Hdm2 Is an Ubiquitin Ligase for the Anti-Apoptotic Protein Ku70.
Authors: Gama Vivian , Gomez Jose A. , Mayo Lindsey D. , Haas Arthur L. , Matsuyama Shigemi 
Published: October 2019

Abstract
Protection of endothelial cells (ECs) from inappropriate apoptosis induced by various stresses (e.g. drugs, parasites, and reactive oxygen species) is necessary to maintain homeostasis. On the other hand, blockade of angiogenesis in malignant tissue is a promising strategy to fight against cancer. However, the molecular mechanism of survival control of ECs is not well understood. Bax is a key mediator of apoptosis in various cell types including ECs. Ku70 is a subunit of the Ku complex involved in DNA repair and is ubiquitously expressed. Recently, we found that the cytosolic form of Ku70 binds Bax and inhibits Bax-mediated apoptosis, and that the decrease of cytosolic Ku70 is necessary to activate Bax. Furthermore, we reported that ubiquitin-dependent Ku70 proteolysis is involved in Ku70 decrease in ECs treated by genotoxic stresses. The remaining important problem was the identification of Ku70 ubiquitin ligase decreasing Ku70 to activate Bax in ECs. Here we report evidences showing that Hdm2 is an ubiquitin ligase of Ku70. It is known that Hdm2 ubiquitinylates p53, a tumor suppressor protein, and that Hdm2 level increases in response to DNA damage. Using purified recombinant proteins, we confirmed that Hdm2 conjugates ubiquitin chains to Ku70. Hdm2 overexpression in human umbilical vein endothelial cells (HUVECs) induced Ku70 decrease. Genotoxic stress (i.e. etoposide treatment) induced Ku70 decrease in Mdm2 (mouse version of Hdm2)-proficient mouse embryonic fibroblasts (MEFs) whereas Ku70 decrease was significantly suppressed in Mdm2-deficient MEFs. We verified Ku70-Hdm2 association by immunoprecipitation and GST pull-downs. Nutlin-3, a well-characterized inhibitor of Hdm2-p53 interaction, effectively disrupted Hdm2-Ku70 interaction, suggesting that Hdm2 uses the same domain to bind both p53 and Ku70. In cancer cells, it has been reported that the survival kinase Akt phosphorylates Hdm2, and that this phosphorylation stimulates nuclear translocation of Hdm2 to ubiquitinylate and inactivate p53 in the nucleus. We investigated whether Akt-dependent phosphorylation of Hdm2 influences its activity to regulate Ku70 level in HUVECs. Vascular endothelial growth factor (VEGF) is known to increase ECs survival through Akt activation. Thus, VEGF was employed as a physiological activator of Akt. VEGF inhibited etoposide-induced Ku70 degradation in HUVECs. Importantly, VEGF stimulated nuclear localization of Hdm2. Constitutively active AKT, which has been reported to induce Hdm2 nuclear translocation, but not the dominant negative AKT, efficiently inhibited Ku70 degradation. Moreover, an Akt phosphorylation resistant mutant of Hdm2 was able to down-regulate Ku70 but not p53 in HUVECs, suggesting a differential mechanism of regulation for these two substrates. In addition, Hdm2 mutant without ubiquitin ligase activity did not decrease Ku70 or p53, supporting the hypothesis that Hdm2 directly modulates these proteins. Taken together, we propose that
Hdm2 acts as a Ku70 ubiquitin ligase, and that VEGF-mediated activation of Akt prevents cytosolic Ku70 ubiquitinylation by Hdm2 probably by inducing Hdm2 translocation into the nucleus.
Our results suggest that Ku70 level in ECs is controlled by the balance of DNA damage-induced factor (Hdm2 and p53) and survival kinase (Akt), and that Ku70 plays an important role in the decision making process of apoptosis induction in ECs.