Isolation of Ku70-binding proteins (KUBs)

Abstract

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.

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype

“…XRCC6BP1 shows amplification frequencies similar to CDK4 within the 12q amplicon and is overexpressed in glioblastoma-derived cell lines and in primary glioblastoma (Fischer et al, 2001). Binding of XRCC6BP1 to Ku70 suggested a role of this protein in NHEJ (Yang et al, 1999). Our preliminary results on the function of XRCC6BP1 in glioblastoma cell lines are consistent with the idea that XRCC6BP1 is involved in DSB repair (data not published).…”

“…A chromosome region on 12q is frequently amplified in glioblastoma and encompasses various genes crucial for the biology of this tumor including genes CDK4 and MDM2 (Reifenberger et al, 1996;Munnia et al, 2001). In addition, the amplified domain on chromosome 12q contains a gene encoding a Ku70-binding protein that was formerly termed KUB3 (Yang et al, 1999) and has been renamed XRCC6BP1. XRCC6BP1 shows amplification frequencies similar to CDK4 within the 12q amplicon and is overexpressed in glioblastoma-derived cell lines and in primary glioblastoma (Fischer et al, 2001).…”

Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer

“…16 Elucidation of the function of CLU in cells after stress is clearly complicated by the recognition of two different forms of the protein which apparently have two separate functions in the cell. A number of studies 1,5,13,16,17 have demonstrated that overexpression of the full-length CLU mRNA that would produce, depending on where the translation would start from and in this case, nCLU, acts as a prodeath signal, inhibiting cell growth and survival. However, other studies 2,6,8,10,[17][18][19] have shown that CLU expression may exert cytoprotective properties.…”

“…8 High levels of nCLU were reported to cause G 1 cell cycle arrest in distinct cell types. 16 The overall function of CLU expression in cell cycle regulation remains unclear. Use of short interfering RNAs (siRNAs) specific to each form of the CLU proteins should allow future studies to delineate cause-effect relationships and reveal whether sCLU or nCLU directly affect cell cycle regulation with or without cell stress.…”

“…In addition to the function of CLU in mediating apoptosis, 1,5,13,16,17 increasing evidence supports an important function for CLU in DNA repair. 23 Interestingly, CLU has been identified as the first stress-inducible protein that is associated with the DNA-dependent protein kinase (DNA-PK) double-strand break (DSB) repair complex.…”