Identification of Domains of Ataxia-telangiectasia Mutated Required for Nuclear Localization and Chromatin Association
2005; Elsevier BV; Volume: 280; Issue: 30 Linguagem: Inglês
10.1074/jbc.m411689200
ISSN1083-351X
AutoresDavid B. Young, Jyoti C Jonnalagadda, Magtouf Gatei, David A. Jans, M. Stephen Meyn, Kum Kum Khanna,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoAtaxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin α1/β1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP)·ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP·ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP·ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5–224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo. Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin α1/β1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP)·ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP·ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP·ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5–224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo. ATM 1The abbreviations used are: ATM, ataxia-telangiectasia mutated; A-T, ataxia-telangiectasia; Gy, gray; DSB, double strand break; NLS, nuclear localization signal; GFP, green fluorescent protein; pEGFP, enhanced green fluorescent protein plasmid; KD, kinase-dead; HIV, human immunodeficiency virus; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; NES, nuclear export signal; MRN, Mre11-Rad50-Nibrin. is homozygously mutated in the germ line of patients with the neurodegenerative and cancer predisposition syndrome, ataxia-telangiectasia (A-T). Cells derived from A-T patients are hypersensitive to agents that cause double strand breaks (DSBs) in DNA, such as ionizing radiation (IR), but retain normal resistance to UV irradiation and other damaging agents. The role of ATM in the DNA damage response is well documented (see Ref. 1Khanna K.K. Lavin M.F. Jackson S.P. Mulhern T.D. Cell Death Differ. 2001; 8: 1052-1065Crossref PubMed Scopus (196) Google Scholar). Loss of ATM function in human and mouse cells cause defects in molecular pathways that are normally activated after DNA DSBs. ATM is reported to be present as inactive dimers in human cells, and exposure to IR induces its autophosphorylation at serine 1981, dimer dissociation, and activation as a kinase (2Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Following DNA damage, ATM accumulates at sites of DNA DSBs as marked by phosphorylation of H2A.X at serine 139. The subsequent signaling cascade that results from ATM activation transduces signals to downstream targets such as p53, MDM2, CHK1, CHK2, BRCA1, and NBS1, which instigate cell cycle arrest and DNA repair. ATM is reported to localize predominantly within the nucleus of most proliferating cells, with small amounts residing in the cytoplasm (3Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsay J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (169) Google Scholar, 4Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Shiloh Y. Tagle D.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (150) Google Scholar), consistent with its role in the DNA DSB response pathway. However, ATM has been reported to localize mainly within the cytoplasm of mouse Purkinje cells, in cells of the human cerebellum, and in a subset of cells in the dorsal root ganglia of mouse (5Barlow C. Ribaut-Barassin C. Zwingman T.A. Pope A.J. Brown K.D. Owens J.W. Larson D. Harrington E.A. Haeberle A.M. Mariani J. Eckhaus M. Herrup K. Bailly Y. Wynshaw-Boris A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 871-876Crossref PubMed Scopus (146) Google Scholar, 6Oka A. Takashima S. Neurosci. Lett. 1998; 252: 195-198Crossref PubMed Scopus (89) Google Scholar). Although it is not clear what function ATM performs within the cytoplasm, it has been demonstrated in mouse that ATM deficiency results in abnormalities of organelles. Many mutations identified to date involve truncation of ATM that results in deletion of the carboxyl-terminal region containing the kinase domain, thereby eliminating its kinase activity. The transport of large proteins such as ATM into the nucleus is a complex process that involves active transport from the cytoplasm to the nucleus in signal-dependent fashion through the action of nuclear localization sequences (NLSs), which are recognized by members of the cellular importin superfamily of transport proteins (7Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (921) Google Scholar, 8Gorlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (289) Google Scholar). The best understood pathways involve those in which cargoes containing lysine-arginine-rich NLSs are recognized by either importin β1 or the importin α1/β1 heterodimer (7Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (921) Google Scholar, 8Gorlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (289) Google Scholar). Subsequent to import into the nucleus through the nuclear envelope-localized nuclear pore complex structures mediated by the importins, release into the nucleoplasm is effected by binding of the monomeric guanine nucleotide-binding protein Ran in activated GTP-bound form to importin β (7Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (921) Google Scholar, 8Gorlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (289) Google Scholar). Sequences reminiscent of known NLSs have been identified within ATM. As a first step to identifying targeting signals within ATM that regulate its localization and activity, we tagged full-length and various subfragments of ATM with green fluorescent protein (GFP) and analyzed their subcellular localization and function. We characterized an importin α1/β1-recognized NLS in the amino terminus of ATM, mutation of which in the context of full-length ATM results in predominantly cytoplasmic localization and inhibits interaction with importins. Importantly, we show that the amino-terminal region of ATM confers association with chromatin and is required for its efficient kinase activity in vivo; only the nuclear fraction of ATM is autophosphorylated in response to IR induced DNA damage. Overall, our results imply that the amino terminus of ATM is crucial for both nuclear localization and chromatin association, thereby facilitating the kinase activity of ATM in vivo. Plasmid Construction—pEGFP·ATM (amino acids 5–1303), encoding the amino-terminal half of the ATM protein fused with GFP, was created by subcloning ATM-(5–1303) as a XhoI/KpnI fragment from pMAT1, which lacks the coding sequence for the first five amino acids (9Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (98) Google Scholar), into pEGFP-C2 (Clontech). pEGFP·ATM-(1303–3056), encoding the carboxyl-terminal half of ATM fused with GFP, was created by subcloning ATM-(1303–3056) as a KpnI fragment from pMAT1 into pEGFP-C2. pSG5GFP·ATM, encoding full-length ATM fused with GFP, was created by subcloning GFP·ATM-(5–1303) as an NheI (end-filled) BamHI fragment from pEGFP·ATM-(5–1303) and subsequently ATM-(1303–3056) as a KpnI fragment from pMAT1 into pSG5 (Stratagene, with an expanded multiple cloning site). Deletion mutants pEGFP·ATM-(5–568), -(677–1303), -(5–224), and -(227–568) were prepared by restriction digestion, end-filling with T4 DNA polymerase, and religation. The NLSm (R386A/K387A) and KD (D2870A/N2875K) mutants of ATM were prepared by site-directed mutagenesis using the QuikChange kit following the supplier's protocol (Stratagene). The NLS-ATM-(1303–3056) construct was prepared by restriction digestion of pEGFP·ATM-(1303–3056) with BsrGI and insertion of an oligonucleotide dimer containing the coding sequence of the SV40 large T-antigen NLS between GFP and ATM-(1303–3056); nuclear localization this fusion protein was confirmed by immunofluorescence assay. The GFP·NES-ATM(NLSm) construct was prepared by restriction digestion of pSG5GFPATM(NLSm) with EagI and insertion of an oligonucleotide dimer containing the coding sequence of the HIV-Rev NES between GFP and ATM(NLSm); cytoplasmic localization of the fusion protein was confirmed by immunofluorescence assay. All of the above GFP·ATM constructs were transferred into pREP4EGFP (pREP4, Invitrogen, with the expression cassette replaced by the pEGFP-C3 expression cassette) using AgeI and KpnI. A detailed description of all constructs is available from the authors upon request. Cell Culture—Mammalian cell lines COS-7 (marmoset, SV40-transformed), HEK293T (human embryonic kidney fibroblast, SV40-transformed), AT5BIVA (human A-T fibroblast cell line, SV40-transformed), AT1ABR (human A-T lymphoblastoid cell line), and HeLa (tumor cell line) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics and incubated at 37 °C with 5% CO2. Lipofectamine-mediated Transfection and Selection of Stable Cell Lines—Cells were seeded into 30-mm Petri dishes containing coverslips for immunofluorescence and grown to 50–80% confluence. Cells were then transfected with 2 μg of plasmid DNA using 6 μl of