Activation of Ras-Ral pathway attenuates p53-independent DNA damage G 2 checkpoint

Abstract

Earlier we have found that in p53-deficient cells the expression of activated Ras attenuates the DMA damage-induced arrest in G1 and G2. 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 G2 arrest induced by ethyl metanesulfonate or doxorubicin. Noteworthy, the activated RalA V23N49 mutant, which cannot interact with RLIP76/RalBP1 protein, one of the best studied Ral effectors, retained the ability to attenuate the DNA damage-induced G 2 arrest. Activation of the Ras-Ral signaling affected neither the level nor the intracellular localization of cyclin B1 and CDC2 but interfered with the CDC2 inhibitory phosporylation at Tyr15 and the decrease in the cyclin B/CDC2 kinase activity in damaged cells. The revealed function of the Ras-Ral pathway may contribute to the development of genetic instability in neoplastic cells.