S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Studies on the effect of DBC1 modification by SUMO2/3 on p53-mediated apoptosis in response to DNA damage
DNA 손상시 유도되는 DBC1의 수모화가 p53에 의해 매개되는 세포사멸에 미치는 영향에 관한 연구
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 8. 정진하.
- SIRT1, a mammalian ortholog of yeast silent interaction regulator 2 (Sir2), is a NAD+-dependent histone III deacetylase. SIRT1 regulates various cellular processes, such as apoptosis, stress response, tumorigenesis, and metabolism. Tumor suppressor p53 is a main target for SIRT1. Under normal conditions, p53 is deacetylated by SIRT1, inactivated, and degraded by MDM2, the major ubiquitin E3 ligase. Under stress conditions (e.g., exposure to UV or etoposide), however, p53 is acetylated by p300/CBP, dissociated from MDM2 for stabilization, and activated, resulting in p53-mediated induction of cell cycle arrest or apoptosis.
Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is conjugated to a variety of cellular proteins. Like ubiquitin, SUMO is conjugated to target proteins by a three enzyme cascade system consisting of SUMO-activating E1 enzyme (SAE1/SAE2), SUMO-conjugating E2 enzyme (Ubc9), and SUMO E3 ligases (PIASs). Conjugated SUMO can be removed by a family of Sentrin-specific proteases (SENPs). This reversible sumoylation process regulates diverse cellular processes, including transcription, nuclear transport, stability, and signal transduction.
Deleted in breast cancer 1 (DBC1) is a tumor suppressor that plays crucial roles in the control of diverse cellular processes, including stress response and energy metabolism. DBC1 is a major inhibitor of SIRT1. Under DNA damage conditions, DBC1 binds to SIRT1 and this tight binding displaces p53 from SIRT1, allowing acetylation and transactivation of p53 for expression of its downstream targets, such as p21, BAX, and PUMA. However, how the function of DBC1 is regulated remained unknown.
Phosphorylation of DBC1 regulates DBC1-SIRT1 interaction and SIRT1 deacetylase activity. Under stress conditions, ATM/ATR kinases are activated and phosphorylates DBC1 at Thr454. This phosphorylation causes tight binding between DBC1 and SIRT1, leading to dissociation of p53 from SIRT1 for subsequent acetylation and transactivation of p53.
In this study, I demonstrated that DBC1 is a target for SUMO modification and that Lys591 serves as the major SUMO acceptor site. Treatment with DNA-damaging agents, such as etoposide and doxorubicin, induced sumoylation of endogenous DBC1. In addition, DBC1 was modified by SUMO2 and SUMO3, but not by SUMO1. Remarkably, this sumoylation of DBC1 promoted its interaction with SIRT1, leading to p53 acetylation.
PIAS3 was found to act as a DBC1-specific SUMO E3 ligase and SENP1 was to serve as DBC1-specific desumoylation enzyme. Interestingly, PIAS3 and SENP1 interacted to the same N-terminal region of DBC1 and therefore competed with each other for binding to DBC1. Etoposide treatment reduced the interaction of DBC1 with SENP1, but promoted that with PIAS3, resulting in an increase in DBC1 sumoylation. Remarkably, the switching from SENP1 to PIAS3 for DBC1 binding was achieved by ATM/ATR-mediated phosphorylation of DBC1. These results demonstrate that PIAS3 and SENP1 antagonistically regulate SUMO modification of DBC1. Consistently, SENP1 knockdown promoted etoposide-induced apoptosis, whereas knockdown of PIAS3 or SUMO2/3 and overexpression of sumoylation-deficient DBC1 mutant inhibited it. Collectively, the present findings indicate that SUMO modification of DBC1 by SUMO2/3 plays a crucial role in p53-mediated apoptosis under DNA damage conditions.