Activation of Ras-Ral pathway attenuates p53-independent DNA damage G2 checkpoint.
Earlier we have found that in p53-deficient cells the expression of activated Ras attenuates the DNA damage-induced 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 G(2) 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 phosphorylation at Tyr(15) 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.
Agapova
Larissa S
LS
Institute of Carcinogenesis, Russian Cancer Research Center, 115478 Moscow, Russia.
Volodina
Julia L
JL
Chumakov
Peter M
PM
Kopnin
Boris P
BP
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D016195:G2 Phase; D005799:Genes, Dominant; D006801:Humans; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009154:Mutation; D012516:Osteosarcoma / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D012190:Retroviridae / Q000378:metabolism; D015398:Signal Transduction; D013936:Thymidine / Q000737:chemistry; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D020726:ral Guanine Nucleotide Exchange Factor / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D016159:Tumor Suppressor Protein p53; D020726:ral Guanine Nucleotide Exchange Factor; D014443:Tyrosine; D004317:Doxorubicin; D004247:DNA; D005020:Ethyl Methanesulfonate; D019869:Phosphatidylinositol 3-Kinases; D016203:CDC2 Protein Kinase; D018631:ras Proteins; D013936:Thymidine