Biomedical subjects

Yosef Shiloh

Publications and source records attributed to Yosef Shiloh.

18 recordsLinked to original sources

PubMed2005

ATM-mediated phosphorylations inhibit Mdmx/Mdm2 stabilization by HAUSP in favor of p53 activation.

The p53 tumor suppressor protein has a major role in protecting genome integrity. Under normal circumstances Mdmx and Mdm2 control the activity of p53. Both proteins inhibit the transcriptional regulation by p53, while Mdm2 also functions as an E3 ubiquitin ligase to target both p53 and Mdmx for proteasomal degradation. HAUSP counteracts the destabilizing effect of Mdm2 by direct deubiquitination of p53. Subsequently, HAUSP was shown to deubiquitinate Mdm2 and Mdmx, thereby stabilizing these proteins. The ATM protein kinase is a key regulator of the p53 pathway in response to double strand breaks (DSBs) in the DNA. ATM fine-tunes p53's response to DNA damage by directly phosphorylating it, by regulating additional post-translational modifications of this protein, and by affecting two p53 regulators: Mdm2 and Mdmx. ATM directly and indirectly induces Mdm2 and Mdmx phosphorylation, resulting in decreased activity and stability of these proteins. We recently provided a mechanism for the reduced stability of Mdm2 and Mdmx by showing that ATM-dependent phosphorylation lowers their affinity for the deubiquitinating enzyme HAUSP. Altogether, the emerging picture portrays an elaborate, but fine-tuned, ATM-mediated control of p53 activation and stabilization following DNA damage. Further insight into the mechanism by which ATM switches the interactions between HAUSP, Mdmx, Mdm2 and p53, to favor p53 activation may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

Adenosine Triphosphate↗

PubMed2005

ATM-dependent phosphorylation of ATF2 is required for the DNA damage response.

Activating transcription factor 2 (ATF2) is regulated by JNK/p38 in response to stress. Here, we demonstrate that the protein kinase ATM phosphorylates ATF2 on serines 490 and 498 following ionizing radiation (IR). Phosphoantibodies to ATF2(490/8) reveal dose- and time-dependent phosphorylation of ATF2 by ATM that results in its rapid colocalization with gamma-H2AX and MRN components into IR-induced foci (IRIF). Inhibition of ATF2 expression decreased recruitment of Mre11 to IRIF, abrogated S phase checkpoint, reduced activation of ATM, Chk1, and Chk2, and impaired radioresistance. ATF2 requires neither JNK/p38 nor its DNA binding domain for recruitment to IRIF and the S phase checkpoint. Our findings identify a role for ATF2 in the DNA damage response that is uncoupled from its transcriptional activity.

Activating Transcription Factor 2↗

PubMed2005

Dissection of a DNA-damage-induced transcriptional network using a combination of microarrays, RNA interference and computational promoter analysis.

BACKGROUND: Gene-expression microarrays and RNA interferences (RNAi) are among the most prominent techniques in functional genomics. The combination of the two holds promise for systematic, large-scale dissection of transcriptional networks. Recent studies, however, raise the concern that nonspecific responses to small interfering RNAs (siRNAs) might obscure the consequences of silencing the gene of interest, throwing into question the ability of this experimental strategy to achieve precise network dissections. RESULTS: We used microarrays and RNAi to dissect a transcriptional network induced by DNA damage in a human cellular system. We recorded expression profiles with and without exposure of the cells to a radiomimetic drug that induces DNA double-strand breaks (DSBs). Profiles were measured in control cells and in cells knocked-down for the Rel-A subunit of NFkappaB and for p53, two pivotal stress-induced transcription factors, and for the protein kinase ATM, the major transducer of the cellular responses to DSBs. We observed that NFkappaB and p53 mediated most of the damage-induced gene activation; that they controlled the activation of largely disjoint sets of genes; and that ATM was required for the activation of both pathways. Applying computational promoter analysis, we demonstrated that the dissection of the network into ATM/NFkappaB and ATM/p53-mediated arms was highly accurate. CONCLUSIONS: Our results demonstrate that the combined experimental strategy of expression arrays and RNAi is indeed a powerful method for the dissection of complex transcriptional networks, and that computational promoter analysis can provide a strong complementary means for assessing the accuracy of this dissection.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2005

Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.

Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.

Ataxia Telangiectasia↗

PubMed2004

In silico identification of transcriptional regulators associated with c-Myc.

The development of powerful experimental strategies for functional genomics and accompanying computational tools has brought major advances in the delineation of transcriptional networks in organisms ranging from yeast to human. Regulation of transcription of eukaryotic genes is to a large extent combinatorial. Here, we used an in silico approach to identify transcription factors (TFs) that form recurring regulatory modules with c-Myc, a protein encoded by an oncogene that is frequently disregulated in human malignancies. A recent study identified, on a genomic scale, human genes whose promoters are bound by c-Myc and its heterodimer partner Max in Burkitt's lymphoma cells. Using computational methods, we identified nine TFs whose binding-site signatures are highly overrepresented in this promoter set of c-Myc targets, pointing to possible functional links between these TFs and c-Myc. Binding sites of most of these TFs are also enriched on the set of mouse homolog promoters, suggesting functional conservation. Among the enriched TFs, there are several regulators known to control cell cycle progression. Another TF in this set, EGR-1, is rapidly activated by numerous stress challenges and plays a central role in angiogenesis. Experimental investigation confirmed that c-Myc and EGR-1 bind together on several target promoters. The approach applied here is general and demonstrates how computational analysis of functional genomics experiments can identify novel modules in complex networks of transcriptional regulation.

Animals↗

PubMed2004

PIVOT: protein interacions visualizatiOn tool.

Protein Interaction VisualizatiOn Tool (PIVOT) is a visualization tool for protein-protein interactions. It allows the user to create personal data sets of interactions by combining information from private and public data sources. The user can gradually access the interactions' data using a clear interactive map that is focused on the researcher's protein of interest, and is reshaped and expanded in response to his/her queries. It also offers several visual enhancements and intelligent queries that help the user efficiently study it. PIVOT allows the user to search the interactions data set for paths connecting proteins that are expected to co-operate. The user can also employ PIVOT to predict unknown interactions among proteins, based on interactions among their homologous proteins in other species.

Database Management Systems↗

PubMed2004

In search of drug treatment for genetic defects in the DNA damage response: the example of ataxia-telangiectasia.

Most genetic disorders are severe diseases that cannot be treated. In the majority of them, enzyme and gene therapy can be significantly curtailed by technical difficulties and the nature of the physiological defects and affected tissues. A rational search for drug treatment for such diseases must be based on understanding the corresponding molecular defects. For example, in a disease stemming from a defective signaling pathway, a drug that can activate redundant pathways could be useful. Screening for such a drug would then depend on the availability of a laboratory assay that faithfully reflects the molecular defect in the corresponding disease, and a technology for applying the assay in a high throughput setup. Deficiencies in various components of the DNA damage response lead to genomic instability syndromes characterized by tissue degeneration, sensitivity to DNA damaging agents, and cancer predisposition. A typical example is ataxia-telangiectasia (A-T), caused by deficiency of the nuclear protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. ATM phosphorylates a multitude of substrates, each of which in turn modulates a branch of the damage response network. A certain redundancy among ATM and related proteins gives hope that activation of ATM-redundant activities might form a basis for drug treatment of A-T. This article describes a high throughput strategy for drug screening for A-T that is based on the above principles. A similar strategy can potentially be applied to drug screening for other genetic disorders.

Animals↗

PubMed2004

Maintaining integrity.

Research on genome stability and integrity now extends far beyond the biochemistry of DNA repair to encompass signal transduction pathways that span numerous aspects of cellular life. Derailed genomic integrity pathways can result in debilitating genetic disorders, premature ageing, predisposition to cancer and degenerative conditions. Current progress in this rapidly expanding field was the subject of an EMBO workshop, Maintenance of Genomic Integrity, that took place in June 2004 in Galway, Ireland. Top

Animals↗

PubMed2003

Requirement of the MRN complex for ATM activation by DNA damage.

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.

Ataxia Telangiectasia↗

PubMed2003

ATM and related protein kinases: safeguarding genome integrity.

Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.

Animals↗

PubMed2003

Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells.

Dissection of regulatory networks that control gene transcription is one of the greatest challenges of functional genomics. Using human genomic sequences, models for binding sites of known transcription factors, and gene expression data, we demonstrate that the reverse engineering approach, which infers regulatory mechanisms from gene expression patterns, can reveal transcriptional networks in human cells. To date, such methodologies were successfully demonstrated only in prokaryotes and low eukaryotes. We developed computational methods for identifying putative binding sites of transcription factors and for evaluating the statistical significance of their prevalence in a given set of promoters. Focusing on transcriptional mechanisms that control cell cycle progression, our computational analyses revealed eight transcription factors whose binding sites are significantly overrepresented in promoters of genes whose expression is cell-cycle-dependent. The enrichment of some of these factors is specific to certain phases of the cell cycle. In addition, several pairs of these transcription factors show a significant co-occurrence rate in cell-cycle-regulated promoters. Each such pair indicates functional cooperation between its members in regulating the transcriptional program associated with cell cycle progression. The methods presented here are general and can be applied to the analysis of transcriptional networks controlling any biological process.

Binding Sites↗

PubMed2002

Contribution of the Atm protein to maintaining cellular homeostasis evidenced by continuous activation of the AP-1 pathway in Atm-deficient brains.

