The cyclopentenone 15-deoxy- 12,14 -prostaglandin J 2 binds to and activates H-Ras Jose ´ Luis Oliva* † , Dolores Pe ´ rez-Sala †‡§ , Antonio Castrillo †¶ , Natalia Martı ´nez*, F. Javier Can ˜ ada ‡ , Lisardo Bosca ´ ¶ , and Jose ´ M. Rojas* § *Unidad de Biologı´a Celular, Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain; ‡ Departamento de Estructura y Funcio ´n de Proteı´nas, Centro de Investigaciones Biolo ´gicas, Consejo Superior de Investigaciones Cientı´ficas, 28006 Madrid, Spain; and ¶ Instituto de Bioquı´mica and Centro Nacional de Investigaciones Cardiovasculares, Centro Mixto Consejo Superior de Investigaciones Cientı ´ficas–Universidad Complutense de Madrid, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain Edited by Edward M. Scolnick, Merck & Co., Inc., West Point, PA, and approved February 7, 2003 (received for review September 26, 2002) The cyclopentenone 15-deoxy- 12,14 -prostaglandin J 2 (15d-PGJ 2 ) in- duces cell proliferation and mitogen-activated protein kinase activa- tion. Here, we describe that these effects are mediated by 15d-PGJ 2 - elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ 2 with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ 2 , whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins. mitogen-activated protein kinase | cell proliferation | posttranslational modification C yclopentenone prostaglandins (CyPG) are naturally occur- ring eicosanoids that display varied biological activities, including antiviral (1) and antitumoral effects (2), modulation of the heat shock response (3), and induction of oxidative stress (4) and apoptosis (5). The CyPG of the J 2 series, such as 15-deoxy-  12,14 -prostaglandin J 2 (15d-PGJ 2 ), arise from the spontaneous dehydration of PGD 2 , whereas PGA 2 is produced by PGE 2 dehydration. CyPG have been detected in vivo in human body fluids (6), foam cells of human atherosclerotic plaques (7), and tissues of patients with sporadic amyotrophic lateral sclerosis (8). 15d-PGJ 2 generation has been found in association with in- creased cyclooxygenase 2 (COX-2) expression during inflam- matory processes (9), where it has been proposed to contribute to the resolution of inf lammation through multiple mechanisms, which may include the inhibition of NF-B activity (10, 11) and the potentiation of macrophage apoptosis (12). An association between COX-2 expression and CyPG generation also has been documented in several cellular models, including colorectal cancer cells (13) and activated macrophages (7). CyPG are potent modulators of cell proliferation. However, the nature of their effect appears to be cell type- and dose-dependent. Although antiproliferative or proapoptotic effects have been most frequently described (14), CyPG have also been found to induce cell proliferation in mesangial (15), breast cancer (16), and COX-2- depleted colorectal cancer cells (13) when used at nanomolar or low micromolar concentrations. It has been reported that 15d-PGJ 2 can cause the activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 12 (17–19), and that phosphatidylinositol 3-kinase (PI3-kinase) inhibitors re- duce 15d-PGJ 2 -elicited cell proliferation (15). These observations are compatible with the hypothesis that 15d-PGJ 2 may interact with the Ras signaling pathway. Ras proteins are critical components of signal transduction leading from cell-surface receptors to the control of cell proliferation, differentiation, or death (20). The mammalian genome contains three ras genes that encode highly related proteins of 21-kDa termed H-Ras, N-Ras, and K-Ras with its two isoforms, K-Ras4A and K-Ras4B, generated from two alternative fourth exons. The three ras genes are concurrently expressed in most mouse and human tissues (21, 22). Ras proteins are membrane-bound GTPases that upon activation interact with effector proteins, mainly Raf, PI3-kinase, and Ral-GDS (23), resulting in the activation of downstream signaling pathways, such as the RafMEKERK, PI3-kinaseAkt, or the Ral-GDSRal A pathway (24, 25) that ultimately lead to the transcriptional activa- tion of genes. We undertook the present study to explore the existence of a 15d-PGJ 2 –Ras activation pathway. We demonstrate that 15d-PGJ 2 itself induces H-Ras activation. Interestingly, this effect is mediated by direct interaction of 15d-PGJ 2 with Cys-184 of H-Ras. Our data also provide evidence for a differential activation of Ras isoforms, because H-Ras was the only Ras isoform able to bind 15d-PGJ 2 effectively. Materials and Methods Cells and Reagents. NIH 3T3 cells were maintained in DMEM (Invitrogen) supplemented with 10% calf serum (Invitrogen). Cos1 cells were maintained in DMEM supplemented with 10% FCS (Invitrogen). For the other reagents see the Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org. DNA Constructs. The plasmids used have been described (26–29). The mutants C118S and C184S of H-Ras were obtained by PCR from pCEFL-KZ-AU5-H-Ras WT, using specific primers provid- ing BglII and NotI sites at the 5' and 3' ends, respectively. The amplified products were subcloned between the BglII and NotI sites of pCEFL-KZ-AU5. Labeling of H-Ras with Biotinylated 15d-PGJ 2 in Vitro. Biotinylated 15d-PGJ 2 was prepared as described (30). H-Ras at 50 nM in 20 mM TrisHCl (pH 7.0), 45 mM NaCl, 5 mM MgCl 2 , 0.1 mM DTT, and 0.14% glycerol was incubated for 2 h at room temperature in the presence of vehicle (DMSO) or 5 M biotinylated 15d-PGJ 2 in a final volume of 50 l. Incorporation of biotin was assessed by Western blot and detection with horseradish peroxidase (HRP)- conjugated streptavidin and ECL (Amersham Pharmacia). Structural Characterization of 15d-PGJ2 -Modified H-Ras. Recombi- nant human WT H-Ras at 5 M in 20 mM TrisHCl (pH 7.0), 45 mM NaCl, 5 mM MgCl 2 , 0.1 mM DTT, and 1.4% glycerol was incubated for 2 h at room temperature in the presence of vehicle (DMSO) or 15d-PGJ 2 at the indicated concentrations in a final This paper was submitted directly (Track II) to the PNAS office. Abbreviations: 15d-PGJ2, 15-deoxy- 12,14 -prostaglandin J2; PG, prostaglandin; CyPG, cyclo- pentenone PGs; MAPK, mitogen-activated protein kinase; COX-2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; PI3-kinase, phosphatidylinositol 3-kinase; HRP, horseradish peroxidase; HA, hemagglutinin. † J.L.O., D.P.-S., and A.C. contributed equally to this work. § To whom correspondence should be addressed. E-mail: dperezsala@cib.csic.es or jmrojas@isciii.es. 4772– 4777 | PNAS | April 15, 2003 | vol. 100 | no. 8 www.pnas.orgcgidoi10.1073pnas.0735842100