Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response.
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.
Bandhakavi
Sricharan
S
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Kim
Young-Mi
YM
Ro
Seung-Hyun
SH
Xie
Hongwei
H
Onsongo
Getiria
G
Jun
Chang-Bong
CB
Kim
Do-Hyung
DH
Griffin
Timothy J
TJ
D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007553:Isotope Labeling; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis / Q000187:drug effects / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011836:Radiation Tolerance / Q000187:drug effects / Q000528:radiation effects; D011839:Radiation, Ionizing; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases
D047908:Intracellular Signaling Peptides and Proteins; D061988:Proteasome Inhibitors; D020543:Proteome; D003513:Cycloheximide; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus