The ubiquitin-interacting motif containing protein RAP80 interacts with BRCA1 and functions in DNA damage repair response.
In this study, we examine the potential role of receptor-associated protein 80 (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), in DNA damage response and double-strand break (DSB) repair. We show that following ionizing radiation and treatment with DNA-damaging agents, RAP80 translocates to discrete nuclear foci that colocalize with those of gamma-H2AX. The UIMs and the region of amino acids 204 to 304 are critical for the relocalization of RAP80 to ionizing radiation-induced foci (IRIF). These observations suggest that RAP80 becomes part of a DNA repair complex at the sites of IRIF. We also show that RAP80 forms a complex with the tumor repressor BRCA1 and that this interaction is mediated through the BRCA1 COOH-terminal repeats of BRCA1. The UIMs are not required for the interaction of RAP80 with BRCA1. Knockdown of RAP80 in HEK293 cells significantly reduced DSB-induced homology-directed recombination (HDR). Moreover, inhibition of RAP80 expression by small interfering RNA increased radiosensitivity, whereas increased radioresistance was observed in human breast cancer MCF-7 cells with overexpression of RAP80. Taken together, our data suggest that RAP80 plays an important role in DNA damage response signaling and HDR-mediated DSB repair. We further show that RAP80 can function as a substrate of the ataxia-telangiectasia mutated protein kinase in vitro, which phosphorylates RAP80 at Ser(205) and Ser(402). We show that this phosphorylation is not required for the migration of RAP80 to IRIF.
Yan
Jun
J
Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC 27713, USA.
Kim
Yong-Sik
YS
Yang
Xiao-Ping
XP
Li
Li-Ping
LP
Liao
Grace
G
Xia
Fen
F
Jetten
Anton M
AM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology; D045744:Cell Line, Tumor; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology; D056488:Histone Chaperones; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000502:physiology; D025801:Ubiquitin / Q000737:chemistry*
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; D025801:Ubiquitin; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases