PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway.
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.
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.|School of Life Sciences, Tsinghua University, Beijing 100084, China.|Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada.
Hongbo
H
Chen
Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China.|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.
Liqiao
L
Han
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.|School of Life Sciences, Tsinghua University, Beijing 100084, China.
Hsiangi
H
Tsai
Department of Laboratory Medicine, The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou 511447, China.
Zhiwei
Z
Wang
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.|School of Life Sciences, Tsinghua University, Beijing 100084, China.
Yanping
Y
Wu
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.
Yanhong
Y
Duo
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.|School of Life Sciences, Tsinghua University, Beijing 100084, China.
Wei
W
Cao
Technology Center of Guangxi Entry-Exit Inspection and Quarantine Bureau, Nanning 530021, China.
Lijun
L
Chen
Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Zhirong
Z
Tan
Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Ning
N
Xu
Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Xianzhang
X
Huang
Department of Laboratory Science, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Junhua
J
Zhuang
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.|School of Life Sciences, Tsinghua University, Beijing 100084, China.
Laiqiang
L
Huang
D021581:Active Transport, Cell Nucleus; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D057809:HEK293 Cells; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D009097:Multienzyme Complexes; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D009097:Multienzyme Complexes; C500712:NOP53 protein, human; D012269:Ribosomal Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C027624:ribosomal protein L11; D016685:Mitomycin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D046988:Proteasome Endopeptidase Complex
DNA damage; PICT-1; nucleolar stress; nucleolus