Volume 11, Issue 8, Pages 614-619 (April 2001) Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals  Mariarosaria Santillo, Paolo Mondola, Rosalba Serù, Tiziana Annella, Silvana Cassano, Ilaria Ciullo, Mario F. Tecce, Giuseppe Iacomino, Simona Damiano, Giovanni Cuda, Roberto Paternò, Valeria Martignetti, Evelina Mele, Antonio Feliciello, Enrico V. Avvedimento  Current Biology  Volume 11, Issue 8, Pages 614-619 (April 2001) DOI: 10.1016/S0960-9822(01)00159-2

Figure 1 Ha-Ras increases ROS, and Ki-Ras decreases ROS. (a) The time course of ROS production induced by Fe2+/ascorbate oxidative stress in 12 h dexamethasone (2 μM)-treated TL+H17 cells and in TL cells, expressing a control plasmid, RSV-NEO, in the absence (TL) or presence (TL*) of dexamethasone. ROS were measured as DCF fluorescence using the oxidation-sensitive probe, 5, 6-carboxy-2′, 7′-dichlorofluorescein diacetate, DCHF-DA. Values are means ± standard errors of a representative experiment performed in triplicate. (b) The effect of H17 expression on H2O2-induced apoptosis in TL cells. Six hours after, the incubation for 1 h with increasing concentrations of H2O2, apoptosis was evaluated by annexin staining. Values are means ± standard errors of a representative experiment performed in triplicate. (c) Immunoblot analysis of endogenous Ras with antibodies to Ha-Ras, Ki-Ras 4B, and Pan-Ras in TL cells. In the bottom panel, the same blots were probed with anti-ERK1/2 antibody (ERK2) as a control. (d)Ki- and Ha-Ras mRNA levels in thyroid cells. An asterisk indicates a sample in which GADPH and Ha-Ras primers were amplified in the same reaction [21]. (e) The time course of basal and stress-induced ROS production in NIH 3T3 fibroblasts transiently transfected with Ki-Ras/GFP or Ha-Ras/GFP and pCMV-lacZ. Control cells were transfected with GFP-IRES plasmid and pCMV-lacZ. ROS levels were normalized for β-galactosidase activity. Values are means ± standard errors of a representative experiment performed in triplicate. (f) The effect of DPI on stress-induced (2 h) ROS levels in NIH 3T3 fibroblasts transiently transfected with Ki- or Ha-Ras/GFP and pCMV-lacZ vectors. The cells were incubated for 30 min in the presence or absence of 20 μM DPI before loading with DCHF-DA. Values, normalized for β-galactosidase activity, are means ± standard errors of a representative experiment performed in triplicate. The expression of the tagged Ras proteins was comparable in the experiments presented. (g) and (h) represent the time course of stress-induced ROS production in cells transfected with Ki-Ras/GFP and Ha-Ras/GFP, (g and h, respectively) alone or with expression vectors encoding Ras-H17 or L61S186 [22] Current Biology 2001 11, 614-619DOI: (10.1016/S0960-9822(01)00159-2)

Figure 2 Ki-Ras stimulates mitochondrial SOD. (a) The effect of Ki-Ras on basal and stress-induced (2 h) ROS levels. ROS levels were measured in control cells (C), stably (NIH 3T3), or transiently (COS7) Ki-Ras- transfected cells. Values are means ± standard errors of three different experiments performed in duplicate. (b) Mn-SOD activity [23] in the same cell lines as indicated above. Values are means ± standard errors of three different experiments performed in duplicate. (c) Immunoblot analysis with antibodies to Mn-SOD in mock transfected NIH 3T3 cells (C), mock transfected NIH 3T3 cells stably expressing Ki-Ras, and in the same cells transiently cotransfected with the trans-dominant-negative mutant of Ha-Ras L61S186 and pEGFP. (d) Northern blot analysis of Mn-SOD mRNA expression [24] in control (C) and Ki-Ras transformed NIH 3T3 cells. The same blot was stripped and reprobed with α-actin cDNA (bottom panel). (e) Time course of ROS production in v-Ki-Ras- and v-Ha-Ras-transformed thyroid cells (PC-KiMol and PC-Ha, respectively, [10]). (f) Immunoblot and densitometric analysis of Mn-SOD from the same cell lines as indicated in (e). In the bottom panel, the same filter was stripped and reprobed with anti-ERK1/2 antibodies as a control. (g) The activity of mitochondrial and (h) cytoplasmic SODs in different thyroid cell lines (PC and TL cells) transformed with Ha and Ki viral genes. Relative units were calculated with SOD activity of control cells set to 1 Current Biology 2001 11, 614-619DOI: (10.1016/S0960-9822(01)00159-2)

Figure 3 Different residues at the COOH terminus of Ki-Ras are required for mitochondrial SOD stimulation and ERK1/2-induced transcription: (a) Inhibition of ERK1/2, but not PI3K, signaling increases basal and stress-induced (2 h) ROS levels in COS7 cells. COS7 cells were incubated for 15 hr with 40 μM PD98059 (Calbiochem), a specific MEK inhibitor, or with 50 μM LY294002 (Calbiochem), a PI3K inhibitor. Under these conditions, the activity of serum-induced ERK1/2 (5 min) and of PI3K induced by insulin (100 μg/ml) were reduced by 80%. Values are means ± standard errors of three different experiments performed in duplicate. (b,c) The inhibition of ERK1/2 signaling abolishes the induction of mitochondrial SOD activity by (b) Ki-Ras or (c) v-Ki-Ras. (b) COS7 cells were transiently transfected with Ki-Ras/GFP, CMV-Mn-SOD, and pCMV-lacZ DNA in the absence and presence of PD98059. Values, normalized for β-galactosidase activity, are means ± standard errors of three different experiments performed in duplicate. (c) The effect of PD98059 on Mn-SOD activity levels in two independent v-Ki-Ras-transformed cell lines TL (1) and PC (2) treated with the inhibitor indicated above for 15 hr. Mn-SOD relative units were calculated by comparing the Mn-SOD activity of the samples to that of control cells set to 1. (d,e) The effects of Ki-Ras COOH tail mutants on Mn-SOD activity and ERK1/2- induced transcription. (d) Mitochondrial SOD activity in cells expressing Ki-Ras COOH mutants. COS7 cells were transiently transfected with HA-tagged Ki-Ras/GFP wild type or the mutant version indicated and pCMV-lacZ plasmid for 24 hr before Mn-SOD activity assay. Values, normalized for β-galactosidase activity, are means ± standard errors of three different experiments performed in duplicate. The HA immunoblot of transfected cells with anti-HA antibodies (clone 12CA5) is shown in the lower panel. (e) ERK1/2-dependent transcription in cells expressing Ki-Ras COOH tail mutants Current Biology 2001 11, 614-619DOI: (10.1016/S0960-9822(01)00159-2)

Figure 4 Dual functions of Ki-Ras mediated by ERK1/2; activation of transcription and mitochondrial SOD. A schematic diagram of Ki-Ras functions. The polybasic stretch is indicated by the six Ks. “C*” indicates the cysteine in the CAAX box, the classical farnesylation site Current Biology 2001 11, 614-619DOI: (10.1016/S0960-9822(01)00159-2)