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. 2006 Apr 7;281(14):9346-50.
doi: 10.1074/jbc.M513265200. Epub 2006 Jan 23.

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

Jeremy S Myers  1 , David Cortez
Affiliations

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

Jeremy S Myers et al. J Biol Chem. .

Abstract

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.

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Figures

Fig. 1
Fig. 1
ATM, ATR, and Mre11 are all required for Chk1 phosphorylation in response to IR. (A) Cells were transfected with non-specific siRNA or siRNA targeting Mre11, ATM, or ATR. Three days after transfection, the cells were exposed to 1mM HU for 5 hours, 50J/m2 UV then incubated for 2 hours, or 5Gy of IR followed by a one hour incubation. Cell lysates were prepared, separated by SDS-PAGE, then immunoblotted with the indicated antibodies. S-Chk1 is a short exposure and L-Chk1 is a long exposure of the same blot. The quantitation of the Chk1 P-S317 signal compared to total Chk1 for this experiment is shown (arbitrary units). These blot are representive of several consistent experiments. (B and C) Cells transfected with non-specific siRNA or siRNA targeting ATR or ATM were exposed to 5Gy or IR then harvested at the indicated times. Cell lysates were prepared, separated by SDS-PAGE and immunoblotted with the indicated antibodies. (*cross-reacting protein band in the Chk1 P-S345 blot). The quantitiation of the Chk1 P-S317 or the Chk1 P-S345 signal compared to total Chk1 for this experiment is shown (arbitrary units).
Fig. 2
Fig. 2
IR-induced Chk1 phosphorylation is defective in cells from A-T patients. A-T or ATM complemented A-T cells (38) were exposed to 5 Gy of IR and harvested at the indicated time points. Cell lysates were prepared, fractionated by SDS-PAGE and immunoblotted with the indicated antibodies. The quantitation of the Chk1 P-S317 signal compared to total Chk1 for this experiment is shown (arbitrary units).
Fig. 3
Fig. 3
IR-induced ATRIP and RPA foci formation is deficient in Mre11 and ATM depleted cells. (A) U2OS cells were exposed to 5 Gy IR and incubated for 1hr. After which cells were fixed in paraformaldehyde, permeabalized, and then immunostained with Mre11, ATRIP and RPA34 antibodies antibodies. After incubation with appropriate rhodamine and fluorescein isothiocyanate (FITC)–conjugated secondary antibodies, fluorescent images were captured on a Zeiss Axioplan microscope. Each image is representative of the immunostaining in the cell population. (B) HeLa cells left untreated (top) or exposed to 5 Gy IR were and incubated for one hour (bottom) immunostained for Mre11, ATRIP, and RPA34. (C) HeLa cells transfected with non-specific siRNA or siRNA targeting Mre11 were immunostained with Mre11 antibody and processed for immunofluorescence as described above. Images were captured using an equal exposure time to show relative Mre11 levels in non-specific and Mre11 siRNA treated cells. (D) HeLa cells transfected with non-specific siRNA or siRNA targeting Mre11 and ATM were exposed to 5 Gy IR. One hour after exposure RPA34 and ATRIP foci were detected. Quantitation of ATRIP and RPA34 foci in HeLa cells transfected with non specific siRNA or siRNA targeting Mre11 and ATM was done in cells processed for immunofluoresecence one hour after exposure to 5 Gy IR. Error bars represent standard error between experiments.
Fig. 4
Fig. 4
Defects in ATRIP and RPA foci in A-T cells are restored by complementation with ATM A-T or ATM complemented A-T cells (38) were exposed to 5 Gy of IR and processed for immunofluorescence with the indicated antibodies one hour after exposure. (A) Representative immunofluorescence images demonstrating ATRIP, RPA34, and Mre11 foci are shown. These images also demonstrate the colocalization of these proteins at IR-induced foci. (B) Quantitation of ATRIP and RPA34 foci in A-T or ATM complemented A-T cells. Error bars represent standard error between experiments.
Fig. 5
Fig. 5
Model of ATR and ATM signaling pathways.
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