Phosphorylation of MCPH1 isoforms during mitosis followed by isoform-specific degradation by APC/C-CDH1.

Microcephalin-1 (MCPH1) exists as 2 isoforms that regulate cyclin-dependent kinase-1 activation and chromosome condensation during mitosis, with MCPH1 mutations causing primary microcephaly. MCPH1 is also a tumor suppressor protein, with roles in DNA damage repair/checkpoints. Despite these important roles, there is little information on the cellular regulation of MCPH1. We show that both MCPH1 isoforms are phosphorylated in a cyclin-dependent kinase-1-dependent manner in mitosis and identify several novel phosphorylation sites. Upon mitotic exit, MCPH1 isoforms were degraded by the anaphase-promoting complex/cyclosome-CDH1 E3 ligase complex. Anaphase-promoting complex/cyclosome-CDH1 target proteins generally have D-Box or KEN-Box degron sequences. We found that MCPH1 isoforms are degraded independently, with the long isoform degradation being D-Box dependent, whereas the short isoform was KEN-Box dependent. Our research identifies several novel mechanisms regulating MCPH1 and also highlights important issues with several commercial MCPH1 antibodies, with potential relevance to previously published data.-Meyer, S. K., Dunn, M., Vidler, D. S., Porter, A., Blain, P. G., Jowsey, P. A. Phosphorylation of MCPH1 isoforms during mitosis followed by isoform-specific degradation by APC/C-CDH1.

Fe65 Ser228 is phosphorylated by ATM/ATR and inhibits Fe65-APP-mediated gene transcription.

Fe65 binds the amyloid precursor protein (APP) and regulates the secretase-mediated processing of APP into several proteolytic fragments, including amyloid ß-peptides (Aß) and APP intracellular domain (AICD). Aß accumulation in neural plaques is a pathological feature of Alzheimer's disease (AD) and AICD has important roles in the regulation of gene transcription (in complex with Fe65). It is therefore important to understand how Fe65 is regulated and how this contributes to the function and/or processing of APP. Studies have also implicated Fe65 in the cellular DNA damage response with knockout mice showing increased DNA strand breaks and Fe65 demonstrating a gel mobility shift after DNA damage, consistent with protein phosphorylation. In the present study, we identified Fe65 Ser(228) as a novel target of the ATM (ataxia telangiectasia mutated) and ATR (ataxia-telangiectasia- and Rad3-related protein) protein kinases, in a reaction that occurred independently of APP. Neither phosphorylation nor mutation of Ser(228) affected the Fe65-APP complex, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Finally, mutation of Ser(228) to alanine (thus blocking phosphorylation) caused a significant increase in Fe65-APP transcriptional activity, whereas phosphomimetic mutants (S(228)D and S(228)E) showed decreased transcriptional activity. These studies identify a novel phosphorylation site within Fe65 and a novel regulatory mechanism for the transcriptional activity of the Fe65-APP complex.

DNA damage, signalling and repair after exposure of cells to the sulphur mustard analogue 2-chloroethyl ethyl sulphide.

Sulphur mustard (SM) is a blistering agent that is directly toxic to the skin and mucosal surfaces of the eye and respiratory system. Symptoms take several hours to develop and the mechanism of action is poorly understood although SM is able to alkylate nucleic acids and proteins. The ability of SM to form adducts with DNA has been documented, although there are limited data demonstrating how cells respond to this insult to repair the damage. This study used the sulphur mustard surrogate 2-chloroethyl ethyl sulphide (CEES) to identify DNA damage repair pathways and signalling events that are activated after exposure to the agent. A dose-dependent increase in DNA damage was observed in TK6 lymphoblastoid cells, which was associated with a loss of cell viability. Using both model human lymphoblastoid cell lines and pharmacological inhibitors, it was found that DNA damage induced by CEES was repaired by base excision repair (BER) and nucleotide excision repair (NER) pathways. Finally, CEES was found to induce the phosphorylation of p53 and Chk2 and these events were mediated by both the ATM ataxia telangiectasia mutated and ATR (ATM and Rad-3 related) protein kinases.

Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage.

