David B. Young

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.

IκB Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex

ABSTRACT Activation of the transcription factor NF-κB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IκB. We recently purified a large multiprotein complex, the IκB kinase (IKK) signalsome, which contains two regulated IκB kinases, IKK1 and IKK2, that can each phosphorylate IκBα and IκBβ. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFκB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IκB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-κB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-κB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-κB activation.

Autophosphorylation of ataxia‐telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia‐telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A‐like activity in vitro. OA did not induce γ‐H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double‐strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A‐like protein phosphatase activity. Moreover, we show that IR induces phosphorylation‐dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

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.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N.

Using the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-J kappa. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A. The region of RBP-2N to which EBNA6A binds has previously been shown to interact with EBNA2.

BCoR-L1 variation and breast cancer

IntroductionBRCA1 is involved in numerous essential processes in the cell, and the effects of BRCA1 dysfunction in breast cancer carcinogenesis are well described. Many of the breast cancer susceptibility genes such as BRCA2, p53, ATM, CHEK2, and BRIP1 encode proteins that interact with BRCA1. BCL6 corepressor-like 1 (BCoR-L1) is a newly described BRCA1-interacting protein that displays high homology to several proteins known to be involved in the fundamental processes of DNA damage repair and transcription regulation. BCoR-L1 has been shown to play a role in transcription corepression, and expression of the X-linked BCoR-L1 gene has been reported to be dysregulated in breast cancer subjects. BCoR-L1 is located on the X chromosome and is subject to X inactivation.MethodsWe performed mutation analysis of 38 BRCA1/2 mutation-negative breast cancer families with male breast cancer, prostate cancer, and/or haplotype sharing around BCoR-L1 to determine whether there is a role for BCoR-L1 as a high-risk breast cancer predisposition gene. In addition, we conducted quantitative real-time PCR (qRT-PCR) on lymphoblastoid cell lines (LCLs) from the index cases from these families and a number of cancer cell lines to assess the role of BCoR-L1 dysregulation in cancer and cancer families.ResultsVery little variation was detected in the coding region, and qRT-PCR analysis revealed that BCoR-L1 expression is highly variable in cancer-free subjects, high-risk breast cancer patients, and cancer cell lines. We also report the investigation of a new expression control, DIDO1 (death inducer-obliterator 1), that is superior to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and UBC (ubiquitin C) for analysis of expression in LCLs.ConclusionOur results suggest that BCoR-L1 expression does not play a large role in predisposition to familial breast cancer.

Erratum to: BCoR-L1 variation and breast cancer

Following the publication of our article [1] we noticed that, due to a production error, the figure legends and images were incorrectly matched. All legends were correctly placed, and cited in the text, but were associated with the wrong image. The figures should therefore appear in the order shown in this correction. Figure 1 BcoR-L1 expression in cancer and normal cell lines. (a) BCoR-L1 expression in cancer and normal cell lines. (b) Mean and standard deviation of BCoR-L1 expression in cancer and normal cell lines. Normal cell lines: ovarian – OSE 64/96, HOSE 17.1; ... Figure 2 BCoR-L1 haplotype sharing family pedigree detailing carriers of the c.516T>C and c.3608-156C>T variants. = breast cancer-positive; c.516T>C and c.3608-156C>T-positive. = breast cancer-negative; c.516T>C and c.3608-156C>T-positive. ... Figure 3 BcoR-L1 expression in lymphoblastoid cell lines (LCLs) from breast cancer families. (a) BCoR-L1 expression in LCLs from breast cancer families (normalised to GAPDH). (b) BCoR-L1 expression in LCLs from breast cancer families (normalised to DIDO-1). (c) ... Figure 4 Variation in control gene expression. (a) Variation in control gene expression in lymphoblastoid cell lines. (b) Variation in control gene expression in cell lines. DIDO-1, death inducer-obliterator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ...

Erratum to: BCoR-L1variation and breast cancer

Following the publication of our article [1] we noticed that, due to a production error, the figure legends and images were incorrectly matched. All legends were correctly placed, and cited in the text, but were associated with the wrong image. The figures should therefore appear in the order shown in this correction. Figure 1 BcoR-L1 expression in cancer and normal cell lines. (a) BCoR-L1 expression in cancer and normal cell lines. (b) Mean and standard deviation of BCoR-L1 expression in cancer and normal cell lines. Normal cell lines: ovarian – OSE 64/96, HOSE 17.1; ... Figure 2 BCoR-L1 haplotype sharing family pedigree detailing carriers of the c.516T>C and c.3608-156C>T variants. = breast cancer-positive; c.516T>C and c.3608-156C>T-positive. = breast cancer-negative; c.516T>C and c.3608-156C>T-positive. ... Figure 3 BcoR-L1 expression in lymphoblastoid cell lines (LCLs) from breast cancer families. (a) BCoR-L1 expression in LCLs from breast cancer families (normalised to GAPDH). (b) BCoR-L1 expression in LCLs from breast cancer families (normalised to DIDO-1). (c) ... Figure 4 Variation in control gene expression. (a) Variation in control gene expression in lymphoblastoid cell lines. (b) Variation in control gene expression in cell lines. DIDO-1, death inducer-obliterator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ...

Correction: BCoR-L1 variation and breast cancer

Following the publication of our article [1] we noticed that, due to a production error, the figure legends and images were incorrectly matched. All legends were correctly placed, and cited in the text, but were associated with the wrong image. The figures should therefore appear in the order shown in this correction. Figure 1 BcoR-L1 expression in cancer and normal cell lines. (a) BCoR-L1 expression in cancer and normal cell lines. (b) Mean and standard deviation of BCoR-L1 expression in cancer and normal cell lines. Normal cell lines: ovarian – OSE 64/96, HOSE 17.1; ... Figure 2 BCoR-L1 haplotype sharing family pedigree detailing carriers of the c.516T>C and c.3608-156C>T variants. = breast cancer-positive; c.516T>C and c.3608-156C>T-positive. = breast cancer-negative; c.516T>C and c.3608-156C>T-positive. ... Figure 3 BcoR-L1 expression in lymphoblastoid cell lines (LCLs) from breast cancer families. (a) BCoR-L1 expression in LCLs from breast cancer families (normalised to GAPDH). (b) BCoR-L1 expression in LCLs from breast cancer families (normalised to DIDO-1). (c) ... Figure 4 Variation in control gene expression. (a) Variation in control gene expression in lymphoblastoid cell lines. (b) Variation in control gene expression in cell lines. DIDO-1, death inducer-obliterator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ...