The oncogenic RAS2(val19) mutation locks respiration, independently of PKA, in a mode prone to generate ROS

Abstract

The RAS2(val19) allele, which renders the cAMP-PKA pathway constitutively active and decreases the replicative life-span of yeast cells, is demonstrated to increase production of reactive oxygen species (ROS) and to elevate oxidative protein damage. Mitochondrial respiration in the mutant is locked in a non-phosphorylating mode prone to generate ROS but this phenotype is not linked to a constitutively active PKA pathway. In contrast, providing RAS2(val19) cells with the mammalian uncoupling protein UCP1 restores phosphorylating respiration and reduces ROS levels, but does not correct for PKA-dependent defects. Thus, the RAS2(val19) allele acts like a double-edged sword with respect to oxidation management: (i). it diminishes expression of STRE element genes required for oxidative stress defenses in a PKA-dependent fashion, and (ii). it affects endogenous ROS production and the respiratory state in a PKA-independent way. The effect of the oncogenic RAS allele on the replicative life-span is primarily asserted via the PKA-dependent pathway since Pde2p, but not UCP1, overproduction suppressed premature aging of the RAS2(val19) mutant.

Fig. 1. Phenotypes of RAS2^val19 cells. (A) Life-span analysis of wild type (open squares) and RAS2^val19 (open circles) grown on SC medium as described in Materials and methods. (B) Iodine–iodide staining of wild-type and RAS2^val19 colonies. Iodine–iodide stains colonies according to their glycogen content. High glycogen content is indicated by a brown appearance. (C) Catalase activities in exponentially growing wild-type and RAS2^val19 cells. Wild-type levels were arbitrarily assigned a value of 100. *Note that catalase activity was often not detectable in the RAS2^val19 mutant unless the assay was scaled up to include more cell material. When doing so, the values obtained corresponded to about 10% of the catalase activity of wild-type cells. (D) Heat shock sensitivity (shift from 30°C to 52°C) of exponentially growing wild-type (open squares) and RAS2^val19 (open circles) cells. (E) Paraquat sensitivity of exponentially growing wild-type and RAS2^val19 cells. Serial dilutions of wild-type and RAS2^val19 cells taken from exponential phase (OD₆₀₀ = 1) cells were dropped on YPD plates with or without (not shown) paraquat (400 µg/ml).

Fig. 2. Indicators of endogenous oxidative stress in wild-type and RAS2^val19 cells. (A) Frequency of petite mutants in wild-type and RAS2^val19 populations. (B) In situ superoxide ion concentrations detected by DHE staining. Relative values are shown and the superoxide concentration of wild-type cells was arbitrarily assigned a value of 100. (C) Protein oxidation levels of specific proteins in total protein extracts (T) and mitochondrial extracts (M) detected by western immunochemical analysis of carbonylated proteins as described previously (Dukan and Nyström, 1998; Aguilaniu et al., 2001). (D) Quantification of carbonyl levels in total protein extracts (T) and mitochondrial extracts (M) of wild-type and RAS2^val19 cells as described (Dukan and Nyström, 1998). The carbonyl levels in wild-type cells were arbitrarily assign a value of 1.0. The standard deviation is shown on top of the bars.

Fig. 3. Respiratory mode, mitochondrial content, membrane potential and energetics of RAS2^val19 cells. (A) RSV measurements on wild-type (black line) and RAS2^val19 (red line) cells growing exponentially in YPD. Cells were placed in an oxygraph chamber and oxygen consumption was followed on-line. TET and CCCP were then added to the measuring chamber and RSV was calculated as explained (Aguilaniu et al., 2001). (B) Western blot quantification of the levels of the e subunit of ATPase in wild-type and RAS2^val19 cells grown exponentially in YPD. (C) Mitochondrial content of exponentially growing wild-type and RAS2^val19 cells detected by staining with MitoTrackerGreen FM. (D) [ATP]/[ADP] ratios in exponentially growing wild-type and RAS2^val19 cells. (E) Membrane potential of wild-type and RAS2^val19 cells measured by Rhodamine 123 fluorescence. The standard deviation is shown on top of the bars.

Fig. 4. Recovery of PKA activity by PDE2 overexpression in RAS2^val19 (RAS^v) cells. (A) Iodine–iodide staining of wild-type and RAS2^val19 cells carrying either the empty vector (top panels) or overexpressing PDE2 (lower panels). (B) Heat shock sensitivity (shift from 30°C to 52°C) of wild-type and RAS2^val19 cells carrying either the empty vector (wt, open squares; RAS2^val19, open circles) or overexpressing PDE2 (wt, filled squares; RAS2^val19, filled circles). (C) Catalase activity in wild-type and RAS2^val19 cells carrying the empty vector (–) and overexpressing PDE2 (+). The catalase activity of Ras2::LEU2 mutant cells carrying the vector is included for comparison.

Fig. 5. Effect of PDE2 overexpression on superoxide concentration and RSV in RAS2^val19 and wild-type cells. (A) In situ superoxide ion concentrations detected by DHE staining in wild-type and RAS2^val19 cells with (+) and without (–) Pde2p overproduction. (B) RSV analysis of RAS2^val19 cells carrying either the empty vector (solid red line) or overexpressing PDE2 (dotted red line). The RSV analysis of a bcy1 mutant is shown by the black line. See Materials and methods for details of the procedures. (C) Glycogen content (inset) and heat sensitivity (shift from 30°C to 52°C) of wild type (open squares), RAS2^val19 cells (open cicrcles) and bcy1 mutants (filled squares).

Fig. 6. Effect of the uncoupling protein UCP1 on respiration in RAS2^val19 cells. (A) Rate of respiration in RAS2^val19 cells with and without ectopic production of UCP1 as indicated on the graph. (B) Effect of the protonophore CCCP on the respiration rate of RAS2^val19 cells carrying the empty vector or producing UCP1. (C) RSV analysis of RAS2^val19 cells with (broken lines) and without (solid lines) ectopic production of UCP1. (D) Superoxide ion concentration in RAS2^val19 cells with (+) and without (–) ectopic expression of UCP1.

Fig. 7. Effect of Pde2p and UCP1 on life-span and protein oxidation. (A) Life-span analysis of wild type (open squares) and RAS^val19 cells carrying empty vector (Yep13) (open circles) or overproducing PDE2 (filled circles). The inset shows western blot analysis of carbonylated proteins in RAS2^val19 cells with and without Pde2p overproduction as indicated. The black arrows indicate polypeptides with reduced carbonylation as a result of Pde2p overproduction. (B) Life-span analysis of RAS^val19 cells without (open circles) and with (filled circles) UCP1. These experiments were conducted on SC medium with appropriate selection as described in Materials and methods. The inset shows western blot analysis of carbonylated proteins in RAS2^val19 cells with and without ectopic production of UCP1 as indicated. The black arrows indicate polypeptides with reduced carbonylation as a result of UCP1 production.