Identification of Domains of Ataxia-telangiectasia Mutated Required for Nuclear Localization and Chromatin Association

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. 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 and of Cell were into containing for immunofluorescence and to Cells were with of DNA using of in of medium and incubated for at 37 °C with The medium was replaced with and the cells were incubated at normal to of AT1ABR lymphoblastoid cells or AT5BIVA cells with cells were in the of and of cells were with in for at with PBS, with in for and with to cell of GFP·ATM and ATM cells were with and as for to with in of ATM and substrates, cells were with in for at and with either H2A.X in ATM in or in at Cells were in and with or were in for at DNA was with in and in Following were with containing and to at °C to fluorescent at using a and were using used H2A.X ATM or were and with to in with and were by assay. were to for full-length ATM or for other proteins and ATM deletion The proteins were transferred to by and the proteins were with in and by used ATM ATM GFP cells, with and were at into in Cells were for following to cells to and to ionizing radiation of or Cells were for a with of medium to for of of Cells were with with and to the of or between and importin proteins was using the by of with was using the in following the (Clontech). were medium the amino acids and to for or and for The of interaction was using a following the (Clontech). The was in in by a small of medium with a of a at was and used to the of and chromatin were as by S. M. M. R. J. 2001; PubMed Scopus Google Scholar). cells were in and in of A The cells were to for and were with of a were by at for at The was transferred to a and The nuclear was in of A with and incubated for to the nuclear proteins were from the nuclear by at for at The containing was transferred to a and The was with of with increasing of and to release proteins and transferred to were by the were analyzed as GFP·ATM between the and our of ATM we created a for expression of ATM in of the GFP·ATM fusion protein was by after expression into various cell and AT5BIVA cell line) The of GFP·ATM protein was within the with of cytoplasmic localization A of cells a predominantly cytoplasmic the nuclear The of GFP·ATM within cells was a with at cells, in cell In of cells GFP·ATM within both the nucleus and cytoplasm, in of cells localization was predominantly in the of the cells GFP·ATM predominantly the cytoplasmic A of GFP·ATM was in all cells The was of the of DNA that protein expression not the of GFP·ATM not In GFP·ATM was from the we to GFP·ATM was to form and phosphorylate substrate in response to DNA as has been reported for the A-T fibroblast cells GFP·ATM were to IR after GFP·ATM was to localization from through the nucleus to that with as a for sites of DNA DSBs A small of ATM were present in in the of DNA damage. Interestingly, could not form following induction of DNA DSBs. As not phosphorylate H2A.X was using ATM. only for ATM and not for the KD data indicate that ATM kinase activity is required for ATM within functional NLSs within ATM, a of deletion mutation constructs was prepared in the pEGFP-C2 of these deletion mutants was confirmed by analysis with all proteins of the GFP·ATM-(5–1303) a subcellular to that of the full-length with nuclear and cytoplasmic localization in the of cells However, the carboxyl-terminal half of ATM-(1303–3056) was within the the of or nuclear localization signals within the amino-terminal half of ATM. the region containing the NLS, we deletion mutation analysis of the amino-terminal half of ATM. to the of amino-terminal and which were predominantly within the nucleus these deletion mutants a of amino acids reminiscent of the SV40 large T-antigen Deletion mutant a of a of these amino sequences form functional site-directed mutagenesis of these was or to acids were mutated to in the construct and amino acids were mutated to in the construct in cells the mutant protein predominantly within the nucleus of cells, that is not for nuclear transport of the construct In the mutant predominantly within the cytoplasm, that is for nuclear import of the the mutation was into the full-length ATM which also predominantly within the cytoplasm of cells that this is the NLS within full-length ATM. The of GFP·ATM(NLSm) within cells was a at cells, in AT5BIVA cells The results indicate that the mutation of this NLS sequence in ATM not in complete of ATM from the as GFP·ATM(NLSm) was still nuclear in of of ATM by and proteins in ATM into the we for interaction between ATM and members of the importin of proteins by with ATM. We were to interaction between ATM and importin β1 in cell prepared from an A-T lymphoblastoid cell line GFP·ATM but not from cells GFP or GFP·ATM(NLSm) to importin by this were because of of importin with As an we used a to ATM with importin or and The fragment of ATM that the NLS, was in in fusion with the binding were with in fusion with the activation We were to show interaction of with to a we for interaction of with the SV40 T-antigen which are known to and of with importin S. Chen Y. Chen J. PubMed Scopus Google Scholar). The of interaction between and importin was with that between and importin The interaction with importin β1 was with that interaction of ATM with importin β1 in is mediated through importin Sequences of ATM for of A-T Cells in to DNA to the function of GFP·ATM with that of GFP·ATM(NLSm) localizes predominantly with the localizes within the is targeted to the nucleus by insertion of an exogenous and in A-T fibroblast cells which are to Cells with GFP·ATM or the above mutants were for their to DNA damage induced by IR at of or Cells GFP·ATM demonstrated a level of with that of cells GFP·ATM(NLSm) for this may be that but amounts of GFP·ATM(NLSm) are present within the nucleus of these this level of GFP·ATM(NLSm) may a damage signal that these cells to Cells and of radiosensitivity at the of results be as phosphorylate localizes within the cytoplasm and may not be to be activated or to phosphorylate substrate in response to a damage Interestingly, cells were also in their to correct mutant of ATM a functional kinase domain and is targeted to the that it is not sufficient to target the kinase domain of ATM to the nucleus of cells to correct these data we that amino-terminal sequences of ATM are required for its activity. Sequences of ATM for in the in of A-T fibroblast cells with of the kinase activity of ATM, we the of A-T lymphoblastoid cells and to phosphorylate of after exposure to IR after GFP·ATM and GFP·ATM(NLSm) were to phosphorylate and could not phosphorylate The level of signal with these mutants was with cells, that the amino terminus of ATM is required for optimal phosphorylation of in vivo. when the above constructs were for ATM activation using the and mutants not show autophosphorylation to ATM. The autophosphorylation of GFP·ATM(NLSm) was that with an for our the autophosphorylation of we immunofluorescence A-T these fusion We that only the nuclear fraction of GFP·ATM or GFP·ATM(NLSm) was autophosphorylated the small of GFP·ATM(NLSm) protein within the nucleus of these cells, this an in the of autophosphorylated GFP·ATM(NLSm) mutant was by GFP·ATM(NLSm) is to and normal phosphorylation of p53, because of the small of ATM that is in the we the HIV-Rev nuclear export signal between GFP and in to ATM from the A-T lymphoblastoid cells GFP·ATM and were for their to correct radiosensitivity and to phosphorylate after DNA damage. We that which within the cytoplasm, a to phosphorylate and radiosensitivity, with GFP·ATM(NLSm) that nuclear localization is required for ATM activity. these results that the amino terminus of ATM is required for optimal ATM activation and the subsequent ATM activity. ATM with and after DNA through ATM has been reported either to with chromatin and after DNA damage Yen 1998; PubMed Scopus Google or to with chromatin only after DNA damage Y. L. L. Shiloh Y. G. J. 2001; PubMed Google Scholar). We to the of this association and to the domain of ATM that is required for association with the of binding we an used to the interaction of recognition and proteins with chromatin S. M. M. R. J. 2001; PubMed Scopus Google Scholar). Using this we that ATM in HeLa cells and GFP·ATM in AT5BIVA cells chromatin and after after DNA damage with with for ATM autophosphorylation only ATM protein from cells to IR of that of the cells not itself ATM. analysis using the deletion mutants of ATM that the nuclear targeted carboxyl-terminal half of ATM was in the fraction only However, the amino-terminal half of ATM was to We the small amino-terminal which a functional NLS, was in the cytoplasmic and but not to with was also in the cytoplasmic and and was in the first chromatin fraction that this region of ATM with chromatin the full-length fragment of ATM was to localize within the but we could not functional NLS in this GFP·ATM and of an is most as a nuclear and is to be in signal pathways in response to DNA DSBs. is that ATM is predominantly in the nucleus of proliferating cells (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). we for the first that ATM nuclear through an NLS that the SV40 ATM NLS was to be for efficient transport of the full-length protein into the as mutation of this NLS to predominantly cytoplasmic localization of ATM in a of data that ATM is into the nucleus in a through the importin α1/β1 heterodimer. for ATM we that ATM(NLSm) could the radiosensitivity of A-T cells, at a level with ATM. it the kinase activity of ATM. In most cells ATM(NLSm) was predominantly within the However, in of cells it nuclear that may be an NLS within the amino sequence of ATM. data that even of nuclear ATM may be sufficient to elicit a response to DNA damage. the HIV-Rev NES between GFP and ATM(NLSm) to ATM from the our data that of radiosensitivity and phosphorylation by NLSm is a small of ATM present in the with this A-T show normal phosphorylation of and the ATM expression D. S. S. M. T. Y. S. S. L. Chessa L. J. 2000; PubMed Google Scholar, M. N. A. D. J. J. PubMed Scopus Google Scholar). it has been demonstrated that ATM protein in a of cell lines and that the of ATM protein not with as by the fraction at IR S. A.M. S.P. Yen T. J. J. 1998; PubMed Scopus Google Scholar). Yen 1998; PubMed Scopus Google also that the of ATM may not be in the cellular DNA damage response. In our data demonstrate that the carboxyl-terminal half of ATM, which localizes within the cytoplasm, is not sufficient for ATM function. fragment of which has kinase activity in not not normal radiosensitivity to A-T cells nor could we kinase activity in vivo. results indicate that carboxyl-terminal of ATM are as a functional Moreover, when the carboxyl-terminal half of ATM was targeted to the nucleus by insertion of exogenous NLS, it was in its to phosphorylate ATM in or to the radiosensitivity of A-T In line with our the kinase domain of the ATM in is inactive and not the damage of cells A.M. T. PubMed Scopus Google Scholar). the amino-terminal sequences of ATM may be required for substrate with this the amino terminus of ATM has been shown to with K.K. Kozlov S. Scott S. Gatei M. Hobson K. Y. D. S.P. Lavin M.F. 1998; PubMed Scopus Google Scholar), M. Scott S.P. N. Lavin M.F. Khanna K.K. 2000; Google Scholar), and A. A. M. J. PubMed Scopus Google in and all of these proteins with ATM in vivo. it is that amino-terminal sequences in ATM with our results that the carboxyl-terminal half of ATM, which the kinase domain, is not sufficient for ATM function and that sequences in the amino terminus are required for its and activity. ATM with through in the as to ATM with chromatin exposure of cells to agents. In a by Y. L. L. Shiloh Y. G. J. 2001; PubMed Google Scholar), a fraction of ATM was reported to to following induction of DNA damage with agents. However, Yen 1998; PubMed Scopus Google that ATM with chromatin and after induction of DNA damage. and our in the in chromatin The we involves proteins in the of with chromatin are with increasing but in the of that and our data that ATM with chromatin through and after DNA damage. In the used by Y. L. L. Shiloh Y. G. J. 2001; PubMed Google at a level increasing in these ATM with chromatin following DNA damage, that ATM association with chromatin from to following DNA damage. Combined, these data may that ATM with chromatin through both and and that the of ATM association with chromatin following DNA damage. We that the amino terminus of ATM with this the which the kinase domain, to with substrate and other A for ATM our data and the data of we a for ATM activity in which ATM is with chromatin as an inactive dimer to its induction of DNA in the DNA ATM with from proteins such as which is to DSBs (2Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, PubMed Scopus Google Scholar, T. Y. L. Y. L. Shiloh Y. EMBO J. 2003; PubMed Scopus Google Scholar, J. J. Jackson S.P. Nature. PubMed Scopus Google Scholar). ATM is chromatin to sites of DNA damage it is to phosphorylate also present at sites of this the rapid and of ATM such as and complex at foci. Interestingly, the complex in as a to other in to the of ATM kinase data using KD ATM indicate that the kinase activity is for ATM after DNA damage. activity may be required to release ATM from sites chromatin that it to sites of damage.

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