Lipofectamine 2000 (Invitrogen) in 1.4 ml of Opti-MEM I (Invitrogen) serum-free medium and incubated for 5 h at 37 °C with 5% CO2. The medium was then replaced with fresh growth medium, and the cells were incubated at least 16 h under normal growth conditions prior to assaying. For stable selection of AT1ABR lymphoblastoid cells or AT5BIVA cells transfected with pREP4-based plasmids, cells were grown in the presence of 200 μg/ml hygromycin B (Roche Applied Science). Fluorescence and Immunofluorescence—For general visualization of GFP-tagged proteins, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, washed with PBS, permeabilized with 0.2% Triton-X100 in PBS for 10 min, washed again, and then counterstained with DAPI to detect cell nuclei. To visualize foci of GFP·ATM and phosphorylated ATM substrate, cells were treated with extraction buffer (20 mm Hepes, 20 mm NaCl, 5 mm MgCl2, 1 mm ATP, 0.1 mm sodium orthovanadate, 1 mm sodium fluoride, and protease inhibitor mixture (Sigma), 0.5% IGEPAL (same chemically as obsolete Nonidet P-40), pH 7.5) on ice for 20 min prior to fixation with 4% paraformaldehyde in PBS. For detection of phosphorylated ATM and substrates, cells were blocked with 0.5% FBS in PBS for 1 h at room temperature and then immunostained with either anti-phosphohistone H2A.X antibody (S139) diluted 1:1000 in blocking solution, anti-phosphoserine 1981 ATM antibody diluted 1:100 in blocking solution, or anti-FLAG antibody diluted 1:600 in blocking solution overnight at 4 °C. Cells were then washed in PBS and counterstained with anti-rabbit or anti-mouse Alexa Fluor 546 (Molecular Probes, cat. no. A-11010/03) secondary antibodies were diluted 1:200 in blocking solution for 1 h at room temperature. DNA was counterstained with DAPI in PBS and then washed in PBS. Following processing, coverslips were mounted with Mowiol containing 0.6% diazobicyclo-octane and allowed to set overnight at 4 °C prior to fluorescent microscopy at room temperature using a Zeiss Axioskop 20 microscope, Zeiss Plan-NEOFLUAR ×100/1,30 oil lens, Zeiss AxioCam MRc digital camera, and MRGrab 1.0 software. Figures were assembled using CorelDraw 8.0. Antibodies used were: anti-phosphohistone H2A.X antibody (S139) (Upstate 05-636), anti-phosphoserine 1981 ATM (Rockland, cat. no. 600-401-400), anti-FLAG M2 (Sigma, F3165), Alexa Fluor 546 or 488 (Molecular Probes, cat. no. A-11010/003/001). Immunoblot Assays—Cells were pelleted and then washed with PBS prior to being lysed directly in sample buffer (5% glycerol, 1.7% SDS, 100 mm dithiothreitol, 0.2% bromphenol blue, 60 mm Tris, pH 6.8) with sonication and boiling. Protein concentrations were determined by Bradford assay. Proteins were subjected to SDS-PAGE, 4% for full-length ATM or 8% for other proteins and some ATM deletion mutants. The separated proteins were transferred to polyvinylidene difluoride membranes by semi-dry transfer (Bio-Rad), and the proteins were detected with antibodies in Blotto and visualized by chemiluminescence (PerkinElmer Life Sciences). Antibodies used were: ATM phosphoserine 1981 (Rockland, 600-401-400), ATM (Genetex, 2C1), GFP (Molecular Probes, A6455), MCM3 (Santa Cruz Biotechnology, sc-9850), BRCA1 (Oncogene, OP92), α-tubulin (Sigma, T9026). Clonogenic Survival Assay—AT5BIVA cells, stably transfected with pREP4GFPATM and mutants, were plated at low density into 6-well Petri dishes in triplicate. Cells were grown for 1 day following plating, to allow cells to adhere, and then subjected to ionizing radiation of 1, 2 or 4 Gy (MDS Nordion, Gammacell 40 Exactor). Cells were grown for a further 13 days with regular changes of growth medium to allow for outgrowth of colonies of surviving cells. Cells were then fixed with methanol, dried, stained with Coomassie Blue stain, and washed to facilitate the counting of colonies (50 or more cells). Yeast Two-hybrid Assays—Interaction between ATM-(227–568) and importin α proteins was determined using the yeast two-hybrid method by growth selection on synthetic dropout media. Small scale co-transformation of yeast with plasmids was carried out using the lithium acetate method in Saccharomyces cerevisiae strain AH109 following the manufacturer's recommendations (Clontech). Transformed yeast were plated on synthetic dropout medium lacking the amino acids Leu and Trp, to select for plasmids, or lacking Leu, Trp, His, and Ade for growth selection. The strength of protein-protein interaction was quantitated using a sensitive colorimetric α-galactosidase