www.sciencemag.org/cgi/content/full/330/6003/517/DC1

Supporting Online Material for

ATM Activation by Oxidative Stress

Zhi Guo, Sergei Kozlov, Martin F. Lavin, Maria D. Person, Tanya T. Paull*

*To whom correspondence should be addressed. E-mail: tpaull@mail.utexas.edu

Published 22 October 2010, Science 330, 517 (2010)

DOI: 10.1126/science.1192912

This PDF file includes:

Materials and Methods Figs. S1 to S12 Tables S1 and S2 References

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Table S1. Summary of ATM mutants and characterization in vitro

Recombinant ATM proteins were expressed and purified with the mutations in the cysteine residues shown above. Some of these sites were chosen for reasons of evolutionary conservation or identification in an A-T patient (1) or tumor (2), or as part of a CXXC motif, while others were identified in a mass spectrometry-based screen for peptides in ATM that undergo oxidation into disulfides, "mass spec". Other were identified using a cyanylation technique that cleaves a polypeptide at sites of disulfide bond formation (3). None of the mutants shown above showed any decrease relative to wild-type protein in activation by H2O2 in vitro. Some were also tested for MRN/DNA activation, while others were not tested, "ND".

Table S2. MS/MS MASCOT scores for cysteine modified peptides in ATM+/- hydrogen peroxide treatment.

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Supplementary Figures

Fig. S1. H2O2 activates ATM in 293T cells. Human 293T cells were treated with 0.5 mM H2O2 for 30 minutes or with 5 Gy of IR. Cell lysates were analyzed by SDS-PAGE and western blotting for phospho-ATM Ser1981 (A), phospho-p53 Ser15 (B), phospho-Chk2 Thr68 (C), phospho-histone H2AX Ser140 (D), phospho-Kap1 Ser824 (E), or the non-phosphorylated proteins as indicated. 293T cells were treated with the ATM inhibitor KU-55933 for 1 hour before the addition of H2O2 and assayed for phospho-ATM Ser1981 (F), phospho-p53 Ser15 (G), or phospho-Chk2 Thr68 (H) as in (A).

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D

Fig. S4. H2O2 stimulates ATP binding by ATM. ATM was treated with 0.8 mM H2O2 and the stimulation in p53 phosphorylation was measured with different concentrations of ATP in the presence of 2.5 mM manganese chloride. Phosphorylation of p53 on serine 15 was analyzed by western blotting and analyzed with antibody directed against phospho-p53 Ser15. The Y axis shows the ratio of the level of p53 phosphorylation in the presence of H2O2 over the level of p53 phosphorylation in the absence of H2O2. Three independent experiments were performed and the average of these is shown here with the error bars indicating the standard deviation. Phosphorylation of p53 on serine 15 was quantified with Odyssey software (Li-Cor).

Fig. S2. SDS-PAGE of purified ATM. WT homodimers (WT-WT), C2991L homodimers (CL-CL), and WT-C2991L heterodimers (WT-CL) of ATM were separated by SDS-PAGE and stained with Coomassie blue (left panel). Wild-type and R3047X ATM protein is shown in the right panel.

Fig. S3. Recombinant ATM purified and stored in the absence of reducing agents is hyperactive. A kinase assay was performed with wild-type ATM as in Fig. 2A, except with ATM that was purified and stored without DTT. Normally ATM is purified and stored in the presence of 1 mM DTT. Concentrations of H2O2 were 0.2 and 2 mM, respectively.

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Fig. S5. Alignment of ATM FATC domains. The position of C2991 is shown with an asterisk.

Fig. S6. The ATM C2991L mutant still forms disulfide-crosslinked dimers. Recombinant ATM protein was exposed to H2O2(0.27, 0.81 and 2.43 mM, respectively) and separated by SDS-PAGE in the absence of reducing agents, followed by western blotting and probing with an antibody directed against ATM, as described for Fig. 2E. "M" and "D" indicate positions of monomer and dimer ATM.

Fig. S7. Induction of wild-type and C2991L ATM in AT1-ABR cells. AT1-ABR cells stably transduced with wild-type or C2991L ATM alleles were induced for 16 hrs with 2 μM CdCl2 and cell lysates were analyzed for ATM expression by western blotting with an antibody directed against ATM.

