Research ArticleDNA damage

ATM directs DNA damage responses and proteostasis via genetically separable pathways

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Sci. Signal.  09 Jan 2018:
Vol. 11, Issue 512, eaan5598
DOI: 10.1126/scisignal.aan5598
  • Fig. 1 Disruption of MRN- and DNA-dependent ATM activation by R2579A/R2580A mutations of ATM.

    (A) Schematic diagram of ATM structure; mutations analyzed in this study are indicated. Features and domains within ATM consist of the nuclear localization signal (NLS), the FRAP/ATM/TRRAP (FAT), the kinase domain (PI3K), and the FAT C-terminal (FATC) domain. (B and C) Kinase assays with dimeric wild-type (WT) or R2579A/R2580A (2RA) ATM (1.35 nM), Mre11/Rad50/Nbs1 (MRN) (9.6 nM), glutathione S-transferase (GST)–p53 substrate (6.25 nM), and linear DNA (~140 nM) probed with antibody to phospho-Ser15 of p53. Data are means ± SD of three independent experiments, showing the fold change in phospho-signal with MRN/DNA relative to the reactions without MRN/DNA. (D) Kinase assays with dimeric WT or 2RA ATM (2.7 nM) and GST-p53 substrate (12.5 nM) in the presence of H2O2 (0.27, 0.81, and 2.4 mM). (E and F) Binding assays with WT or 2RA ATM and biotinylated MRN (E) or Mre11/Rad50 (MR) (F) (20 nM; incubated with 50 nM ATM and isolated with streptavidin-coated magnetic beads), as indicated. Bound proteins were visualized by Western blotting with antibodies against ATM, Nbs1, or Rad50. (G and H) Kinase assays as in (A) and (B) except with triple-mutant ATM (R2579A/R2580A/C2991L, “2RA + CL”). (I and J) Kinase assays as in (A) and (B) except using heterodimeric ATM (WT/2RA and 2RA/CL) as indicated.

  • Fig. 2 Separation-of-function mutations in ATM dictate responses to DNA damage and oxidative stress.

    Human U2OS osteosarcoma cells were depleted for endogenous ATM using short hairpin RNA (shRNA) and induced to express various ATM alleles as indicated. (A) Cells were induced for ATM expression with doxycycline (10 ng/ml) and exposed to camptothecin (CPT) (10 μM) for 1 hour. ATM activity was examined using antibodies directed against phospho-Kap1 Ser824, and ATM levels were assessed with anti-ATM antibody. (B) U2OS cells were treated as in (A) except that expression was induced with doxycycline (1 μg/ml). ATM activity was assessed with antibodies directed against ATM, phospho-ATM Ser1981, Kap1, phospho-Kap1 Ser824, Chk2, and phospho-Chk2 Thr68 as indicated. (C) U2OS cells expressing shRNA against ATM (shATM) and either vector (CTRL) or various ATM alleles as in (B) were treated with 10 μM CPT for 1 hour, and the levels of phosphorylated Kap1 were determined, in comparison to total Kap1 protein, and normalized with the phosphorylated signal from WT ATM–expressing cells. NS, not significant. (D) U2OS cells were depleted of endogenous ATM and induced for recombinant ATM expression as in (B) but were exposed to IR (10 Gy) followed by 1-hour recovery. Phosphorylation was assessed as in (B). (E) U2OS cells were treated as in (B) but exposed to 100 μM H2O2 or arsenite for 1 hour in the presence of doxycycline (1 μg/ml). Phosphorylation was assessed as in (B). (F) U2OS cells expressing various ATM alleles as in (B) were treated with 100 μM H2O2 for 1 hour in serum-free medium, and the amount of phosphorylated Chk2 was quantitated in comparison to total Chk2 protein and normalized with the phosphorylated signal from WT ATM–expressing cells. Data are means ± SD of three independent experiments. *P < 0.05 and **P < 0.005.

  • Fig. 3 ATM deficient in activation via MRN exhibits defects in survival of DNA damage and in DSB resection.

