Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response

Mol Cell Proteomics. 2010 Feb;9(2):403-14. doi: 10.1074/mcp.M900326-MCP200. Epub 2009 Nov 23.

Abstract

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed down-regulation of nuclear abundance of many proteins involved in translation and RNA modification. Unexpectedly, mTOR inhibition also down-regulated several proteins functioning in chromosomal integrity and up-regulated those involved in DNA damage responses (DDRs) such as 53BP1. Consistent with these proteomic changes and DDR activation, mTOR inhibition enhanced interaction between 53BP1 and p53 and increased phosphorylation of ataxia telangiectasia mutated (ATM) kinase substrates. ATM substrate phosphorylation was also induced by inhibiting protein synthesis and suppressed by inhibiting proteasomal activity, suggesting that mTOR inhibition reduces steady-state (abundance) levels of proteins that function in cellular pathways of DDR activation. Finally, rapamycin-induced changes led to increased survival after radiation exposure in HeLa cells. These findings reveal a novel functional link between mTOR and DDR pathways in the nucleus potentially operating as a survival mechanism against unfavorable growth conditions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism*
  • Cell Nucleus / radiation effects
  • Cycloheximide / pharmacology
  • DNA Damage*
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Isotope Labeling
  • Proteasome Endopeptidase Complex / metabolism
  • Proteasome Inhibitors
  • Protein Biosynthesis / drug effects
  • Protein Biosynthesis / radiation effects
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / metabolism*
  • Proteome / metabolism
  • Proteomics / methods*
  • Radiation Tolerance / drug effects
  • Radiation Tolerance / radiation effects
  • Radiation, Ionizing
  • Reproducibility of Results
  • Signal Transduction / drug effects
  • Signal Transduction / radiation effects
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases

Substances

  • Intracellular Signaling Peptides and Proteins
  • Proteasome Inhibitors
  • Proteome
  • Cycloheximide
  • MTOR protein, human
  • Protein Serine-Threonine Kinases
  • TOR Serine-Threonine Kinases
  • Proteasome Endopeptidase Complex
  • Sirolimus