Human (h)PTIP plays important but poorly understood roles in cellular responses to DNA damage. hPTIP interacts with 53BP1 tumour suppressor but only when 53BP1 is phosphorylated by ATM after DNA damage although the mechanism(s) and significance of the interaction of these two proteins are unclear. Here, we pinpoint a single ATM-phosphorylated residue in 53BP1--Ser25--that is required for binding of 53BP1 to hPTIP. Binding of phospho-Ser25 to hPTIP in vitro and in vivo requires two closely apposed pairs of BRCT domains at the C-terminus of hPTIP and neither pair alone can bind to phospho-Ser25, even though one of these BRCT pairs in isolation can bind to other ATM-phosphorylated epitopes. Mutations in 53BP1 and in hPTIP that prevent the interaction of the two proteins, render cells hypersensitive to DNA damage and weaken ATM signalling. The C-terminal BRCT domains of hPTIP are also required for stable retention of hPTIP at sites of DNA damage but this appears to be independent of binding to 53BP1. Thus, the BRCT domains of hPTIP play important roles in the cellular response to DNA damage.

M2I-1 disrupts the in vivo interaction between CDC20 and MAD2 and increases the sensitivities of cancer cell lines to anti-mitotic drugs via MCL-1s.

BACKGROUND: Drugs such as taxanes, epothilones, and vinca alkaloids are widely used in the treatment of breast, ovarian, and lung cancers but come with major side effects such as neuropathy and loss of neutrophils and as single agents have a lack of efficacy. M2I-1 (MAD2 inhibitor-1) has been shown to disrupt the CDC20-MAD2 interaction, and consequently, the assembly of the mitotic checkpoint complex (MCC). RESULTS: We report here that M2I-1 can significantly increase the sensitivity of several cancer cell lines to anti-mitotic drugs, with cell death occurring after a prolonged mitotic arrest. In the presence of nocodazole or taxol combined with M2I-1 cell death is triggered by the premature degradation of Cyclin B1, the perturbation of the microtubule network, and an increase in the level of the pro-apoptotic protein MCL-1s combined with a marginal increase in the level of NOXA. The elevated level of MCL-1s and the marginally increased NOXA antagonized the increased level of MCL-1, a pro-survival protein of the Bcl-2 family. CONCLUSION: Our results provide some important molecular mechanisms for understanding the relationship between the mitotic checkpoint and programmed cell death and demonstrate that M2I-1 exhibits antitumor activity in the presence of current anti-mitotic drugs such as taxol and nocodazole and has the potential to be developed as an anticancer agent.

Environmental Xenoestrogens Super-Activate a Variant Murine ER Beta in Cholangiocytes.

High systemic levels of oestrogens are cholestatic and primary biliary cholangitis (PBC)-which is characterized by hepatic ductular inflammation-is thought to be triggered by exposure to xenobiotics such as those around landfill sites. Xenoestrogens may be a component of this chemical trigger. We therefore hypothesized that xenoestrogens are present at higher levels in the proximity of landfill sites. To test this hypothesis, soil samples were collected, extracts prepared and biological oestrogenic activity examined using cell-based reporter gene assays. Extracts from several sample sites around a landfill site contained a chemical(s) which activated the human ERα in a dose-dependent manner. Extracts from 3 separate control sampling sites were absent of any detectable activity. The mouse ERα and 2 variant mouse ERß cDNAs were cloned and extracts from sample sites around a landfill site also activated these receptors. One variant murine ERß was constitutively active when expressed in cholangiocytes, was readily inactivated by ICI182780 and activated in a dose-responsive, ICI182780-inhibitable manner by oestrogen. However, when this receptor was activated by extracts from landfill site soils, ICI182780 failed to antagonize activation. ERß was readily detectable in murine cholangiocytes and exposing mice acutely to a pooled ER activating soil extracts also gave rise to a mild cholestatic injury. These data indicate that the environment around landfill sites may contain higher levels of xenoestrogens; that these chemicals have "super-activating" characteristics with a variant ERß and therefore these chemicals could be a component of a xenobiotic insult that triggers PBC.

Hepatic effects of tartrazine (E 102) after systemic exposure are independent of oestrogen receptor interactions in the mouse.