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Fig. S8. The C2991L mutation blocks apoptosis in lymphoblasts induced by oxidative stress, as measured by propidium iodide staining. AT1-ABR cells inducibly expressing either wild-type or C2991L ATM alleles were treated with H2O2 (25 μM) or camptothecin (5 μg/ml). The cleavage of nuclear DNA during apoptosis was measured using propidium iodide (PI) staining. The cells expressing the C2991L allele does not show a significant increase in PI staining after H2O2 exposure.

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Fig. S9. The C2991L mutation blocks apoptosis in lymphoblasts induced by oxidative stress, as measured by Annexin V staining. AT1-ABR cells inducibly expressing either wild-type or C2991L ATM alleles were treated with H2O2 (25 µM) or camptothecin (5 µg/ml). The loss of membrane integrity during apoptosis was measured using FITC-annexin V staining. The cells expressing the C2991L allele does not show a significant increase in Annexin V staining after H2O2 exposure.

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Fig. S10. The C2991L mutation blocks ATM oxidative activation in human cells. (A) Human 293 cells stably expressing wild-type or C2991L alleles of ATM were treated with H2O2 (100 μM) or bleomycin (10 μg/ml). Levels of ATM and phosphorylation of p53 and Chk2 were assayed as in Fig. 1A. (B, C) Three independent experiments as in (A) were performed and the average is shown with the error bars indicating the standard deviation. The Y axis indicates the level of phosphorylated protein in the treated sample relative to the untreated sample.

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Fig. S11. The Chk2 phosphorylation seen in R3047X lymphocytes is dependent on ATM. (A, B) R3047X A-T lymphocytes were treated with camptothecin (10 μg/ml) or H2O2 (12.5 μM) with the ATM inhibitor KU-55933 as indicated. Cell lysates were analyzed by SDS-PAGE and western blotting for phospho-Chk2 Thr68, or non-phosphorylated Chk2. (C, D, E) Wild-type lymphocytes was analyzed as described for (A, B).

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Fig. S12. Model for ATM activation via oxidation, in which ATM converts from a non-covalent, inactive dimer into a disulfide-linked, active dimer containing several disulfide bonds (red lines). This activation pathway is different from MRN/DNA-dependent activation in which MRN facilitates monomer formation, although we hypothesize that these two activated forms may share common conformations of the kinase domain. Substrate is shown as a grey shape, "sub"; Rad50 is shown as a blue octagon with connecting coiled-coils; Nbs1 is shown as a purple diamond; Mre11 is shown as a yellow oval. The diagram is not meant to indicate the exact stoichiometry of MRN. Both subunits of ATM in the active dimer are shown binding substrate, but this aspect of the model has not yet been tested.

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Supplementary Methods

Tissue culture

Primary human fibroblasts (GM08399, Coriell Cell Repository) were cultured in

Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal

bovine serum (FBS) (Invitrogen) and 2 mM L-glutamine. 293T cells (CRL-12688, ATCC)

were cultured in DMEM supplemented with 10% animal serum complex (Gemini). All

cells were grown in a CO2 (5%) incubator, at 37°C. AT1-ABR cell lines inducibly

expressing WT or C2991L ATM were established using the ATM cDNA expression

vector pMAT1 essentially as described previously (4). ATM cDNA was expressed under

the control of the heavy-metal inducible methallothionein promoter in the pMEP4 vector

(Invitrogen). Mutagenesis of the pMAT1 construct was performed using the QuikChange

II XL mutagenesis kit (Stratagene) to create the C2991L mutant. Transfection of A-T

lymphoblastoid cell line (AT1ABR) was performed using Lipofectamine 2000 reagent

(Invitrogen). 2x106 exponentially growing AT1ABR cells were transfected with 5 µg of

pMAT1 and C2991L ATM DNA. 0.2 mg/mL of Hygromycin B (Boehringer Mannheim)

was added 48 h post-transfection to start selection for resistant cells. After 3-4 weeks of

hygromycin selection, cells containing a stable replicating episomal vector were obtained.

Complemented AT1ABR cells were grown in RPMI 1640 (11875, Invitrogen)

supplemented with 15% FBS (10438, Invitrogen) and Hygromycin B (0.2 mg/mL). Cells

were subcultured (1:2) 24 hrs before induction of ATM with 2 µM CdCl2 for 16 hours.

ATLD cells expressing wild-type Mre11 or GFP were a generous gift from Matthew

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Weitzman. ATLD cells were cultured in DMEM (11995, Invitrogen) supplemented with

15% FBS and plasmocin (5 µg/ml, Invivogen). R3047X A-T lymphocytes (GM15785,

Coriell) and wild-type lymphocytes (GM00131, Coriell) were grown in RPMI 1640

supplemented with 15% FBS.

For H2O2 and bleomycin treatment (experiments shown in Fig. 1 and Supp. Fig. 1),

cells were grown to near confluence, the growth medium was changed to sterile PBS

buffer containing various concentrations of H2O2 or bleomycin, and cells were incubated

at 37 °C for 30 minutes before harvesting. The ATM inhibitor (Ku-55933, Calbiochem)

was made as a 10 mM stock (1000X) in dimethylsulfoxide (DMSO). The inhibitor was

added to the medium at 1X concentration 1h before H2O2 or bleomycin, with the same

volume of DMSO added to the control dishes. AT1-ABR cells (Fig. 4, A-D) were

induced for ATM expression as described above, harvested, and resuspended in serum-

free media. H2O2 (25 µM) or camptothecin (10 µg/ml, C9911, Sigma) treatment were

done in 37°C CO2 incubator for 1h. R3047X and wild-type lymphocytes (Fig. 4, E, F and

Supp. Fig. 8) were changed to serum free media for H2O2 (12.5 µM) or camptothecin (10

µg/ml) treatment (37°C, 30 minutes).

Flag-tagged ATM stable cell lines were obtained by integrating linearized Flag-

ATM pcDNA3 plasmid (5) into 293 cells (CRL-1573, ATCC) and selecting with 400

μg/ml G418 (Invitrogen). Flag-ATM stable cell lines were maintained with DMEM

supplemented with 10% FBS and 200 μg/ml G418. Phosphorylation of p53 and Chk2

was performed as described for the primary wild-type fibroblasts in the manuscript.

Western Blotting

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Cells were washed with PBS buffer and lysed in cell lysis buffer (9803, Cell Signaling).

The supernatant from the cell lysate (10 µg) was separated by SDS-PAGE and analyzed

by western blotting. Protein concentrations were determined with the BCA protein assay

kit (Pierce). When detection of γ-H2AX was required, 5X SDS loading buffer was added

directly to the whole cell lysate. Proteins were transferred to PVDF-FL membrane

(Millipore) and probed with antibodies directed against phospho-specific ATM and its

substrates: ATM (GTX70103, Genetex); phospho-ATM Ser1981 (AF-1655, R&D

Systems); Chk2 (GTX70295, Genetex); phospho-Chk2 Thr68 (2661S, Cell Signaling);

H2AX (ab20669, Abcam); γ-H2AX (GTX80694, Genetex); p53 (GTX70214, Genetex);

phospho-p53 Ser15 for cell lysates (9286S, Cell Signaling). R3047X ATM was

inmmunoprecipitated with ATM antibody (GTX70105, Genetex) prior to western

blotting with phospho-ATM Ser1981 antibody. Western blots shown are representative of

several independently performed experiments.

Apoptosis assays

AT1-ABR cells inducible expressing WT or C2991L ATM were treated with H2O2 (25

µM) or camptothecin (5 µg/ml) for 4 hrs after 16 hr CdCl2 induction as described above.

Caspase 3 activity was measured with cleavable fluorescence substrate PhiPhiLux-G2D2

(A304RG-5, OncoImmunin). The loss of membrane integrity and cleavage of nuclear

DNA during apoptosis were measured with FITC annexin V apoptosis detection kit or

with propidium iodide (556547, BD) according to the manufacturer's instructions. After

treatment, cells were analyzed by flow cytometry on FASCalibur (BD).

ATM mutagenesis and purification

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Mutations in the human ATM gene were made using the Quikchange XL method

(Stratagene) in a Flag-ATM/pCDNA3 construct or HA-ATM/pCDNA3 (5). Recombinant

ATM purification was performed as previously described (6). A construct to express

biotinylated ATM was prepared by insertion of an N-terminal BirA recognition site into

Flag-tagged ATM to make pTP1056. This plasmid was transfected with a BirA

expression plasmid (gift from Mauro Modesti) into 293T cells as previously described (6).

Details of cloning and sequences of mutations are available upon request.

ATM kinase assays with purified components

ATM activity was tested in vitro as described previously (7), with 0.36 nM ATM, 2.2 nM

MRN, 50 nM GST-p53 and 10 ng linear DNA in a 40 µl reaction. Products were

separated by SDS-PAGE, and analyzed by western blot, probing with antibody to

phosphorylated Ser15 of p53 (PC461, Calbiochem). In experiments where H2O2 or

diamide was used, no additional DTT was added to the reactions. Western blots shown

are representative of several independently perfomed experiments.

ATM oxidation in vitro

Oxidized ATM was treated with reducing (50 mM betamercaptoethanol) or non-reducing

SDS loading buffer, separated by SDS-PAGE, and analyzed by western blotting using a

monoclonal antibody directed against ATM (GTX70103, Genetex).

The stoichiometry of oxidized ATM was measured by glycerol gradient

sedimentation essentially as described (7). 10-20% glycerol gradients (12 ml) in Tris-HCl

buffer (25mM, pH 8.0, 100mM NaCl) were used to separate 1 µg of biotinylated ATM or

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high molecular weight protein markers (Bio-Rad). Ultracentrifugation was performed at

29,000 RPM using a SW41 rotor (Beckman) at 4°C for 20 hours. 500 µl fractions were

taken from the top of gradient and filtered through a PVDF membrane with a dot-blot

device (Bio-Rad). Biotinylated ATM on the membrane was detected with a streptavidin

fluorescence conjugate (Molecular Probes).

Substrate binding

0.2 µg of biotinylated ATM or an equal volume of buffer A (25 mM Tris-HCl pH 8.0,

10% glycerol, 100 mM NaCl, 1 mM DTT) was incubated with streptavidin-coated

magnetic beads (Promega) at 4°C for 30 minutes. The beads were then incubated with

50 ng of GST-p53 each in the presence or absence of H2O2 (0.27 mM) at 30°C for 30

minutes. After washing 3 times, the bound GST-p53 was eluted by boiling the beads with

1X SDS loading buffer and analyzed by western blotting using an anti-GST antibody

(GE). The blots were quantified using the Odyssey system (Li-Cor).

Mass spectrometry

One µg of ATM was treated with 0.5 mM H2O2 at 30 °C for 30 minutes, then

separated by non-reducing SDS-PAGE and stained with Coomassie blue G250. A gel

slice containing oxidized ATM was incubated with 50 mM iodoacetamide to alkylate free

sulfhydryls at room temperature for 1 h. Tris(2-carboxyethyl)phosphine (TCEP) was

added to reduce disulfide bonds (room temperature, 1 h) followed by the addition of 10

mM N-ethyl maleimide (NEM, room temperature, >2 hr). Freshly made trypsin solution

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(Promega sequencing grade modified trypsin, 20 (µg/ml), in 50 mM ammonium

bicarbonate) was added and incubated at 37 °C overnight.

A Dionex (Sunnyvale, CA) Ultimate HPLC was used to separate the digest

peptides using a gradient of 5-50% B (water:acetonitrile: formic acid: trifluoroacetic acid

10:90:0.1:0.01) in 60 minutes, with a 75 µm ID x 15 cm column (Dionex Pepmap100)

flowing at 240 nl/min. The eluant from the column was deposited with 90 sec deposition

time onto 48 of 192 well MALDI target using the Probot spotting robot. 0.5 µl of α-

cyano-4-hydroxy cinnamic acid matrix (LaserBio Labs, France) at 5 mg/ml concentration

in 65% acetonitrile and 35% water) was deposited over the LC spots. MS and MS/MS

were acquired on a MALDI-TOF/TOF (ABI 4700 Proteomics Analyzer, Foster City, CA).

MS were acquired in a region of the gradient previously established to contain the tryptic

fully cleaved C2991 peptide and a missed cleavage peptide. Then MS/MS were acquired

manually of the Cys modified peptides, with 7-12,000 laser shots for the cysteine residue

modified by propionamide, trioxidation, NEM or NEM plus water. The data was

processed using GPS Explorer v3.5. MASCOT V2.0 was used for the database search,

using the supplied ATM sequence and aforementioned modifications. No cysteine

carbamidomethylation was observed in any of the samples.

References cited:

1. D. Watters et al., Oncogene 14, 1911 (1997). 2. C. Greenman et al., Nature 446, 153 (2007). 3. J. Wu, J. T. Watson, Protein Sci 6, 391 (1997). 4. N. Zhang et al., Proc Natl Acad Sci U S A 94, 8021 (1997). 5. C. J. Bakkenist, M. B. Kastan, Nature 421, 499 (2003). 6. J. H. Lee, T. T. Paull, Methods Enzymol 408, 529 (2006). 7. J. H. Lee, T. T. Paull, Science 308, 551 (2005).