Martin F. Lavin

Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer

First described over 80 years ago, ataxia-telangiectasia (A-T) was defined as a clinical entity 50 years ago. Although not encountered by most clinicians, it is a paradigm for cancer predisposition and neurodegenerative disorders and has a central role in our understanding of the DNA-damage response, signal transduction and cell-cycle control. The discovery of the protein A-T mutated (ATM) that is deficient in A-T paved the way for rapid progress on understanding how ATM functions with a host of other proteins to protect against genome instability and reduce the risk of cancer and other pathologies.

ATM Activation by Oxidative Stress

Stress, DNA Damage, and ATM The protein kinase ATM (ataxia-telangiectasia mutated) is a key component of the signaling pathway through which cells are protected from DNA damage. ATM becomes activated within a protein complex at sites of double-stranded breaks in DNA. ATM is also activated in response to increased production of reactive oxygen species (ROS). Such activation was thought to reflect DNA damage caused by ROS, but Guo et al. (p. 517) showed that ATM was in fact directly activated by ROS. A cysteine residue in ATM contributes to the formation of disulfide-linked dimers of activated ATM on exposure to ROS in vitro. Experiments using mutated forms of the enzyme suggested that two distinct mechanisms regulated ATM activity. The protein kinase ATM is a sensor for reactive oxygen species. The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide–cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells.

The complexity of p53 stabilization and activation.

A number of proteins are activated by stress stimuli but none so spectacularly or with the degree of complexity as the tumour suppressor p53 (human p53 gene or protein). Once stabilized, p53 is responsible for the transcriptional activation of a series of proteins involved in cell cycle control, apoptosis and senescence. This protein is present at low levels in resting cells but after exposure to DNA-damaging agents and other stress stimuli it is stabilized and activated by a series of post-translational modifications that free it from MDM2 (mouse double minute 2 but used interchangeably to denote human also), a ubiquination ligase that ubiquitinates it prior to proteasome degradation. The stability of p53 is also influenced by a series of other interacting proteins. In this review, we discuss the post-translational modifications to p53 in response to different stresses and the consequences of these changes.

ATM-dependent phosphorylation of nibrin in response to radiation exposure

Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). There are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50 (refs 1,2). This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (IR) and is observed in brightly staining nuclear foci after a longer period of time. The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. Here we demonstrate that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. We also show that ATM physically interacts with and phosphorylates nibrin on serine 343 both in vivo and in vitro. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. Our data provide a biochemical explanation for the similarity in phenotype between A-T and NBS.

ATM associates with and phosphorylates p53: mapping the region of interaction

The human genetic disorder ataxia-telangiectasia (AT) is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects and cancer predisposition. The gene mutated in this syndrome, ATM (for AT mutated), encodes a protein containing a phosphatidyl-inositol 3-kinase (PI-3 kinase)-like domain. ATM also contains a proline-rich region and a leucine zipper, both of which implicate this protein in signal transduction. The proline-rich region has been shown to bind to the SH3 domain of c-Abl, which facilitates its phosphorylation and activation by ATM (Refs 4,6). Previous results have demonstrated that AT cells are defective in the G1/S checkpoint activated after radiation damage and that this defect is attributable to a defective p53 signal transduction pathway. We report here direct interaction between ATM and p53 involving two regions in ATM, one at the amino terminus and the other at the carboxy terminus, corresponding to the PI-3 kinase domain. Recombinant ATM protein phosphorylates p53 on serine 15 near the N terminus. Furthermore, ectopic expression of ATM in AT cells restores normal ionizing radiation (IR)-induced phosphorylation of p53, whereas expression of ATM antisense RNA in control cells abrogates the rapid IR-induced phosphorylation of p53 on serine 15. These results demonstrate that ATM can bind p53 directly and is responsible for its serine 15 phosphorylation, thereby contributing to the activation and stabilization of p53 during the IR-induced DNA damage response.

DNA-DEPENDENT PROTEIN KINASE CATALYTIC SUBUNIT : A TARGET FOR AN ICE-LIKE PROTEASE IN APOPTOSIS

Radiosensitive cell lines derived from X‐ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA‐dependent protein kinase, DNA‐PK, suggesting that DNA‐PK plays an important role in DNA double‐strand break repair. Another enzyme implicated in DNA repair, poly(ADP‐ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA‐PKcs, the catalytic subunit of DNA‐PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA‐binding component of the enzyme, remains intact. Degradation of DNA‐PKcs was accompanied by loss of DNA‐PK activity. One cell line resistant to etoposide‐induced apoptosis failed to show degradation of DNA‐PKcs. Protease inhibitor data implicated an ICE‐like protease in the cleavage of DNA‐PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA‐PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA‐PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA‐PKcs is a critical target for proteolysis by an ICE‐like protease during apoptosis.

ATM activation and DNA damage response.

Well before the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) was described it was evident from the clinical, molecular and cellular phenotype of A-T that this gene would play a central role in the DNA damage response. Mutation of ATM causes defective cell cycle checkpoint activation,a reduced capacity for repair of DNA double strand breaks and abnormal apoptosis, all of which contribute to the major features of A-T including genome instability, increased cancer risk and neurodegeneration. While the exact mechanism of activation remains unknown, it is clear that the Mre11 complex plays an important role both in the recruitment of ATM to the sites of DNA damage and in the efficient activation of ATM. Although ATM responds to agents that produce double strand breaks in DNA, other stimuli are also capable of ATM activation. The description of autophosphorylation on S1981 of ATM and the ensuing transition from an inactive dimer to an active monomer represents a major milestone in our understanding of the activation process. However, it is now evident that more than one autophosphorylation event is required and not surprisingly this process is also attenuated by phosphatases and other modifications such as acetylation are also implicated. This is further complicated by a recent report that autophosphorylation at S1987 (the mouse site corresponding to S1981) is dispensable for Atm activation in an Atm mutant mouse model. Use of cell extracts and in vitro approaches in the reconstruction of activation complexes have shed further light on what it takes to activate ATM. The aim here is to examine the evidence for the involvement of these various steps in ATM activation and attempt to put together a comprehensive picture of the overall process and its significance to DNA damage signalling.

Involvement of novel autophosphorylation sites in ATM activation.

ATM kinase plays a central role in signaling DNA double‐strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia‐telangiectasia cells. We conclude that there are at least three functionally important radiation‐induced autophosphorylation events in ATM.

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks.

The recognition and repair of DNA double-strand breaks (DSBs) is a complex process that draws upon a multitude of proteins. This is not surprising since this is a lethal lesion if left unrepaired and also contributes to genome instability and the consequential risk of cancer and other pathologies. Some of the key proteins that recognize these breaks in DNA are mutated in distinct genetic disorders that predispose to agent sensitivity, genome instability, cancer predisposition and/or neurodegeneration. These include members of the Mre11 complex (Mre11/Rad50/Nbs1) and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human genetic disorder A-T. The mre11 (MRN) complex appears to be the major sensor of the breaks and subsequently recruits ATM where it is activated to phosphorylate in turn members of that complex and a variety of other proteins involved in cell-cycle control and DNA repair. The MRN complex is also upstream of ATM and ATR (A-T-mutated and rad3-related) protein in responding to agents that block DNA replication. To date, more than 30 ATM-dependent substrates have been identified in multiple pathways that maintain genome stability and reduce the risk of disease. We focus here on the relationship between ATM and the MRN complex in recognizing and responding to DNA DSBs.

Ataxia Telangiectasia Mutated (ATM) Signaling Network Is Modulated by a Novel Poly(ADP-ribose)-dependent Pathway in the Early Response to DNA-damaging Agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling.