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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
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Activation of Ras-Ral pathway attenuates p53-independent DNA damage G2 checkpoint

Larissa S Agapova1, Julia L Volodina, Peter M Chumakov

  • 1Institute of Carcinogenesis, Russian Cancer Research Center, 115478 Moscow, Russia.

The Journal of Biological Chemistry
|June 23, 2004
PubMed

Insights

Activated Ras signaling, specifically through the Ras-RalGEFs-Ral pathway, weakens the G(2) cell cycle arrest following DNA damage. This Ras-Ral pathway activation impacts CDC2 activity, potentially contributing to genetic instability in cancer cells.

Area of Science:

  • Cellular biology
  • Molecular oncology
  • Signal transduction

Background:

  • p53-deficient cells exhibit attenuated G(1) and G(2) DNA damage-induced arrest when activated Ras is expressed.
  • Ras signaling pathways play a crucial role in cell cycle regulation and DNA damage response.

Purpose of the Study:

  • To investigate the specific role of Ras-mediated signaling in the attenuation of the G(2) checkpoint following DNA damage in human cell lines.
  • To elucidate the involvement of the Ras-RalGEFs-Ral pathway in modulating the G(2) arrest response.

Main Methods:

  • Utilized human cell lines (MDAH041 fibroblasts, Saos-2 osteosarcoma cells).
  • Introduced various H-Ras mutants and RalA mutants (activated and dominant-negative) via transduction.
  • Assessed the impact on DNA damage-induced G(2) arrest using ethyl methanesulfonate and doxorubicin treatments.
  • Analyzed cyclin B1 and CDC2 levels, localization, and CDC2 kinase activity, including phosphorylation status.

Main Results:

  • Activation of the Ras-RalGEFs-Ral pathway attenuated DNA damage-induced G(2) arrest.
  • A specific RalA mutant (V23N49), unable to interact with RLIP76/RalBP1, still inhibited the G(2) arrest, suggesting effector-independent roles.
  • Ras-Ral signaling did not alter cyclin B1/CDC2 levels or localization but interfered with CDC2 inhibitory phosphorylation (Tyr15) and reduced cyclin B/CDC2 kinase activity.

Conclusions:

  • The Ras-Ral pathway is a key mediator in attenuating the DNA damage-induced G(2) checkpoint.
  • This pathway's interference with cell cycle regulation mechanisms, particularly CDC2 activity, may promote genetic instability in neoplastic cells.

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