Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response

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.

Fig. 1.

Rapamycin induces abundance changes within nuclear proteome of HeLa cells. a, experimental design for quantitative proteomics analysis. For additional details, see “Experimental Procedures.” b, functional classification of nuclear proteins that changed in nuclear abundance 2-fold or more upon rapamycin treatment. c, normalized -fold changes in the levels of rapamycin-regulated proteins. The labels w, endo, and gfp in parentheses indicate Western blotting, immunostaining of endogenous proteins, and GFP fluorescence microscopy, respectively, which were used to validate mass spectrometry measurements.

Fig. 2.

Independent validation of proteomic changes across rapamycin-affected functional categories. a, validation of mass spectrometric results performed by immunostaining of endogenous (endo) or recombinant proteins (myc) or using GFP fluorescence (gfp) of transiently expressed proteins. HeLa cells were stained using antibody specific to the indicated proteins (endo) or transiently transduced to express GFP-tagged proteins (gfp) and visualized by fluorescence microscopy. b, validation of mass spectrometric results of nuclear abundance changes in HeLa cells by Western blotting. HeLa cell extracts were separated into cytoplasm (cyto) and nuclear (nuc) fractions as described under “Experimental Procedures,” and their cellular equivalents were loaded on SDS-PAGE prior to Western blotting. S6K1 and fibrillarin levels were monitored for assessing purity of cytoplasmic and nuclear fractions, respectively. VCP, valosin-containing protein; snRNP, small nuclear ribonucleoprotein.

Fig. 3.

mTOR inhibition activates cellular DNA damage response pathways. a, time course of the effects of rapamycin on nuclear (nu) and cytoplasmic (cyt) levels of 53BP1 in HeLa cells. -Fold changes for relative intensities of 53BP1 bands are given below the 53BP1 blot. b, nuclear and cytoplasmic levels of 53BP1 upon rapamycin treatment. The amount of cytoplasmic protein loaded was increased and nuclear protein amounts were proportionally decreased to test for translocation of 53BP1 by rapamycin. c, rapamycin enhances interaction between 53BP1 and p53. Immunoprecipitates (IP) were isolated from HeLa cells using either anti-p53 or anti-53BP1 antibody, and the amounts of associated 53BP1 and p53, respectively, were analyzed by Western blotting. d and e, rapamycin (3 h) and mTOR knockdown enhance phosphorylation of 53BP1 and H2AX in HeLa cells. f, rapamycin and mTOR silencing induced an increase in 53BP1 foci formation. HeLa cells treated with rapamycin/vehicle for 3 h or transduced with mTOR/control siRNA were monitored for the number of 53BP1 foci microscopically; error bars indicate calculated standard deviation from independent experiments. g, rapamycin induces phosphorylation of 53BP1 and H2AX in ATM^+/+ cells within 3 h but not in ATM^−/− cells. h, rapamycin (rapa) induced H2AX phosphorylation in WI38 and 293T cells. i, rapamycin inhibits extraction of nuclear Ku70 by salt treatment in HeLa nuclear preparations. HeLa cells treated with rapamycin/methanol for 3 h were processed for Ku70 or histone H1 (control) extractability from nuclear fractions under increasing NaCl concentrations as indicated. Numbers below blots indicate relative band intensities as determined by the Image J software. exp., exposure; sh, short hairpin.

Fig. 4.

Rapamycin-induced changes are mimicked by protein synthesis inhibition. a, effect of protein synthesis inhibitor cycloheximide (CHX; 50 μm, 90 min) on γ-H2AX and 53BP1 phosphorylation in HeLa cells. b, order of appearance of phosphorylation of 53BP1 and H2AX versus down-regulation of rapamycin-sensitive chromosomal integrity maintenance proteins upon cycloheximide treatment. Numbers indicate -fold changes in band intensities relative to those at no cycloheximide. c, effect of proteasomal inhibitor MG132 on rapamycin-induced phosphorylation of H2AX and down-regulation of the chromosomal integrity proteins. d, effect of mTOR knockdown, S6K1 knockdown, and 24 h of rapamycin treatment on levels of SNF2L2, TERT, NCAPD3, SMC2, and γ-H2AX.

Fig. 5.

Pretreatment with rapamycin enhances resistance of HeLa cells to radiation. HeLa cells treated with rapamycin/vehicle were exposed immediately to increasing doses of ionizing radiation (a) or after 3 (b) or 24 h (c) of pretreatment with rapamycin. Cell viability was measured as described under “Experimental Procedures.” The effective doses of Gy to reduce the number of cell colonies by 50% were 3.44 ± 0.43 (rapamycin−) and 3.75 ± 0.32 Gy (rapamycin+) for co-treatment, 3.32 ± 0.24 (rapamycin−) and 4.88 ± 0.26 Gy (rapamycin+) for 3-h pretreatment, and 2.79 ± 0.19 (rapamycin−) and 4.39 ± 0.21 Gy (rapamycin+) for 24-h pretreatment. d, rapamycin-induced resistance to radiation (when cells were pretreated for 3 or 24 h) correlated with reduced accumulation of γ-H2AX in response to ionizing radiation. Numbers below the blots show -fold change in protein levels as determined by the Image J software. exp., exposure.