Maintenance of genome stability is essential for keeping cellular homeostasis. The DNA damage response is a central component in maintaining genome integrity. Among of the most cytotoxic DNA lesions are double strand breaks (DSBs) caused by ionizing radiation or radiomimetic chemicals. ATM is missing or inactivated in patients with ataxia-telangiectasia. Ataxia-telangiectasia patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Additional features are immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. Disruption of the mouse Atm locus creates a murine model of ataxia-telangiectasia that exhibits most of the clinical features of the human disease but very mild neuronal abnormality. The ATM protein is a multifunctional protein kinase, which serves as a master regulator of cellular responses to DSBs. There is growing evidence that ATM may be involved in addition to the DSB response in other processes that maintain processes in cellular homeostasis. For example, mounting evidence points to increased oxidative stress in the absence of ATM. Here we report that the AP-1 pathway is constantly active in the brains of Atm-deficient mice not treated with DNA damaging agents. A canonical activation (increased phosphorylation of mitogen-activated protein kinase kinase-4, c-Jun N-terminal kinase, and c-Jun) of the AP-1 pathway was found in Atm-deficient cerebra, whereas induction of the AP-1 pathway in Atm-deficient cerebella is likely to mediate elevated expression of c-Fos and c-Jun. Although Atm(+/+) mice are capable of responding to ionizing radiation by activating stress responses such as the AP-1 pathway, Atm-deficient mice display higher basal AP-1 activity but gradually lose their ability to activate AP-1 DNA-binding activity in response to ionizing radiation. Our results further demonstrate that inactivation of the ATM gene results in a state of constant stress.

Animals↗

PubMed2002

Ionizing radiation induces ataxia telangiectasia mutated kinase (ATM)-mediated phosphorylation of LKB1/STK11 at Thr-366.

The serine/threonine protein kinase LKB1 functions as a tumour suppressor, and mutations in this enzyme lead to the inherited Peutz-Jeghers cancer syndrome. We previously found that LKB1 was phosphorylated at Thr-366 in vivo, a residue conserved in mammalian, Xenopus and Drosophila LKB1, located on a C-terminal non-catalytic moiety of the enzyme. Mutation of Thr-366 to Ala or Asp partially inhibited the ability of LKB1 to suppress growth of G361 melanoma cells, but did not affect LKB1 activity in vitro or LKB1 localization in vivo. As a first step in exploring the role of this phosphorylation further, we have generated a phosphospecific antibody specifically recognizing LKB1 phosphorylated at Thr-366 and demonstrate that exposure of cells to ionizing radiation (IR) induced a marked phosphorylation of LKB1 at Thr-366 in the nucleus. Thr-366 lies in an optimal phosphorylation motif for the phosphoinositide 3-kinase-like kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia-related kinase (ATR), which function as sensors for DNA damage in cells and mediate cellular responses to DNA damage. We demonstrate that both DNA-PK and ATM efficiently phosphorylate LKB1 at Thr-366 in vitro and provide evidence that ATM mediates this phosphorylation in vivo. This is based on the finding that LKB1 is not phosphorylated in a cell line lacking ATM in response to IR, and that agents which induce cellular responses via ATR in preference to ATM poorly induce phosphorylation of LKB1 at Thr-366. These observations provide the first link between ATM and LKB1 and suggest that ATM could regulate LKB1.

AMP-Activated Protein Kinase Kinases↗

PubMed2002

Ubiquitination capabilities in response to neocarzinostatin and H(2)O(2) stress in cell lines from patients with ataxia-telangiectasia.

The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50-70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H(2)O(2) as the stress, in view of preexisting evidence of the effects of H(2)O(2) on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an approximately 45% increase in endogenous ubiquitin conjugates by 2-4 h after exposure to H(2)O(2). Both cell types showed a rapid 50-150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an 'aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.

Antibiotics, Antineoplastic↗

PubMed2002

ATM-dependent activation of the gene encoding MAP kinase phosphatase 5 by radiomimetic DNA damage.

Cellular responses to DNA damage are mediated by an extensive network of signaling pathways. The ATM protein kinase is a master regulator of the response to double-strand breaks (DSBs), the most cytotoxic DNA lesion caused by ionizing radiation. ATM is the protein missing or inactive in patients with the pleiotropic genetic disorder ataxia-telangiectasia (A-T). A major response to DNA damage is altered expression of numerous genes. While studying gene expression in control and A-T cells following treatment with the radiomimetic chemical neocarzinostatin (NCS), we identified an expressed sequence tag that represented a gene that was induced by DSBs in an ATM-dependent manner. The corresponding cDNA encoded a dual specificity phosphatase of the MAP kinase phosphatase family, MKP-5. MKP-5 dephosphorylates and inactivates the stress-activated MAP kinases JNK and p38. The phosphorylation-dephosphorylation cycle of JNK and p38 by NCS was attenuated in A-T cells. Thus, ATM modulates this cycle in response to DSBs. These results further highlight ATM as a link between the DNA damage response and major signaling pathways involved in proliferative and apoptotic processes.

Ataxia Telangiectasia↗

PubMed2002

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage.

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.

Animals↗

PubMed2001

Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.

Adenosine Diphosphate Ribose↗

PubMedDate not supplied

ATM: ready, set, go.

Explore the source record for details and available documents.

DNA Damage↗