    (A to C) U2OS cells depleted for endogenous ATM (shATM) or treated with a control shRNA (shCTRL) were induced to express vector only (CTRL) or various ATM alleles [doxycycline (1 μg/ml)] as indicated and were analyzed for cell survival after treatment with IR (A), CPT (B), or arsenite (C) as indicated. Data are means ± SE of three independent experiments. P values (inset) were assessed by Student’s t test. (D) DNA end resection at Asi SI–induced breaks in cells expressing various ATM alleles. Endogenous ATM in U2OS–ER–Asi SI cells was depleted by shRNA treatment, and cells were complemented with induced expression of ATM alleles [doxycycline (1 μg/ml)] as indicated, 3 days before treatment with 4-hydroxytamoxifen (600 nM for 4 hours), which induces nuclear translocation of the Asi SI enzyme (98). Harvested genomic DNA was either digested with restriction enzymes to distinguish between single-stranded DNA (ssDNA) and double-stranded DNA or mock-digested as described previously (99). Quantitation of single-stranded DNA intermediates generated by resection was performed by real-time polymerase chain reaction. Data are means ± SD of three experiments. *P < 0.05 and ***P < 0.0005. DSB, double-strand break; nt, nucleotides; DA, D2889A. (E and F) U2OS cells depleted of endogenous ATM with shRNA and inducibly expressing various ATM alleles [doxycycline (1 μg/ml)] were synchronized in G1–early S with aphidicolin (2 μg/ml; 17 hours). The intra-S cell cycle checkpoint was analyzed by quantification of the percentage of G2-M cells 17 hours after removal of aphidicolin and treatment with CPT (1 μM) (E) or arsenite (100 μM) (F) compared with untreated group, as indicated. Data are means ± SE of three independent experiments. **P < 0.005.

  • Fig. 4 The mechanism of ATM activation determines the functional response of ATM in cell cycle checkpoint regulation and in ROS homeostasis.

    (A and B) Reactive oxygen species (ROS) levels were measured in U2OS cells depleted of endogenous ATM with shRNA (Fig. 2) and inducibly expressing various ATM alleles [doxycycline (1 μg/ml)] using the general ROS indicator 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (A) or a probe for superoxide levels, dihydroethidium (DHE) (B). Fluorescence was analyzed by flow cytometry using 10,000 cells per cell line and normalized with data from cells expressing WT ATM. (C) Cells expressing various ATM alleles as indicated were analyzed for membrane potential–dependent mitochondrial mass using MitoTracker Red staining, followed by analysis using flow cytometry with 10,000 cells per cell line and normalization with data from cells expressing WT ATM. Data are means ± SD of three independent experiments. *P < 0.05 and **P < 0.005. (D) U2OS Flp-In T-REx cells expressing WT, CL, or 2RA alleles were infected with retrovirus containing the mKeima mitochondria-targeted pH indicator, and pH changes in the mitochondria were measured by comparison of the emission when excited at 561 or 488 nm. The y axis shows the ratio of the emission values, normalized to the cells expressing the WT allele. Quantitation of the mean (± SD) emission ratios (561 nm/488 nm) each from four independent experiments with 10,000 cells is analyzed per cell line. ***P < 0.0005. (E) Acridine orange was used to stain acidic vesicular organelles (AVOs) in U2OS cells expressing WT, CL, or 2RA alleles, with either no treatment, CPT (5 μM), or arsenite treatment (100 μM) as indicated. The autophagy inhibitor spautin-1 (10 μM) was used to confirm that acridine orange reflects autophagy-dependent vesicles. (F) Acridine orange staining in U2OS cells expressing WT, CL, or 2RA alleles with either no treatment, CPT (5 μM), or arsenite treatment (100 μM) as indicated was quantified by fluorescence-activated cell sorting, showing here the fold increase in the percentage of cells with 585-nm emission relative to untreated cells. Data are means ± SD of three biological replicates with about 5000 cells measured per replicate. *P < 0.05. (G and H) U2OS cell lines expressing WT or mutant cell lines as indicated were treated with CPT (5 μM) or arsenite (100 μM) for 120 min, followed by 48-hour recovery, and then blotted for LC3-II and LC3-I. Data are means ± SD of three independent biological replicates, plotted as fold increase relative to the untreated controls. *P < 0.05, **P < 0.005.

  • Fig. 5 Phosphoproteomic analysis of A-T patient lymphoblast cells expressing an oxidation-deficient ATM mutant shows defects in global phosphorylation patterns.

    (A) AT1ABR lymphoblast cells deficient in WT ATM were complemented with inducible expression of WT, CL, 2RA, or 2RA + CL ATM alleles. Phosphopeptides were enriched and analyzed by mass spectrometry (MS). A histogram of total phosphopeptide counts (log2 values) in each cell line is shown after normalizing raw values for each cell line to the parental AT1ABR cell line. (B) Pairwise comparisons of the phosphoproteomic raw data from AT1ABR cells with or without expression of ATM. The raw intensity of each phosphopeptide was analyzed in pairwise comparisons of each cell line, and Spearman’s rank correlation coefficient, ρ, was calculated for each pairwise comparison. (C) Hierarchical clustering of all the phosphopeptides. Phosphopeptide levels were normalized to the parental AT1ABR cell line. Each vertical line within a cell line is a phosphopeptide and varies from green to black to red to represent decreased, equal, or increased levels of the phosphopeptides compared to the parental AT1ABR cell line, respectively. (D) Phosphopeptides from the phosphoproteome or the C2991 Dependent Cluster [indicated by the yellow box in (C)] were analyzed with motif-x. Sequences contained six residues N-terminal and C-terminal of each phosphorylation event. Fold enrichment is shown relative to the abundance of motifs in the proteome. (E and F) Empirical cumulative distribution functions of the ratio of phosphopeptide intensities of cells expressing WT ATM or cells expressing the CL allele, comparing the phosphoproteome and the predicted ATM phosphoproteome (E) or the predicted CK2 phosphoproteome (F). (G and H) Results from Kolmogorov-Smirnov tests showing the predicted number of phosphopeptides in the data set for each kinase and the P value of the observed changes in the CL- and 2RA + CL–expressing cell lines relative to cells expressing the WT allele. The red line marks P = 0.05.

  • Fig. 6 Loss of ATM oxidative stress activation causes aggregation of CK2β.

    (A) In vitro kinase assay with immunoprecipitated CK2. HEK-293T cells stably expressing V5-tagged CK2β were treated with 10 μM ATM inhibitor (KU-55933) or an equivalent amount of dimethyl sulfoxide (DMSO) for 16 hours, and CK2β was immunoprecipitated with magnetic anti-V5 beads. CK2 was incubated with 1.67 mM ATP, 5 fCi of [γ-32P]ATP, and 1.15 μg of GST-CK2 substrate for 1 hour, and 32P-labeled substrate was analyzed by PhosphorImager. (B) Normalized amount of immunoprecipitated (IP) CK2β-V5 from (A) with the levels of CK2β-V5 normalized to levels in mock-treated cells. Quantitation was performed on the LI-COR system using Image Studio version 4.0. *P < 0.05. (C to E) Distribution profile of CK2 subunits CK2α (C), CK2α′ (D), and CK2β (E) in HEK-293T cells after sucrose gradient sedimentation. HEK-293T cells expressing CK2α-V5, CK2α′-V5, or CK2β-V5 were treated with 10 μM KU-55933 or an equivalent amount of DMSO for 16 hours, harvested, and lysed in the absence of detergent. One milligram of lysate in a total volume of 500 μl of lysis buffer was added to the top of a sucrose gradient made with 1-ml layers of 50% to 5% sucrose in 5% increments. After ultracentrifugation, 500-μl fractions were collected and analyzed by Western blotting. (F and G) Distribution profile of CK2α (F) and CK2β (G) from the AT1ABR cells expressing WT or CL ATM alleles, analyzed by sucrose gradient sedimentation. Lysates were analyzed as in (C) to (E) except with induced AT1ABR cells expressing WT or CL alleles of ATM as indicated. (H) Sucrose gradient sedimentation pattern of V5-tagged CK2β stably expressed in U2OS cells treated with 10 μM KU-55933 or an equivalent amount of DMSO for 16 hours or treated with a combination of KU-55933 and 1 mM N-acetylcysteine (NAC), as indicated. (I) Analysis of detergent-resistant aggregates in U2OS cells treated with 10 μM KU-55933 or DMSO or a combination of KU-55933 and 1 mM NAC, as indicated. Aggregate fractions were isolated and compared with total lysate using Western blotting for stably expressed V5-tagged CK2β. (J) Analysis of detergent-resistant aggregates in U2OS Flp-In T-REx cells expressing WT, CL, or 2RA ATM treated with 25 μM arsenite as shown in (I). (K) Aggregate fractions were isolated from U2OS cells expressing shCTRL or ATM shRNA and probed for CK2β, ATM, and β-actin as indicated from two independent cultures. (L) Means ± SD of CK2β abundance in aggregates, quantified from three independent experiments [including the replicates shown in (K)]. **P < 0.005.

  • Fig. 7 Loss of ATM oxidative stress activation causes global protein aggregation.

