular response. The signaling initiated by p21Ras that results in the assembly of the kinase cascade, particularly the ERK pathway, has been 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.117 * Corresponding authors. Fax: +55 11 30697557. E-mail addresses: hpmonte@uol.com.br (H.P. Monteiro), arairj@yahoo.com.br (R.J. Arai). Biochemical and Biophysical Research Communications 369 (2008) 1001–1006 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc