PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

// Hongbo Chen 1, 2, 6, * , Liqiao Han 3, 1, * , Hsiangi Tsai 1, 2, * , Zhiwei Wang 4 , Yanping Wu 1, 2 , Yanhong Duo 1 , Wei Cao 1, 2 , Lijun Chen 5 , Zhirong Tan 3 , Ning Xu 3 , Xianzhang Huang 3 , Junhua Zhuang 3 , Laiqiang Huang 1, 2 1 The Shenzhen Key Lab of Gene and Antibody Therapy, Center for Biotechnology & Biomedicine, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China 2 School of Life Sciences, Tsinghua University, Beijing 100084, China 3 Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China 4 Department of Laboratory Medicine, The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou 511447, China 5 Technology Center of Guangxi Entry-Exit Inspection and Quarantine Bureau, Nanning 530021, China 6 Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada * These authors have contributed equally to this work Correspondence to: Xianzhang Huang, email: huangxz020@163.com Junhua Zhuang, email: zjh2208@163.com Laiqiang Huang, email: huanglq@tsinghua.edu.cn Keywords: PICT-1, nucleolus, DNA damage, nucleolar stress Received: June 13, 2016      Accepted: October 19, 2016      Published: November 04, 2016 ABSTRACT PICT-1 is an essential ribosome biogenesis factor whose loss induces p53 accumulation and apoptosis. Here, we show that DNA damage changes PICT-1 localization and decreases PICT-1 protein levels via the proteasome pathway. Two important phosphatidylinositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and the Ku70 subunit of DNA-dependent protein kinase (DNA-PK), co-localize and interact with PICT-1 in the nucleolus. Computational prediction of phosphorylation sites and detection using an anti-phospho-substrate antibody suggest that PICT-1 might be a substrate of PIKKs. PICT-1 S233 and T289 were identified as the key phosphorylation sites in this pathway, as mutating both to alanine abolished UVB-induced increase of PICT-1 phosporylation. Inhibition of PIKKs or ATM (with wortmannin and KU55933, respectively) prevented the agglomeration and degradation of PICT-1, suggesting that ATM is a key regulator of PICT-1. PICT-1(S233A, T289A) demonstrated marked resistance to DNA damage-induced agglomeration and loss of PICT-1. Phosphomimetic PICT-1 (S233D, T289D) showed a different nuclear distribution and was more rapidly degraded after DNA damage than wild-type PICT-1. Furthermore, both phosphorylation and degradation of PICT-1 released RPL11 from the nucleolus to the nucleoplasm, increased binding of RPL11 to MDM2, and promoted p53 accumulation and apoptosis in an ATM-dependent manner after DNA damage. These data indicate that PICT-1 is a major nucleolar sensor of the DNA damage repair response and an important upstream regulator of p53 via the RPL11-MDM2-p53 pathway.