assay following the manufacturer's recommendations (Clontech). The assay was carried out in 96-well plates, in triplicates, by combining a small aliquot of cell-free culture medium with a fixed volume of assay buffer. After a prescribed preincubation time, absorbance at 410 nm was recorded and used to calculate the concentration of α-galactosidase. Chromatin Fractionation—S1, S2, and P chromatin fractions were isolated as described by Kreitz et al. (10Kreitz S. Ritzi M. Baack M. Knippers R. J. Biol. Chem. 2001; 276: 6337-6342Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, 3 × 106 cells were washed in PBS and resuspended in 0.4 ml of cold hypertonic buffer A (20 mm Hepes, 20 mm NaCl, 5 mm MgCl2, 1 mm ATP, protease inhibitor mixture (Sigma), 1 mm NaF, 1 mm NaOV3, pH 7.5). The cells were allowed to swell on ice for 15 min and were then broken with 30 strokes of a Dounce homogenizer. Nuclei were pelleted by centrifugation at 325 × g (2000 rpm) for 20 min at 4 °C. The supernatant (cytosolic proteins) was transferred to a fresh tube and placed on ice. The nuclear pellet was resuspended in 0.4 ml of buffer A with 0.5% IGEPAL and incubated on ice for 15 min to lyse the nuclear envelope. Free nucleosolic proteins were separated from the nuclear matrix by centrifugation at 13400 × g (12000 rpm) for 15 min at 4 °C. The supernatant, containing nucleosolic proteins, was transferred to a fresh tube and placed on ice. The pellet was washed with 50 μl of buffer B (20 mm Hepes, 0.5 mm MgCl2, 1 mm ATP, 0.3 m sucrose, protease inhibitor mixture (Sigma), 1 mm NaF, 1 mm NaOV3, pH 7.5) with increasing concentrations of NaCl (0.1, 0.2, and 0.4 m) to release structure bound proteins and transferred to fresh tubes on ice. Protein concentrations were determined by the Bradford method. Samples were analyzed as described under "Immunoblot Assays." GFP·ATM Is Distributed between the Nucleus and Cytoplasm—To initiate our studies of ATM localization, we created a plasmid for expression of GFP-tagged ATM in mammalian cells. Localization of the GFP·ATM fusion protein was determined by fluorescence microscopy after transient expression into various cell types including HEK293T, COS-7, and AT5BIVA (an ATM-deficient cell line) cells. The majority of GFP·ATM protein was localized within the nucleus, with variable levels of cytoplasmic localization being observed. A minority of cells displayed a predominantly cytoplasmic accumulation around the nuclear periphery (Fig. 1A). The distribution of GFP·ATM within cells was determined statistically over a larger sample, with at least 200 transfected cells, repeated over three independent experiments in three cell lines. In ∼60–70% of log phase cells GFP·ATM localized within both the nucleus and cytoplasm, whereas in 25–40% of cells localization was predominantly nuclear; in the remaining 5% of the cells GFP·ATM localized predominantly around the cytoplasmic periphery (Fig. 1B). A similar distribution of GFP·ATM was seen in all three cells lines. The observed nuclear/cytoplasmic ratio was independent of the amount of DNA transfected, indicating that protein expression levels did not impact the observed intracellular distribution of GFP·ATM (data not shown). In addition, GFP·ATM was excluded from the nucleoli. Next we wished to determine whether GFP·ATM was able to form foci and phosphorylate substrate in response to DNA DSBs, as has been reported for the endogenous wild type protein. When A-T fibroblast cells (AT5BIVA) expressing GFP·ATM were exposed to IR (30 min after 4 Gy IR), GFP·ATM was seen to change localization from being evenly distributed through the nucleus to forming distinct foci that co-localized with phospho-H2A.X (S139) as a marker for sites of DNA DSBs (Fig. 1C). A small number of ATM foci were sometimes present in nuclei in the absence of DNA damage. Interestingly, GFP·ATM(KD) could not form foci following induction of DNA DSBs. As expected, GFP·ATM(KD) did not phosphorylate H2A.X (Ser-139). This experiment was repeated using FLAG-tagged ATM. Once again foci formation occurred only for wild type ATM and not for the KD mutant. These data indicate that ATM kinase activity is required for ATM foci formation. Identifying Nuclear Localization Signals within ATM—To map functional NLSs within ATM, a series of deletion mutation constructs was prepared in the pEGFP-C2 vector (Clontech) (Fig. 2A). Expression of these GFP-tagged deletion mutants was confirmed by immunoblot analysis (Fig. 2B), with all proteins being of the predicted size. Ectopically expressed GFP·ATM-(5–1303) exhibited a subcellular distribution similar to that of the full-length protein, with mixed nuclear and cytoplasmic localization in the majority of cells (Fig. 2C). However, the carboxyl-terminal half of ATM-(1303–3056) was localized exclusively within the cytoplasm. This implied the existence of one or more nuclear localization signals within the amino-terminal half of ATM. To narrow down the region containing the NLS, we performed deletion mutation analysis of the amino-terminal half of ATM. This led to the identification of two non-overlapping amino-terminal fragments, ATM-(5–224) and ATM-(227–568), which were localized predominantly within the nucleus (Fig. 2C). Both these deletion mutants contained a short motif of basic amino acids reminiscent of the SV40 large T-antigen NLS. Deletion mutant ATM-(5–224) contained a cluster of three (+) charged basic residues (23RKK28), whereas ATM-(227–568) contained a cluster of four (+) charged basic residues (385KRKK388). To determine whether these amino acid sequences form functional NLSs, site-directed mutagenesis of these motifs was performed, changing core lysine or arginine residues to alanine. Amino acids 23RKK25 were mutated to 23RAA25 in the construct GFP·ATM-(5–224), and amino acids 385KRKK388 were mutated to 385KAAK388 in the construct GFP·ATM-(227–568). When expressed in cells the 23RAA25 mutant protein remained localized predominantly within the nucleus of transfected cells, indicating that 23RKK25 is not important for nuclear transport of the GFP·ATM-(5–224) construct (Fig. 2D). In contrast, the 385KAAK388 mutant localized predominantly within the cytoplasm, indicating that 385KRKK388 is critical for nuclear import of the GFP·ATM-(227–568) construct. Subsequently, the 385KAAK388 mutation was introduced into the full-length ATM construct, GFP·ATM(NLSm), which also localized predominantly within the cytoplasm of transfected cells (Fig. 2D), indicating that this is the dominant NLS within full-length ATM. The distribution of GFP·ATM(NLSm) within cells was determined statistically over a larger sample, at least 200 transfected cells, repeated over three independent experiments in AT5BIVA cells (Fig. 2E). The results indicate that the mutation of this NLS sequence in ATM does not result in complete absence of ATM from the nucleus, as GFP·ATM(NLSm) was still faintly nuclear in about 18–25% of cells. Nuclear Import of ATM Is Mediated by Importin α1 and Importin β1—To identify proteins involved in transporting ATM into the nucleus, we assayed for interaction between ATM and members of the importin family of proteins by co-immunoprecipitation with ATM. We were able to detect interaction between ATM and importin β1 in whole cell extracts prepared from an A-T lymphoblastoid cell line stably expressing GFP·ATM but not from cells stably expressing GFP or GFP·ATM(NLSm) (Fig. 3A). Attempts to detect importin α by this technique were inconclusive because of close migration of importin α (58 KDa) with immunoglobulin. As an alternative strategy we used a yeast two-hybrid assay to assess whether ATM interacts directly with importin α1, α3, or α5 and β1. The fragment of ATM that contains the NLS, ATM-(227–568), was expressed in yeast in fusion with the GAL4 binding domain. Yeast were co-transformed with plasmids expressing importins in fusion with the GAL4 activation domain. We were able to show interaction of ATM-(227–568) with importins α1 and, to a lesser extent, α5 (Fig. 3B). To establish positive controls we assayed for interaction of p53 with the SV40 T-antigen (Clontech, yeast two-hybrid controls), which are known to interact very strongly, and of BRCA1 with importin α1 (11Chen C.F. Li S. Chen Y. Chen P.L. Sharp Z.D. Lee W.H. J. Biol. Chem. 1996; 271: 32863-32868Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The extent of interaction between ATM-(227–568) and importin α1 was comparable with that seen between BRCA1 and importin α1. The interaction with importin β1 was negligible compared with GAD424 vector alone, suggesting that interaction of ATM with importin β1 detected in co-immunoprecipitation experiments is mediated through importin α1. Amino-terminal Sequences of ATM Are Required for Survival of Complemented A-T Cells in Response to IR-induced DNA Damage—We chose to compare the protective function of GFP·ATM with that of GFP·ATM(NLSm) (which localizes predominantly with the cytoplasm), GFP·ATM-(1303–3056) (which localizes within the cytoplasm), GFP·NLS-ATM-(1303–3056) (which is artificially targeted to the nucleus by insertion of an exogenous NLS), and GFP·ATM(KD) in stably transfected A-T fibroblast cells (AT5BIVA), which are exquisitely sensitive to IR. Cells complemented with GFP·ATM or the above mentioned mutants were assayed for their ability to survive DNA damage induced by IR at doses of 1, 2, or 4 Gy. Cells expressing GFP·ATM demonstrated a similar, although slightly higher, level of survival compared with that of cells expressing GFP·ATM(NLSm) (Fig. 4A). An explanation for this observation may be that low, but sufficient, amounts of GFP·ATM(NLSm) are present within the nucleus of these cells. Even this low level of GFP·ATM(NLSm) may provide a damage signal that allows these cells to survive. Cells expressing GFP·ATM(KD) and GFP·ATM-(1303–3056) exhibited no correction of radiosensitivity at the higher dose of 4 Gy. These results would be expected, as GFP·ATM(KD) cannot phosphorylate substrate, whereas GFP·ATM-(1303–3056) localizes exclusively within the cytoplasm and may therefore not be able to be activated or to phosphorylate substrate in response to a damage signal. Interestingly, cells expressing GFP-NLS·ATM-(1303–3056) were also impaired in their ability to correct radiosensitivity. This mutant of ATM contains a functional kinase domain and is artificially targeted to the nucleus, indicating that it is not sufficient to simply target the isolated kinase domain of ATM to the nucleus of cells to correct radiosensitivity. From these data we infer that amino-terminal sequences of ATM are required for its biological activity. Amino-terminal Sequences of ATM Are Required for Substrate Phosphorylation in Vivo—To assess whether the differences in survival of complemented A-T fibroblast cells correlate with restoration of the kinase activity of ATM, we examined the ability of A-T lymphoblastoid cells stably expressing GFP·ATM, GFP·ATM(KD), GFP·ATM(NLSm), and GFP·NLS-ATM-(1303–3056) to phosphorylate Ser-15 of p53 after exposure to IR (30 min after 4 Gy). GFP·ATM and GFP·ATM(NLSm) were able to phosphorylate p53 (Fig. 4B), whereas GFP·ATM(KD) and GFP·NLS-ATM-(1303–3056) could not phosphorylate p53. The residual level of signal seen with these mutants was comparable with GFP-vector only-expressing cells, suggesting that the amino terminus of ATM is required for optimal phosphorylation of substrates in vivo. Furthermore, when the above mentioned constructs were tested for ATM activation using phospho-Ser-1981 antibody, the GFP· ATM(KD) and GFP·NLS-ATM-(1303–3056) mutants did not show any detectable autophosphorylation relative to GFP· ATM. The autophosphorylation of GFP·ATM(NLSm) was considerably weaker than that seen with GFP·ATM. To find an explanation for our observation concerning the reduced autophosphorylation of GFP·ATM(NLSm), we performed immunofluorescence assays on A-T fibroblasts expressing these fusion proteins. We found that only the nuclear fraction of GFP·ATM or GFP·ATM(NLSm) was autophosphorylated (Fig. 4C). Considering the relatively small amount of GFP·ATM(NLSm) protein within the nucleus of these cells, this observation would explain why an apparent substantial reduction in the amount of autophosphorylated GFP·ATM(NLSm) mutant was seen by immunoblot analysis. To determine whether GFP·ATM(NLSm) is able to confer radioresistance and normal phosphorylation of p53, perhaps because of the small amount of ATM that is localized in the nucleus, we introduced the HIV-Rev nuclear export signal (NES) between GFP and ATM(NLSm), GFP· NES-ATM(NLSm), in order to remove ATM from the nucleus. A-T lymphoblastoid cells stably expressing GFP·ATM (wild type), GFP·ATM(KD), GFP·ATM(NLSm), and GFP-NES-ATM-(NLSm) were compared for their ability to correct radiosensitivity and to phosphorylate p53 on Ser-15 after IR-induced DNA damage. We found that GFP·NES-ATM(NLSm), which localized exclusively within the cytoplasm, had a substantially reduced ability to phosphorylate p53 on Ser-15 (Fig. 4D) and increased radiosensitivity, compared with GFP·ATM(NLSm) (Fig. 4E), suggesting that nuclear localization is required for ATM activity. Taken together, these results suggest that the amino terminus of ATM is required for optimal ATM activation and the subsequent ATM activity. ATM Associates with Chromatin before and after IR-induced DNA Damage through Its Amino-terminal region—Previously ATM has been reported
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