    (A) U2OS cells depleted for endogenous ATM and inducibly expressing WT, CL, or 2RA alleles with or without concurrent arsenite treatment (25 μM) were lysed, and detergent-resistant aggregate fractions were isolated and analyzed by MS using label-free quantification. Three biological replicates were analyzed for each cell line, and proteins enriched by ≥1.5-fold in cells expressing the CL or 2RA alleles compared to the level in cells expressing WT ATM are shown (only those with P < 0.05 by a t test). (B) Fold enrichment over WT for proteins identified in (A) for each target. (C) Gene ontology analysis of proteins enriched in aggregate fraction of cells expressing the CL allele with arsenite treatment (Panther Gene Ontology database). The analysis included Bonferroni correction for multiple testing; all results shown had P < 0.05. (D) Comparison of TANGO and WALTZ scores for 497 polypeptides isolated from U2OS cells expressing the CL allele with arsenite treatment in (A) compared to the entire proteome. Polypeptide length is shown in amino acids. n = 3 biological replicates per sample; P values by two-tailed t test. CI, confidence interval.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/512/eaan5598/DC1

    Fig. S1. Recombinant ATM protein expression.

    Fig. S2. Arsenite and peroxide treatments do not induce DSBs in ATM-inducible U2OS cell lines.

    Fig. S3. The 2RA ATM mutant is deficient in CPT-induced KAP1 phosphorylation.

    Fig. S4. Expression of kinase-deficient ATM sensitizes cells to exogenous stress.

    Fig. S5. U2OS cells expressing wild-type and mutant alleles of ATM have equivalent ratios of cells in S phase.

    Fig. S6. Mitochondrial dysfunction in AT1ABR cells expressing CL ATM.

    Fig. S7. The mKeima mitochondria-targeted pH probe responds to induction or repression of mitophagy.

    Fig. S8. ATM depletion reduces macroautophagy responses of human cells in response to DNA damage or oxidative stress.

    Fig. S9. ATM mutant expression does not alter the predicted phosphorylation targets of CK1 or CDK.

    Fig. S10. CK2 subunit levels are not reduced in A-T cells expressing the CL ATM allele.

    Fig. S11. Expression of CL ATM causes Rad50 accumulation in protein aggregates.

    Table S1. Phosphorylated peptides observed in AT1ABR cells with inducibly expressed wild-type, CL, 2RA, or 2RA/CL ATM alleles.

    Table S2. Proteins detected in aggregate fractions.

  • Supplementary Materials for:

    ATM directs DNA damage responses and proteostasis via genetically separable pathways

    Ji-Hoon Lee, Michael R. Mand, Chung-Hsuan Kao, Yi Zhou, Seung W. Ryu, Alicia L. Richards, Joshua J. Coon, Tanya T. Paull*

    *Corresponding author. Email: tpaull{at}utexas.edu

    This PDF file includes:

    • Fig. S1. Recombinant ATM protein expression.
    • Fig. S2. Arsenite and peroxide treatments do not induce DSBs in ATM-inducible U2OS cell lines.
    • Fig. S3. The 2RA ATM mutant is deficient in CPT-induced KAP1 phosphorylation.
    • Fig. S4. Expression of kinase-deficient ATM sensitizes cells to exogenous stress.
    • Fig. S5. U2OS cells expressing wild-type and mutant alleles of ATM have equivalent ratios of cells in S phase.
    • Fig. S6. Mitochondrial dysfunction in AT1ABR cells expressing CL ATM.
    • Fig. S7. The mKeima mitochondria-targeted pH probe responds to induction or repression of mitophagy.
    • Fig. S8. ATM depletion reduces macroautophagy responses of human cells in response to DNA damage or oxidative stress.
    • Fig. S9. ATM mutant expression does not alter the predicted phosphorylation targets of CK1 or CDK.
    • Fig. S10. CK2 subunit levels are not reduced in A-T cells expressing the CL ATM allele.
    • Fig. S11. Expression of CL ATM causes Rad50 accumulation in protein aggregates.
    • Legends for tables S1 and S2

    [Download PDF]

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Phosphorylated peptides observed in AT1ABR cells with inducibly expressed wild-type, CL, 2RA, or 2RA/CL ATM alleles.
    • Table S2 (Microsoft Excel format). Proteins detected in aggregate fractions.

    [Download Tables S1 and S2]


    Citation: J.-H. Lee, M. R. Mand, C.-H. Kao, Y. Zhou, S. W. Ryu, A. L. Richards, J. J. Coon, T. T. Paull, ATM directs DNA damage responses and proteostasis via genetically separable pat. Sci. Signal. 11, eaan5598 (2018).

    © 2018 American Association for the Advancement of Science

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