Isolation of Ku70-binding proteins (KUBs).
DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.
Yang
C R
CR
Department of Radiation Oncology and Department of Pharmacology and the Ireland Cancer Center,Laboratory of Molecular Stress Responses, Case Western Reserve University, BRB-326 East,10900 Euclid Avenue, Cleveland, OH 44106-4942, USA.
Yeh
S
S
Leskov
K
K
Odegaard
E
E
Hsu
H L
HL
Chang
C
C
Kinsella
T J
TJ
Chen
D J
DJ
Boothman
D A
DA
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D051152:Clusterin; D004265:DNA Helicases; D015342:DNA Probes / Q000235:genetics; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014407:Tumor Cells, Cultured
C585461:ATP23 protein, human; D034961:Antigens, Nuclear; C495242:CLU protein, human; D002352:Carrier Proteins; C495243:Clu protein, mouse; D051152:Clusterin; D015342:DNA Probes; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C081647:high affinity DNA-binding factor, S cerevisiae; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen