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

Oncotarget. 2016 Dec 13;7(50):83241-83257. doi: 10.18632/oncotarget.13082.

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.

Keywords: DNA damage; PICT-1; nucleolar stress; nucleolus.

MeSH terms

  • Active Transport, Cell Nucleus
  • Apoptosis* / drug effects
  • Apoptosis* / radiation effects
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Calcium-Binding Proteins / metabolism
  • Cell Line, Tumor
  • Cell Nucleolus / drug effects
  • Cell Nucleolus / metabolism*
  • Cell Nucleolus / pathology
  • Cell Nucleolus / radiation effects
  • DNA Damage*
  • DNA Repair
  • HEK293 Cells
  • Humans
  • Mitomycin / pharmacology
  • Multienzyme Complexes
  • Mutation
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Binding
  • Proteolysis
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Ribosomal Proteins / metabolism*
  • Signal Transduction
  • Time Factors
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Ultraviolet Rays

Substances

  • CIB1 protein, human
  • Calcium-Binding Proteins
  • Multienzyme Complexes
  • NOP53 protein, human
  • Ribosomal Proteins
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • ribosomal protein L11
  • Mitomycin
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Proteasome Endopeptidase Complex