ATR 의존적 인산화에 의한 CHFR Ub-ligase의 활성 조절 연구

Abstract

CHFR is an E3 ubiquitin ligase and checkpoint protein that regulates cell cycle progression and tumorigenesis. Recently, it has been reported that CHFR is involved in cellular responses to DNA damage and plays a role in maintaining genome integrity. Functions of CHFR as a cell cycle checkpoint have been revealed by studies of its downstream targets such as PLK1, Aurora A and HDAC1. However, it still remains elusive how CHFR is regulated. In this study, I showed that CHFR is phosphorylated by ATR in response to DNA damage. ATR is a new CHFR-interacting protein and CHFR is phosphorylated by ATR in vitro. ATR is the master regulator of DNA damage response including cell cycle arrest, DNA repair and apoptosis. ATR activates the signaling pathway in response to DNA damage and replication stress. Therefore, I examined whether CHFR is phosphorylated upon DNA damage. In response to UV irradiation and doxorubicin treatment, CHFR is phosphorylated in vivo and it is ATR-dependent. There are five ATR consensus motifs (SQ or TQ) on CHFR. In order to narrow down the phosphorylation site, in vitro kinase assay was performed using CHFR deletion mutants. It was shown that the N-terminal region of CHFR is responsible for the phosphorylation. In addition, I found that CHFR is phosphorylated at threonine 130 in vivo upon UV irradiation. Furthermore, ATR-mediated phosphorylation of CHFR affects its checkpoint function. The phosphorylation-deficient mutant of CHFR does not exert anti-proliferative activity upon UV irradiation. Taken together, these results indicate that ATR-dependent phosphorylation is a new regulatory mechanism of CHFR for its checkpoint activity in response to DNA damage.