Molecular Mechanism for the Downregularion of KU70 in Apoptotic Cells.

Bax is a pro-apoptotic protein that plays a key role in apoptosis. Ku70 has been known as a DNA repair protein in the nucleus. Recently, we found that the cytosolic form of Ku70 has an anti-apoptotic activity to suppress the cytotoxic activity of Bax. Previously, we reported that apoptotic stimuli decreased Ku70 levels in human cancer cell lines such as HeLa cells and human epithelial kidney (HEK) 293T cells, and that the Ku70 disappearance is a caspase-independent event in the early phase of apoptosis in these cell lines. Based on these results, we hypothesized that the down regulation of cytosolic Ku70 levels is one of the mechanisms to trigger Bax-mediated cell death. In this study, (1) we examined whether Ku70 level decrease is a common apoptotic event in various cell types including primary normal (non-cancerous) cells, cancer cells and cells from rat kidney, and (2) we also investigated the molecular mechanism of Ku70 degradation in apoptotic cells.

The effects of apoptotic stimuli on the cellular Ku70 levels were examined in 32D (EpoR wt) cells (IL-3-deprivation-induced apoptosis), the primary mouse mast cells (IL-3-deprivation-induced apoptosis), Human Umbilical Vein Endothelial Cells (HUVEC) (doxorubicin treatment), a human megakaryocytic cell line (DAMI cells) (etoposide treatment) and rat kidney cells (ischemia/reperfusion treatment). Ku70 levels were monitored by Western blot analysis. In all the systems examined, the decrease of Ku70 levels was confirmed, supporting our hypothesis that the down regulation of Ku70 is required for the induction of Bax-mediated cell death.

To examine the possibility that apoptotic stimuli affect the transcription of Ku70 mRNA, real-time RT-PCR analysis was performed. Apoptotic stimuli did not change Ku70 mRNA levels in HeLa cells (staurosporin treatment) and DAMI cells(etoposide treatment), suggesting that apoptotic stimuli control Ku70 levels by post-translational modification. Although, there is no previous report of the ubiquitination of Ku70, we found that a large proportion of cellular Ku70 proteins are constitutively ubiquitinated in HeLa cells, HEK293T cells, DAMI cells, and in rat kidney cells. Higher molecular weight bands corresponding to ubiquitinated Ku70 become detectable only when Ku70 proteins are enriched by immunoprecipitation. MG132, a proteosome inhibitor, increased the accumulation of ubiquitinated Ku70, suggesting that ubiquitinated Ku70 proteins are subjected to proteosome-dependent degradation. Furthermore, in response to apoptotic stimuli, the levels of ubiquitinated Ku70 increased, probably accounting at least in part, for the decrease of non-modified Ku70 seen during apoptosis.

In summary, we found that (1) apoptotic stimuli decrease cellular Ku70 levels in various cell types including cancer and normal cells, and that (2) ubiquitination of Ku70 is one of the mechanisms to down regulate Ku70 levels in apoptotic cells. Ku70 has multiple functions to protect cells from apoptosis and DNA damage. Our results indicate that the stability and probably the function of Ku70 can be regulated by ubiquitination. Identification of the ubiquitin ligase and the de-ubiquitinating enzyme will be needed to understand the precise mechanism of how stress response of cells regulates Ku70 function maintaining cell survival and genomic stability.