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Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest
Pierre-Marie Girard1, Enriqueta Riballo, Adrian C Begg
1MRC Cell Mutation Unit, University of Sussex, Brighton, East Sussex, BN1 9RR, UK.
Abstract:
Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and 'leaky' A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiation-induced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.
Insights
Nijmegen Breakage Syndrome (NBS) cells exhibit impaired G1/S arrest, similar to ataxia telangiectasia (A-T) cells with low ATM. Nbs1 protein facilitates ATM-dependent phosphorylation of substrates crucial for cell cycle checkpoints.
Area of Science:
- Cell biology
- Molecular genetics
- Cancer research
Background:
- Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) are genetic disorders characterized by genomic instability.
- Cell lines from NBS and A-T patients display defects in cell cycle checkpoints, particularly during the S phase.
- Previous studies suggested Nbs1's checkpoint function is S phase-specific, as NBS cells showed less G1/S defect than A-T cells.
Purpose of the Study:
- To investigate the impact of residual Nbs1 function in NBS cells by comparing them with 'leaky' A-T cells expressing low levels of ATM.
- To evaluate the dose-dependent cell cycle response and ATM substrate phosphorylation in NBS and A-T cell lines after irradiation.
- To elucidate the role of Nbs1 in facilitating ATM kinase activity and downstream substrate phosphorylation.
Main Methods:
- Comparison of cell cycle arrest (G1/S, G2/M, S phase) in NBS and A-T (AT-5762ins137) cell lines following gamma irradiation.
- Analysis of radiation-induced phosphorylation of ATM-dependent substrates (Chk2, RPAp34, p53-Ser15) using Western blotting.
- Assessment of ATM kinase activation in NBS and A-T cells in vitro and in vivo post-irradiation.
Main Results:
- NBS and 'leaky' A-T cells exhibited nearly identical radiation-induced cell cycle responses, including normal G2/M arrest, dose-dependent G1/S arrest, and an A-T-like S phase defect.
- Phosphorylation of ATM substrates Chk2, RPAp34, and p53-Ser15 was similarly impaired in NBS and AT-5762ins137 cells in a dose-dependent manner.
- NBS cells demonstrated normal ATM kinase activation following irradiation, contrasting with the impaired substrate phosphorylation.
Conclusions:
- Checkpoint assays may vary in sensitivity to low levels of ATM activity.
- Nbs1 plays a crucial role in facilitating ATM-dependent phosphorylation of multiple downstream substrates, including those essential for G1/S arrest.
- The findings suggest that Nbs1's function extends beyond S phase specificity, contributing to G1/S checkpoint control through ATM pathway modulation.