The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.Cells respond to DNA damage by activating specific signaling pathways that culminate into cell cycle checkpoints, in which cell cycle progression is arrested and DNA repair is effected (1). These checkpoints are regulated, in part, by members of the phosphoinositide kinase (PIK)

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G 2 /M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser 219 -Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser 219 potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser 219 completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/ TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.Mutations in the ATM gene are responsible for the rare autosomal human recessive disorder ataxia-telangiectasia (A-T), 1 characterized by progressive neurological degeneration, telangiectasia, growth retardation, premature aging, immunodeficiency, mitotic checkpoint defect, hypersensitivity to ionizing radiation, gonadal atrophy, genomic instability, and predisposition to cancer (1). ATM encodes a protein kinase that is activated by ionizing DNA damage and is critical for maintaining genome stability, telomere maintenance, and induction of cell cycle checkpoints by double strand DNA breaks (2-4). ATM has been shown to bind and/or phosphorylate many key regulators, including p53, ␤-adaptin, c-Abl, Chk1-2, Brca1, and Nijmegen breakage syndrome protein (5-15). Identification of these ATM target proteins opens new avenues for understanding the physiological function of ATM as well as for explaining the pleiotropic pheno...

A characteristic series of immunological abnormalities are observed in the human genetic disorder ataxiatelangiectasia (A-T). The recent cloning of a gene mutated in this syndrome provides additional evidence for a defect in intracellular signaling in A-T. We have investigated the possibility that signaling through the B cell antigen receptor is one manifestation of the A-T defect. In response to cross-linking of the B cell receptor, several A-T cell lines were defective in their mitogenic response; in addition Ca 2؉ mobilization from internal stores was either absent or considerably reduced in these cell lines in response to cross-linking. The defect in signaling was not due to difference in expression of surface immunoglobulin. The defective response in A-T cells was also evident in several arms of the intracellular cascade activated by B cell cross-linking. Tyrosine phosphorylation of phospholipase C␥1, a key step in activation of the enzyme, was reduced or negligible in some A-T cell lines. This defect in signaling was also seen at the level of Lyn tyrosine kinase activation and its association with and activation of phosphatidylinositol 3-kinase. Our results provide evidence for a role for the ATM gene product in intracellular signaling which may account at least in part for the abnormalities in B cell function in A-T. The human genetic disorder ataxia-telangiectasia (A-T)1 is characterized by immunodeficiency, neurological abnormality, abnormal development, radiosensitivity, cell cycle anomalies, and cancer predisposition (1-3). Furthermore, a variety of immunological abnormalities characterize this syndrome, including hypogammaglobulinemia, selective deficiency of serum IgA and IgE (4, 5), abnormalities in IgG subclasses (6), depressed blastogenic response (7), faulty development or complete absence of the thymus (8), failure to produce virus-specific histocompatibility-restricted cytotoxic T lymphocytes (9), and an overall poor response to skin test antigens (10).The gene responsible for the defect in A-T, ATM, has been cloned recently (11) and shown to possess a carboxyl-terminal domain homologous to phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme, a heterodimer composed of a catalytic subunit (p110) and a regulatory subunit (p85), plays a central role in transmitting signals from the cell surface to the nucleus (12). The ATM protein is related to TOR1 and TOR2 proteins of yeast (13) and their mammalian counterparts FRAP (14) and RAFT1 (15) through the PI 3-kinase domain, and to a second group of proteins not only through this domain but also through an adjacent region of weaker homology (16). The latter group includes Mei-41 of Drosophila melanogaster, rad3p, Mec1p and Tel1p of yeast, and DNA-dependent protein kinase from human cells (17)(18)(19)(20). These proteins are involved in cell cycle control and response to DNA damage.It is evident that the ATM gene is involved not only in the response to DNA damage but also in regulating a number of cellular processes important in differentiation and...

Cells from patients with the hereditary multisystem disorder ataxia-telangiectasia (A-T) are hypersensitive to the cytotoxic action of DNA-breaking agents, such as X-rays, bleomycin and neocarzinostatin (NCS). A defect in the repair of a certain DNA lesion induced by all three agents may underlie this hypersensitivity. This DNA lesion may be a certain type of DNA strand break. Most of the previous experiments done with X-rays and bleomycin failed to show any retardation in the rejoining of DNA strand breaks in A-T cells. However, since both A-T homozygous and heterozygous cells are particularly hypersensitive to NCS, we studied the time course of strand breakage induction and repair in A-T skin fibroblast strains treated with NCS, using the sensitive method of alkaline or neutral elution. A linear dose response was obtained for the induction by NCS of single-strand breaks and double-strand breaks. A-T cells did not respond with a higher initial extent of strand breakage compared with normal cells. NCS is an appropriate agent for studying the kinetics of rejoining strand breaks, due to its rapid action in the cells; this action, which is completed within 2--4 min, was studied by monitoring strand break induction, inhibition of DNA synthesis and decrease in cellular survival. The time course of strand break rejoining found after NCS treatment was very similar to that found following X-irradiation: with both single- and double-strand breaks, a rapid phase of rejoining was first noticed (t 1/2 approximately 5 min for single-strand breaks and 20--25 min for double-strand breaks). This was followed by a second, slow phase that continued for several hours. No difference could be detected between normal and A-T cells either with regard to the time course of rejoining or the fraction of non-rejoined breaks remaining several hours after treatment.

Ten of 18 children in a highly inbred Arab kindred suffered from either ataxia telangiectasia (AT) or a variant syndrome consisting of ataxia, microcephaly, and congenital cataract (AMC). Four of the nine afflicted children were treated in our unit when they developed lymphomas (both Hodgkin's and non-Hodgkin's including Burkitt's). They were given chemotherapy (either standard COMP or low-dose ABV/CVPP). The children with non-Hodgkin's lymphomas died of sepsis after receiving full-dose COMP. Low-dose ABV/CVPP brought about a 20-month remission in one child with nodular sclerosing Hodgkin's lymphoma and both AT and AMC, but she developed a preleukemic syndrome and her parents refused further treatment; she too died. A fourth child, also with nodular sclerosing Hodgkin's lymphoma, is currently in complete remission after ABV/CVPP. Treatment of lymphomas in patients with AT is extraordinarily difficult and has potential side effects so grave as to necessitate careful monitoring and individualized protocols.