Konstantin S. Leskov, Timothy J. Kinsella, David A. Boothman*
Department of Radiation Oncology, Ireland Comprehensive Cancer Center
and Case Western Reserve University School of Medicine, 10900 Euclid Avenue,
BRB-326E, Cleveland, Ohio 44106-4942
ABSTRACT
We investigated the structure, function and intracellular localization of the nuclear form of Clusterin (nCLU), an apoptosis-associated protein induced by low doses of ionizing radiation (at least 2 cGy). Our analyses indicate that nCLU contains two coiled-coil domains, one on the N-terminus and the other within the C-terminal part of the protein. We demonstrated that the C-terminal coiled-coil domain binds to Ku70 protein via a leucine zipper-like mechanism. Our results also indicate that the C-terminal coiled-coil domain directly interacts with the N-terminal coiled-coil domain providing a clue for the intra-molecular folding of nCLU. There has been a controversy on whether nCLU overexpression leads to apoptosis or prevents it. Our functional analysis of nCLU domains demonstrated that the over-expression of GFP-fused nCLU and the C-terminal coiled-coil domain caused cell death as determined by Annexin V binding in the GFP-positive cell population. Other domains of nCLU did not increase apoptotic index. These data strongly argue that nCLU can cause cell death via its C-terminal leucine coiled-coil domain. Overexpression of nCLU, as well as the C-terminal coiled-coil domain in Ku70 knock-out cells also lead to increased cell death among the GFP-nCLU positive cells. These data suggest that nCLU binding to Ku70 is not necessary for nCLU-induced cell death. Our data are consistent with a model that upon induction by ionizing radiation, the C-terminal coiled-coil domain of nCLU dissociates from the N-terminal coiled-coil domain and binds to hydrophobic domains of Ku70 as well as other target proteins via its leucine zipper-like motif. We are currently identifying other key proteins that interact with nCLU and secretory CLU (sCLU), as well as determining the exact mechanism of death caused by accumulation of nCLU. These data will also be invaluable to better understand the potential bystander effects of secretory CLU (sCLU), which is induced by low doses of IR (~2 cGy in wt p53 cells and 0.5 cGy in mp53 cells) and may have potential bystander effects in IR-exposed or distant non-treated cells.
Supported by DOE grant #DE-FG02-99EQ62724 to D. A. B.