PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway
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Hongbo Chen1,2,6,*, Liqiao Han3,1,*, Hsiangi Tsai1,2,*, Zhiwei Wang4, Yanping Wu1,2, Yanhong Duo1, Wei Cao1,2, Lijun Chen5, Zhirong Tan3, Ning Xu3, Xianzhang Huang3, Junhua Zhuang3, Laiqiang Huang1,2
1The 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
2School of Life Sciences, Tsinghua University, Beijing 100084, China
3Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China
4Department of Laboratory Medicine, The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou 511447, China
5Technology Center of Guangxi Entry-Exit Inspection and Quarantine Bureau, Nanning 530021, China
6Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada
*These authors have contributed equally to this work
Xianzhang Huang, email: [email protected]
Junhua Zhuang, email: [email protected]
Laiqiang Huang, email: [email protected]
Keywords: PICT-1, nucleolus, DNA damage, nucleolar stress
Received: June 13, 2016 Accepted: October 19, 2016 Published: November 04, 2016
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.
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