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. Author manuscript; available in PMC: 2008 May 21.

Published in final edited form as: Cancer Res. 2007 Jul 9;67(14):6647–6656. doi: 10.1158/0008-5472.CAN-07-0924

RAP80 is a substrate for phosphorylation by ATM kinase. A. Schematic view of the location the potential ATM phosphorylation sites (arrows; dashed arrows indicate sites not conserved between human and mouse) in human RAP80. Comparison of a partial human and mouse RAP80 amino acid sequence is shown below. The most likely ATM phosphorylation sites conserved between mouse and human are highlighted. B. Phosphorylation of GST-RAP80(168–405) protein by ATM kinase or ATM(KD) was examined as described in Materials and Methods. C. Identification of ATM phosphorylation sites in RAP80. Phosphorylation of WT and several mutant GST-RAP80(168–405) proteins by ATM kinase was examined. D. Phosphorylation of RAP80 by ATM is not required for its translocation to IRIF. MCF-7 cells were transfected with pEGFP-RAP80 plasmid DNA encoding wild type RAP80 or RAP80 in which the ATM-phosphorylation sites were mutated. After 48 h incubation cells were treated with γ-irradiation (10 Gy) and 3 h later stained with anti-γ-H2AX antibody and DAPI. Localization of EGFP-RAP80 and γ-H2AX was examined by confocal microscopy.

RAP80 is a substrate for phosphorylation by ATM kinase. A. Schematic view of the location the potential ATM phosphorylation sites (arrows; dashed arrows indicate sites not conserved between human and mouse) in human RAP80. Comparison of a partial human and mouse RAP80 amino acid sequence is shown below. The most likely ATM phosphorylation sites conserved between mouse and human are highlighted. B. Phosphorylation of GST-RAP80(168–405) protein by ATM kinase or ATM(KD) was examined as described in Materials and Methods. C. Identification of ATM phosphorylation sites in RAP80. Phosphorylation of WT and several mutant GST-RAP80(168–405) proteins by ATM kinase was examined. D. Phosphorylation of RAP80 by ATM is not required for its translocation to IRIF. MCF-7 cells were transfected with pEGFP-RAP80 plasmid DNA encoding wild type RAP80 or RAP80 in which the ATM-phosphorylation sites were mutated. After 48 h incubation cells were treated with γ-irradiation (10 Gy) and 3 h later stained with anti-γ-H2AX antibody and DAPI. Localization of EGFP-RAP80 and γ-H2AX was examined by confocal microscopy.