RAP80 responds to DNA damage induced by both ionizing radiation and UV irradiation and is phosphorylated at Ser 205.
Receptor-associated protein (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), was recently found to be associated with breast cancer-1 (BRCA1) and to translocate to ionizing radiation-induced foci (IRIF). In this study, we show that the BRCT mutant of BRCA1, R1699W, which is associated with increased risk of breast cancer, is unable to interact with RAP80. Previously, we showed that ataxia-telangiectasia mutated protein kinase (ATM) can phosphorylate RAP80 in vitro at Ser(205), but whether this site is a target of ATM in whole cells was not established. To address this question, we generated an anti-RAP80Ser205(P) antibody that specifically recognizes RAP80 phosphorylated at Ser(205). Our data show that RAP80 becomes phosphorylated at Ser(205) in cells exposed to ionizing irradiation and that RAP80Ser205(P) translocates to IRIF. We show that this phosphorylation is mediated by ATM and does not require a functional BRCA1. The phosphorylation occurs within 5 minutes after irradiation, long before the translocation of RAP80 to IRIF. In addition, we show that UV irradiation induces translocation of RAP80 to DNA damage foci that colocalize with gamma-H2AX. We further show that this translocation is also dependent on the UIMs of RAP80 and that the UV-induced phosphorylation of RAP80 at Ser(205) is mediated by ATM- and RAD3-related kinase, not ATM. These findings suggest that RAP80 has a more general role in different types of DNA damage responses.
Yan
Jun
J
Cell Biology Section, Laboratory of Respiratory Biology, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709, USA.
Yang
Xiao-Ping
XP
Kim
Yong-Sik
YS
Jetten
Anton M
AM
D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D056488:Histone Chaperones; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011839:Radiation, Ionizing; D012694:Serine / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C466234:UIMC1 protein, human; D012694:Serine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases