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Residual Ataxia Telangiectasia Mutated Protein Function in Cells from Ataxia Telangiectasia Patients, with 5762ins137 and 7271T→G Mutations, Showing a Less Severe Phenotype

2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês

10.1074/jbc.m103160200

1083-351X

Grant S. Stewart, James I. Last, Tatjana Stanković, Neva E. Haites, Alexa Kidd, Philip J. Byrd, A. Malcolm R. Taylor,

Microtubule and mitosis dynamics

We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM5762ins137 splice site or a 7271T→G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinasein vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T→G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T→G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T→G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients. We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM5762ins137 splice site or a 7271T→G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinasein vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T→G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T→G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T→G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients. ataxia telangiectasia lymphoblastoid cell lines gray c-Jun N-terminal kinase glutathione S-transferase polyacrylamide gel electrophoresis antibody ataxia telangiectasia mutated Ataxia telangiectasia (A-T)1 is a human autosomal recessive disorder in which affected individuals exhibit a diverse range of clinical symptoms affecting multiple organ systems (1Sedgwick R.P. Boder E. Vinken P.J. Bruyn G.W. Klawans H.L. Handbook of Neurology. Elsevier Science Publishers, Amsterdam1991: 347-422Google Scholar). The principal manifestation of A-T presents as a progressive truncal and limb ataxia as a consequence of degeneration of the cerebellum. This ultimately results in the patients being wheelchair-bound from the early teenage years. Other clinical signs of A-T are the presence of oculocutaneous telangiectasia, selective immunodeficiency, an increased sensitivity to ionizing radiation and other radiomimetic chemicals, and a marked prevalence of tumors of the lymphoid system (reviewed in Ref.2Shiloh Y. Annu. Rev. Genet. 1997; 31: 635-662Crossref PubMed Scopus (428) Google Scholar). These features define classical A-T, which results from total absence of functional ATM protein. At the gene level, the vast majority of classical A-T patients in the UK are compound heterozygotes for null mutations, most of which are truncating ATM mutations (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The clinical diagnosis is unambiguous in these A-T patients. Whether or not clinical and cellular heterogeneity exists within the group of classical A-T patients with two null mutations is unknown, as no careful correlation studies have been undertaken. Across all A-T patients there is a degree of variation between them in both the severity and expression of the clinical features. In particular, the degree of telangiectasia, the extent of immunodeficiency, the susceptibility to recurrent sinopulmonary infections, longevity, the rate of neuronal degeneration, and the development of tumors show the greatest degree of heterogeneity among patients. In some cases this variation presents as a clearly milder form of A-T. These individuals have a combination of the following: a slightly later age of onset of the clinical symptoms, a slower rate of disease progression, an extended life span, when compared with most classical A-T patients, and a reduced or absent hypersensitivity to ionizing radiation (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 4McConville C.M. Stankovic T. Byrd P.J. McGuire G.M. Yao Q.-Y. Lennox G.G. Taylor A.M.R. Am. J. Hum. Genet. 1996; 59: 320-330PubMed Google Scholar, 5Gilad S. Chessa L. Khosravi R. Russell P. Galanty Y. Piane M. Gatti R.A. Jorgensen T.J. Shiloh Y. Bar-Shira A. Am. J. Hum. Genet. 1998; 62: 551-561Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Given the predominance of null mutations underlying the classical form of ataxia telangiectasia (6Gilad S. Khosravi R. Shkedy D. Uziel T. Ziv Y. Savitsky K. Rotman G. Smith S. Chessa L. Jorgensen T.J. Harnik R. Frydman M. Sanal O. Portnoi S. Goldwicz Z. Jaspers N.G.J. Gatti R.A. Lenoir G. Lavin M. Tatsumi K. Wegner R.D. Shiloh Y. Bar-Shira A. Hum. Mol. Genet. 1996; 5: 433-439Crossref PubMed Scopus (257) Google Scholar), it is highly likely that milder, "variant" forms arise from less severe mutations that retain some normal protein function. In support of this, we have previously reported two different ATM gene mutations that are specifically associated with a milder clinical syndrome (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 4McConville C.M. Stankovic T. Byrd P.J. McGuire G.M. Yao Q.-Y. Lennox G.G. Taylor A.M.R. Am. J. Hum. Genet. 1996; 59: 320-330PubMed Google Scholar). The first mutation, present in the heterozygous state in 15% of A-T families in the United Kingdom, is an intronic missense mutation that activates a cryptic splice donor/acceptor site resulting in the insertion of 137 nucleotides of intronic sequence at position 5762. It appears, however, that this mutation is "leaky" and also allows normal splicing to occur, albeit at a reduced level. It was proposed that the presence of some normal ATM protein was sufficient to reduce the severity of the disease (4McConville C.M. Stankovic T. Byrd P.J. McGuire G.M. Yao Q.-Y. Lennox G.G. Taylor A.M.R. Am. J. Hum. Genet. 1996; 59: 320-330PubMed Google Scholar). In a similar context, a leaky splice mutation (3576G→A) was identified in four Italian A-T patients with a milder phenotype (5Gilad S. Chessa L. Khosravi R. Russell P. Galanty Y. Piane M. Gatti R.A. Jorgensen T.J. Shiloh Y. Bar-Shira A. Am. J. Hum. Genet. 1998; 62: 551-561Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), which again allows residual amounts of normally spliced ATM transcript. In contrast, the second ATM mutation we reported, associated with a mild variant A-T phenotype (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), is a T to G transition at position 7271, which has so far been found in three A-T families, and homozygous in one, in the UK. In this case the genotype-phenotype relationship is thought to result from some normal activity retained by the mutant ATM protein, which appears to be expressed at levels comparable to that in a normal individual, at least in affected individuals that are homozygous for this mutation (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The mechanism for the milder clinical course in both the 5762ins137 and the 7271T→G patients is currently unknown. Our current study, therefore, was devised to try and identify cellular pathways dependent on ATM that may be important for moderating the symptoms arising from loss of ATM function. Lymphoblastoid cell lines (LCLs) and skin fibroblast strains were derived in our laboratory from normal individuals and patients with ataxia telangiectasia. LCLs were routinely maintained in RPMI medium supplemented with 10% fetal calf serum, glutamine, penicillin, and streptomycin. Whole cell extracts (from ∼4 × 107 cells) were prepared as described (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Briefly, cells were sonicated in UTB buffer (9 m urea, 150 mmβ-mercaptoethanol, 50 mm Tris/HCl (pH 7.5)), and cellular debris was removed by centrifugation. Proteins were fractionated in 6% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose, and immunoblots were performed with p53 (donated by D. P. Lane), p53 phosphoserine 15 (New England Biolabs), p21 (Santa Cruz Biotechnology), MDM2 (2A10), ATM (FP8r) (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), hMre11, Nbs1, and hRad50 (7Dolganov G.M. Maser R.S. Novikov A. Tosto L. Chong S. Bressan D.A. Petrini J.H. Mol. Cell. Biol. 1996; 16: 4832-4841Crossref PubMed Scopus (190) Google Scholar, 8Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates III, J.R. Hays L. Morgan W.F. Petrini J.H. Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar) antisera. To verify that equivalent amounts of each sample were loaded, the filters were additionally probed with an actin monoclonal antibody (AC74, Sigma). Band density was quantified using scanning densitometry. LCL cells (∼4 × 107) were lysed on ice for 30 min in TGN buffer (50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 1% (v/v) Tween 20, 0.2% (v/v) Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaF, 1 mm sodium orthovanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µg/ml pepstatin A). Cellular debris was pelleted for 30 min at 4 °C (13,000 rpm). 1 mg of whole cell extract was pre-cleared with 30 µg of agarose-coupled anti-mouse IgG-agarose beads pre-washed in TGN buffer for 1 h at 4 °C on a rotator. Cleared lysates were incubated with 10 µg of Ab-2 or Ab-3 anti-ATM antibody (Oncogene Science) for 2 h at 4 °C on a rotator. Immune complexes were precipitated for 1–2 h by rolling at 4 °C with 30 µg of agarose-coupled anti-mouse IgG beads (or protein A beads) pre-washed in TGN buffer. Bead-bound immunoprecipitates were washed twice with TGN buffer, once with TGN buffer supplemented with 0.5 m LiCl, and then twice with kinase buffer (50 mm Hepes (pH 7.5), 150 mmNaCl, 6 mm MgCl2, 4 mmMnCl2, 10% (v/v) glycerol, 1 mmdithiothreitol, 100 µm sodium orthovanadate). Each immunoprecipitate was resuspended in 30 µl of kinase buffer supplemented with 20 µm cold ATP (Sigma), 1 µg of PHAS-I (Stratagene), and 10 µCi of [γ-32P]ATP (10 mCi/ml, 3000 Ci/mmol) (Amersham Pharmacia Biotech) and incubated at 30 °C for 20 min. The kinase reaction was stopped by the addition of SDS sample buffer and boiled for 5 min. Proteins were fractionated on a biphasic (6 and 12.5%) SDS-polyacrylamide gel. Proteins were visualized by silver staining, and the gel was dried and then subjected to autoradiography. LCLs (∼4 × 107) were lysed on ice for 30 min in lysis buffer (10 mm Tris/HCl (pH 7.5), 100 mm NaCl, 5 mm EDTA, 0.5% (v/v) Nonidet P-40, 0.5 mmphenylmethylsulfonyl fluoride, 2 mm sodium orthovanadate, 10 µg/ml leupeptin), and cellular debris was pelleted for 5 min. The lysate was precleared for 1–2 h at 4 °C with either protein G-agarose beads (Sigma) or agarose-coupled anti-mouse IgG beads (Sigma). Cleared lysates were incubated on ice for 1 h with Nbs1 monoclonal antibody 9H4 ascites fluid or anti-ATM antiserum (NT1 (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar)). Immune complexes were precipitated for 1–2 h by rolling at 4 °C with protein G-agarose or agarose-coupled anti-mouse IgG beads. Bead-bound immunoprecipitates were washed four times with lysis buffer, boiled in SDS sample buffer, and loaded on SDS 6% polyacrylamide gels. Proteins were analyzed by immunoblotting using standard methods and detected as described above. For phosphatase treatment, following washing with lysis buffer, immunoprecipitates were washed a further two times in phosphatase buffer (50 mm Tris-HCl (pH 7.5), 2 mmMnCl2, 0.1 mm EDTA, 5 mmdithiothreitol, 0.01% Brij 35) and then incubated with 400 units of λ-phosphatase (New England Biolabs) at 30 °C for 30 min. The reaction was stopped by boiling in SDS sample buffer. Exponentially growing LCL cells (∼4 × 106 per time point) were irradiated with 3 Gy of 60Co-gamma rays (about 1 Gy/min) and subsequently incubated at 37 °C. Cells were harvested at the time points indicated, washed three times in ice-cold phosphate-buffered saline, and whole cell extracts made (see above). 20 µg of whole cell extract from each time point were routinely loaded onto a 10% SDS-polyacrylamide gel and analyzed by immunoblotting. To verify protein equal loading, the filters were additionally probed with an anti-actin monoclonal antibody (AC74). Band density was quantified using scanning densitometry. LCL cells (∼4 × 107) were irradiated with 20 Gy of 60Co-gamma rays (about 2 Gy/min) at room temperature and subsequently incubated for 1 h at 37 °C. The cells were pelleted, washed in ice-cold phosphate-buffered saline, and resuspended in lysis buffer (20 mm Tris/HCl (pH 7.6), 0.5% (v/v) Triton X-100, 250 mm NaCl, 3 mm EGTA, 3 mm EDTA, 200 µm phenylmethylsulfonyl fluoride, 2 mm sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mm dithiothreitol, 50 mm NaF). The cells were lysed for 45 min, and cellular debris was pelleted for 15 min at 4 °C. 250 µg of whole cell extract was used per immunoprecipitation. 0.5 µg of rabbit anti-JNK antibody (Santa Cruz Biotechnology, sc-474) or an equivalent amount of nonspecific rabbit IgG (Sigma) was added to each immunoprecipitation and then incubated for 2 h at 4 °C. Immune complexes were precipitated for 2 h by rolling at 4 °C with protein G-agarose beads (Sigma). Bead-bound immunoprecipitates were washed once with lysis buffer and then twice with kinase buffer (20 mm Hepes (pH 7.5), 20 mm β-glycerophosphate, 10 mmMgCl2, 10 mm MnCl2, 1 mm dithiothreitol, 50 µm sodium orthovanadate). Each immunoprecipitate was resuspended in 30 µl of kinase buffer supplemented with 1 µm cold ATP (Sigma), 2 µg of GST-c-Jun (Stratagene), and 1 µCi of [γ-32P]ATP (10 mCi/ml, 3000 Ci/mmol) (Amersham Pharmacia Biotech) and incubated at 30 °C for 30 min. 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Petrini J.H. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (673) Google Scholar, 18Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (694) Google Scholar, 19Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar). In an attempt to understand the underlying reason for the milder clinical phenotypes exhibited by patients with the 5762ins137 and 7271T→G mutations, we initially carried out Western blotting to evaluate the level of ATM protein expression in these patients and then, subsequently, assayed the residual protein for kinase activity. The level of residual ATM protein expression in patients with the 5762ins137 ATM gene mutation ranged from 3 to 60% of the normal level of expression (Fig. 1,A and B). Patients 1-3, 40-4, 52-4, 59-4, and 79-3 all had a second truncating mutation (TableI), consistent with the suggestion that the residual protein was solely wild type protein derived from the leaky 5762ins137 mutation (4McConville C.M. Stankovic T. Byrd P.J. McGuire G.M. Yao Q.-Y. Lennox G.G. Taylor A.M.R. Am. J. Hum. Genet. 1996; 59: 320-330PubMed Google Scholar). The second mutation in patient 62-4 was a deletion of three amino acids at position 2546, which has been found to be associated with a classical A-T phenotype (4McConville C.M. Stankovic T. Byrd P.J. McGuire G.M. Yao Q.-Y. Lennox G.G. Taylor A.M.R. Am. J. Hum. Genet. 1996; 59: 320-330PubMed Google Scholar). The secondATM gene mutation for the remaining patients has not been determined. However, the high level of ATM expression in patient 45-3 suggested that the second mutation was an expressed missense point mutation.Table IMutations in A-T cell lines used in assaysPatientMutation 1Mutation 2Cell lines with 5762ins137 mutations1–35762ins137Stop19303801delGStop126814-45762ins137Stop1930ND38-35762ins137Stop1930ND40-45762ins137Stop19308787ins14Stop293344-45762ins137Stop1930ND45-35762ins137Stop1930ND52-45762ins137Stop19309139C→TStop304759-45762ins137Stop19306412delAGStop214462-45762ins137Stop19307636del9delSRI79-35762ins137Stop19302282delCTStop763135-35762ins137Stop1930NDCell lines with 7271T→G mutation109 II-17271T→GV2424G7271T→GV2424G109 II-57271T→GV2424G7271T→GV2424G109 II-67271T→GV2424G7271T→GV2424G46, II-17271T→GV2424G3910del7Stop130446, II-27271T→GV2424G3910del7Stop1304136, II-17271T→GV2424GNDCell lines with 7636del926-47636del9DelSRIND39-47636del9DelSRI136del4Stop 46Cell lines from classical A-T with two truncating ATM mutations92-32249ins9ter7522249ins9ter752113-32639del200ter8802639del200ter880118-3794ins4Stop 2662839del83Stop 947 Open table in a new tab In contrast, the 7271T→G mutation was expressed at levels comparable to normal in cells from patients 109 II-1, 109II-5, and 109 II-6, homozygous for the mutation and approximately half that of normal in patient 46 II-2 who was heterozygous for the 7271 mutation and had a second null mutation, 3910del7 (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The normal level of ATM protein in the 7271 heterozygous patient 136 II-1 suggested that the second unknown mutation was also an expressed missense point mutation that did not affect the stability of the protein (Fig. 1 C). To evaluate the kinase activity of the residual ATM protein expressed in the 5762ins137 patients, an immunoprecipitation in vitrokinase assay was carried out in which the amount of protein was increased to compensate for the reduced level of expression. A ratio of the level of immunoprecipitated ATM to phosphorylated PHAS-I substrate was calculated and expressed as a percentage of the activity found in normal cells. Three of four 5762ins137 patients tested, 40-4, 45-3, and 135-3, exhibited the same specific activity of ATM kinase as normals. The fourth patient, 59-4, expressed ATM protein at levels that were too low to obtain any ATM kinase activity above background (data not shown). Patient 39-4, who was compound heterozygous for a truncating null ATM allele 136del4 and the 2546delSRI (7636del9) mutation exhibited no detectable ATM kinase activity, in keeping with the classical A-T phenotype exhibited by this patient (Fig.2 A). A similar assay was carried out using cells from patients with the 7271T→G mutation, except that similar amounts of protein were used in each immunoprecipitate given that the level of mutant protein in these patients was comparable to that found in normal cells (3Stankovic T. Kidd A.M. Sutcliffe A. McGuire G.M. Robinson P. Weber P. Bedenham T. Bradwell A.R. Easton D.F. Lennox G.G. Haites N. Byrd P.J. Taylor A.M.R. Am. J. Hum. Genet. 1998; 62: 334-345Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The mutant protein expressed in cells from the homozygous 7271 patients 109II-1, 109 II-5, and 109 II-6 showed only ∼4–6% of normal ATM kinase activity when using PHAS-I as a substrate (Fig. 2 B). In addition, this mutant ATM protein was also able to undergo weak autophosphorylation (data not shown). The kinase activity of the mutant ATM protein in the 7271T→G heterozygous patient 46 II-2 was too low to detect over and above nonspecific background phosphorylation of PHAS-I observed in the negative control (data not shown). In contrast, the kinase activity in the heterozygous patient 136 II-1 was ∼10–15% of normal ATM activity suggesting that the second expressed mutant allele, in addition to the 7271T→G mutation, significantly contributed to the residual ATM function in these cells (data not shown). Therefore, the residual ATM protein expressed in cells derived from both the 5762ins137 and the 7271T→G patients retained detectable in vitro protein kinase activity. In addition, the secondATM mutation present in the cells may also affect the level of remaining activity. To assess the activity of the residual ATM protein in these variant A-T patients in vivo, we examined the ATM-dependent phosphorylation of the double strand break repair proteins, Nbs1 and hMre11, after DNA damage. The phosphorylation of Nbs1 (17Lim D.S. Kim S.T. Xu B. Maser R.S. Lin J. Petrini J.H. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (673) Google Scholar, 20Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 21Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar, 22Zhao S. Weng Y.C. Yuan S.S. Lin Y.T. Hsu H.C. Lin S.C. Gerbino E. Song M.H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.Y. Nature. 2000; 405: 473-477Crossref PubMed Scopus (435) Google Scholar) and hMre11 (23Dong Z. Zhong Q. Chen P.L. J. Biol. Chem. 1999; 274: 19513-19516Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) can be detected by a change in protein mobility on an SDS-PAGE gel. The bandshift of Nbs1 and hMre11 proteins is clearly evident in normal cells but not in cells from a classical A-T after exposure to 50 Gy of γ-radiation (Fig.3, A and B). To confirm that the mobility shift of both hMre11 and Nbs1 was specifically due to phosphorylation, immunoprecipitates of hMre11 and Nbs1 following exposure of normal and classical A-T cells to ionizing radiation were treated with λ protein phosphatase. Phosphatase treatment resulted in normal mobility of hMre11 and Nbs1 (Fig.3 C), therefore confirming that retardation of the hMre11 and Nbs1 protein after DNA damage was specifically due to phosphorylation. The three 5762ins137 cell lines analyzed showed a small detectable level of Nbs1 but not hMre11 phosphorylation following DNA damage (Fig.3 A). Immunoprecipitation and phosphatase treatment of Nbs1 in one of the 5762ins137 cell lines (40-4) following DNA damage demonstrated a small amount of damage-induced Nbs1 phosphorylation (Fig. 3 C). However, immunoprecipitation of hMre11 in this cell line following exposure to ionizing radiation failed to show any damage-induced mobility shift (Fig. 3 C). In contrast, phosphorylation of Nbs1 and hMre11 was observed at a higher level in irradiated cells derived from patients that were either homozygous or heterozygous for the 7271T→G mutation, albeit at reduced levels when compared with normal (Fig. 3 B). Thus in both the 5762ins137 and the 7271 cells, the ATM protein did retain some normal activityin vivo as well as in vitro, although it was more evident in patients with the 7271T→G mutant protein. ATM activation results in the induction of p53 and certain downstream responsive genes. A-T cells exhibit a defective induction of p53 and GADD45 after exposure to ionizing radiation (24Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2930) Google Scholar). However, in keeping with the heterogeneity that is characteristic of A-T, the kinetics of the damage-induced p53 accumulation in these cells does show some variability. It would appear that the defect in cells from classical A-T patients is either a delay in the accumulation of stable p53 or in both the timing of peak p53 accumulation and also a reduction in the maximal level of detectable p53 protein (24Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2930) Google Scholar, 25Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar, 26Canman C.E. Wolff A.C. Chen C.Y. Fornace Jr., A.J. Kastan M.B. Cancer Res. 1994; 54: 5054-5058PubMed Google Scholar, 27Artuso M. Esteve A. Bresil H. Vuillaume M. Hall J. Oncogene. 1995; 11: 1427-1435PubMed Google Scholar). Cell lines derived from 10 normal individuals were analyzed for the induction of p53, p21, and MDM2 following exposure to γ-radiation. The peak of maximum p53 induction occurred at 4 h post-irradiation with the fold p53 induction over basal levels varying between 6- and 15-fold. The induction of the p53-responsive gene products p21 and MDM2 reached a peak at 2–4 h post-irradiation (data not shown). When compared with normal the p53 induction kinetics in cells from 6 classical A-T patients showed significantly delayed and reduced accumulation when compared with normal (data not shown). The up-regulation of p21 and MDM2 in these cells was also defective. Seven LCLs with the 5762ins137 ATM mutation and two LCLs with the 7636del9 ATM mutation were analyzed for their ability to accumulate p53, p21, and MDM2 after exposure to ionizing radiation. Although some variation of p53 responses was observed within the 5762ins137 LCLs tested, cells from all seven patients exhibited a p53 induction profile that was intermediate between normal and classical A-T LCLs, characteristically showing a rapid accumulation of p53 within the first 2 h post-irradiation. The p21 and MDM2 accumulation in the 5762ins137 LCLs showed normal induction kinetics (Fig. 4). In contrast the two 7636del9 LCLs exhibited a p53, p21, and MDM2