Activation of Ras-Ral Pathway Attenuates p53-independent DNA Damage G2 Checkpoint

Abstract

Earlier we have found that in p53-deficient cells the expression of activated Ras attenuates the DNA damageinduced arrest in G 1 and G 2 . In the present work we studied Ras-mediated effects on the G 2 checkpoint in two human cell lines, MDAH041 immortalized fibroblasts and Saos-2 osteosarcoma cells. The transduction of the H-Ras mutants that retain certain functions (V12S35, V12G37, and V12C40 retain the ability to activate Raf or RalGDS or phosphatidylinositol 3-kinase, respectively) as well as the activated or dominant-negative mutants of RalA (V23 and N28, respectively) has revealed that the activation of Ras-RalGEFs-Ral pathway was responsible for the attenuation of the The proteins of the Ras family, which include H-, K-, and N-Ras, function as key regulators of the signal transduction pathways controlling cell proliferation, survival, migration, and differentiation (1-5). Various extracellular signals reaching cell-surface receptors stimulate the conversion of the Ras proteins from the inactive GDP-bound to the active GTP-bound form. In the GTP-bound form Ras stimulates downstream effectors, which, in turn, affect activities of numerous proteins, including the large group of transcription factors (1-5).Mutations that constitutively activate Ras proteins are characteristic of many human tumors. In fact, they are found in 95-98% of pancreatic cancers, 40 -50% of colon and thyroid tumors, 25-30% of lung tumors, leukemias, and some other malignancies (6 -8). Substantial experimental data indicate that such aberrant Ras activation plays a critical role in oncogenesis causing permanent stimulation of cell proliferation, increased cell motility, and inhibition of apoptosis and angiogenesis (4, 9 -15), i.e. the features that are responsible for tumor growth and invasion (16). These effects are mediated mainly via three groups of Ras effectors: (i) Raf serine/threonine kinases (c-Raf-1, A-Raf, and B-Raf), (ii) PI3Ks 1 lipid kinases, and (iii) RalGEFs for the small GTPases RalA and RalB (RalGDS, Rgl/Rsb2, Rgl2/Rlf, and RGL3) (1-5, 10, 17-20).The Raf kinases phosphorylate and activate Mek1 and Mek2 dual specificity kinases, which, in turn, phosphorylate and activate Erk1/p44 and Erk2/p42 mitogen-activated protein kinases playing key roles in the stimulation of cell division (21,22). Frequent occurrence in human melanomas and thyroid papillary cancers of either Ras or B-Raf mutations indicate that Raf activation can substitute the oncogenic effect of Ras activation in the course of development of some types of human tumors (23,24

Repression of Sestrin Family Genes Contributes to Oncogenic Ras-Induced Reactive Oxygen Species Up-regulation and Genetic Instability

“…Earlier, we reported that the rate of Ras-induced mutagenesis is higher in p53-deficient cells as compared with isogenic cells expressing functional p53 (11). In part, the synergy in the induction of chromosome instability by oncogenic RAS in the p53-null background is explained by the ability of Ras to attenuate the p53-independent components of the DNA damage-induced G 1 and G 2 phase checkpoints (11,16). In the present study, we found a connection between up-regulated ROS and inhibited expression of sestrin family genes, which might represent an additional factor enhancing Ras-induced mutagenesis in p53-deficient cells.…”

“…23; provided by J. Downward, Cancer Research UK, London, United Kingdom); pLXSN-neo/RalA-V23 with constitutively active V23 RalA mutant and lentiviral pLV-CMV/RalA-N28 expressing dominant-negative N28 RalA mutant (16); and lentiviral pLSLP vectors bearing short hairpin RNAs (shRNA) against p53 or human SESN1 and SESN2 genes (21,24). Complementary 60-bp hairpin oligonucleotides containing 19-nucleotide regions corresponding to the human SESN3 gene (5 ¶-GACGAGGAGAAGAGCATTT-3 ¶ and 5 ¶-CCAGAGAGAGATCCAGAAA-3 ¶) were designed according to siRNA-scale algorithm 5 and cloned into pLSLP vector for shRNA, as described earlier (24).…”

“…Their derivatives with tetracycline-repressible expression of activated N-RasD13 mutant (MDAH041/ tet-Ras, REF52/tet-Ras, and Rat1/tet-Ras cell lines, respectively) were described by us earlier (11,22). The cell sublines with constitutive expression of various H-Ras, N-Ras, RalA, and Rac1 mutants were previously described (11,16). Briefly, the retroviral DNA constructs were transfected into Phoenix-ampho packaging cells using LipofectAMINE PLUS reagent (Invitrogen).…”

“…We have previously found that the mutagenic effect of Ras is especially prominent in p53-deficient cells (11) and can be connected with both attenuation of DNA damage cell cycle checkpoints and up-regulation of reactive oxygen species (ROS; refs. 15,16). In fact, expression of Ras oncoproteins increases ROS content (15,(17)(18)(19)(20) and interferes with DNA damage-induced arrest in G 1 and G 2 (11,16), although the mechanisms of these effects are poorly understood.…”

The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster

“…Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (2,13,17,23,30,41).…”