Tartrazine is a food colour that activates the transcriptional function of the human oestrogen receptor alpha in an in vitro cell model. Since oestrogens are cholestatic, we hypothesised tartrazine will cause periportal injury to the liver in vivo. To test this hypothesis, tartrazine was initially administered systemically to mice resulting in a periportal recruitment of inflammatory cells, increased serum alkaline phosphatase activity and mild periportal fibrosis. To determine whether an oestrogenic effect may be a key event in this response, tartrazine, sulphonated metabolites and a food additive contaminant were screened for their ability to interact with murine oestrogen receptors. In all cases, there were no interactions as agonists or antagonists and further, no oestrogenicity was observed with tartrazine in an in vivo uterine growth assay. To examine the relevance of the hepatic effects of tartrazine to its use as a food additive, tartrazine was orally administered to transgenic NF-κB-Luc mice. Pre- and concurrent oral treatment with alcohol was incorporated given its potential to promote gut permeability and hepatic inflammation. Tartrazine alone induced NF- κB activities in the colon and liver but there was no periportal recruitment of inflammatory cells or fibrosis. Tartrazine, its sulphonated metabolites and the contaminant inhibited sulphotransferase activities in murine hepatic S9 extracts. Given the role of sulfotransferases in bile acid excretion, the initiating event giving rise to periportal inflammation and subsequent hepatic pathology through systemic tartrazine exposure is therefore potentially associated an inhibition of bile acid sulphation and excretion and not on oestrogen receptor-mediated transcriptional function. However, these effects were restricted to systemic exposures to tartrazine and did not occur to any significant effect after oral exposure.

Fe65 Is Phosphorylated on Ser289 after UV-Induced DNA Damage.

Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser228 as a specific site of phosphorylation, targeted by the ATM and ATR protein kinases, with phosphorylation inhibiting the Fe65-dependent transcriptional activity of the amyloid precursor protein (APP). The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD) and cytotoxic amyloid ß (Aß) peptides. Given that the accumulation of Aß peptides in neural plaques is a pathological feature of Alzheimer's disease (AD), it is essential to understand the mechanisms controlling Aß production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aß peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser289 as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser289 affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser289 phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser228 site.

Checkpoint kinase 1 is activated and promotes cell survival after exposure to sulphur mustard.

Sulphur mustard (SM) is a vesicating agent that has been used several times as a weapon during military conflict and continues to pose a threat as an agent of warfare/terrorism. After exposure, SM exerts both acute and delayed long-term toxic effects principally to the skin, eyes and respiratory system. These effects are thought to be mediated, at least in part, by direct interaction of SM with DNA, forming a myriad of DNA lesions and initiating effects on cell cycle and cell death pathways. Previous studies have demonstrated that a complex network of cellular DNA damage response pathways are utilised in cells exposed to SM, consistent with SM causing multiple forms of DNA damage. The present study focused on the role of Checkpoint kinase 1 (CHK1), a protein with putative roles in homologous recombination repair, p53 activation and the initiation of cell cycle checkpoints after certain forms of DNA damage. The data showed that SM caused robust activation of CHK1, monitored by multi-site phosphorylation analysis and that this activation was dependent on the ataxia telangiectasia and Rad3-related (ATR) protein kinase. Furthermore, specific inhibition of CHK1 increased SM toxicity in multiple human cell lines, with concomitant increases in markers of apoptosis, DNA damage and mitosis. Finally, the effect of CHK1 inhibition on SM toxicity was much more marked in cells with non-functional p53.

Whole genome expression analysis in primary bronchial epithelial cells after exposure to sulphur mustard.

Sulphur mustard (SM) is a highly toxic chemical agent and poses a current threat to both civilians and military personnel in the event of a deliberate malicious release. Acute SM toxicity develops over the course of several hours and mainly affects the skin and mucosal surfaces of the eyes and respiratory system. In cases of acute severe exposure, significant lung injury can result in respiratory failure and death. Systemic levels of SM can also be fatal, frequently due to immunodepletion and the subsequent development of secondary infections. Whilst the physical effects associated with SM exposure are well documented, the molecular mechanisms mediating these changes are poorly understood, hindering the development of an effective therapeutic strategy. To gain a better understanding of the mechanism of SM toxicity, this study investigated whole genome transcriptional changes after SM in primary human bronchial epithelial cells, as a model for inhalation exposure. The analysis revealed >400 transcriptional changes associated with SM exposure. Pathways analysis confirmed the findings of previous studies suggesting that DNA damage, cell cycle arrest, cell death and inflammation were important components of SM toxicity. In addition, several other interesting observations were made, suggesting that protein oxidation as well as effects on the mitotic apparatus may contribute to SM toxicity.

DNA damage responses in cells exposed to sulphur mustard.

Sulphur mustard (SM) is a blistering agent that causes debilitating damage to the skin, eyes and respiratory system. In cases of severe exposure, immunodepletion can occur as well as death, due to secondary infections. The toxicity of SM is thought to be mediated in part by the alkylation of nucleic acids and proteins, although the exact mechanisms are not clear. In addition, although the first known use of SM was in military conflict nearly 100 years ago, there are still no effective treatments or preventative measures. In order to develop treatments it is necessary to have a detailed understanding of the cellular biochemical changes induced by SM as well as information on the mechanisms that cells employ to protect against SM toxicity. We have previously demonstrated that the homologous recombination (HR) DNA repair pathway promotes cell survival after SM. This study investigated the role of other DNA repair pathways in the cellular response to SM, specifically base excision repair (BER), nucleotide excision repair (NER) and non-homologous end joining (NHEJ) as well as studying the activation and regulation of DNA damage signalling pathways. Our data confirmed that HR is the major repair pathway protecting against acute SM toxicity, with NER and NHEJ also contributing to cell survival. In addition, this study demonstrated the dose- and time-dependent activation of DNA damage signalling pathways after SM in human TK6 lymphoblastoid cells, in particular the phosphorylation of CHK1, CHK2 and p53. These phosphorylation events were orchestrated by a combination of the ATM and ATR protein kinases.

The role of homologous recombination in the cellular response to sulphur mustard.

Sulphur mustard (SM) is a blistering agent that has been used several times as a weapon during military conflict. Interest in this compound persists due to its ease of production and potential threat as an agent of warfare/terrorism. In addition, there are increasing reports of long-term health effects in individuals previously exposed to this compound, including an increased incidence of certain cancers. It is therefore important to elucidate the toxic mechanisms of SM and how the cell responds to any damage produced. This will allow for better healthcare planning in the event of an exposure and aid in the development of a therapeutic strategy, which is currently lacking. SM is a bifunctional alkylating agent, producing both DNA monoadducts and crosslinks, although the cellular response to these lesions is not well understood. This study aimed to investigate the DNA repair pathways employed by cells exposed to SM. It was found that DNA double strand breaks were generated after SM exposure and cells lacking the homologous recombination DNA repair pathway were more sensitive to the toxicity of SM than wild type cells. Finally, we demonstrate that chemical activation of the HR protein RAD51 offers cellular protection against SM toxicity and thus could be a novel target for therapeutic intervention.

Human PTIP facilitates ATM-mediated activation of p53 and promotes cellular resistance to ionizing radiation.

Mus musculus Pax2 transactivation domain-interacting protein (Ptip) is an essential gene required for the maintenance of genome stability, although its precise molecular role is unclear. Human PTIP (hPTIP) was recently isolated in a screen for proteins, translated from cDNA pools, capable of interacting with peptides phosphorylated by the ATM (ataxia telangiectasia-mutated)/ATR (ataxia telangiectasia-related) protein kinases. hPTIP was described as a 757-amino acid protein bearing four BRCT domains. Here we report that instead full-length endogenous hPTIP contains 1069 amino acids and six BRCT domains. hPTIP shows increased association with 53BP1 in response to ionizing radiation (IR) but not in response to other DNA-damaging agents. Whereas translocation of both 53BP1 and hPTIP to sites of IR-induced DNA damage occurs independently of ATM, IR-induced association of PTIP and 53BP1 requires ATM. Deletion analysis identified the domains of 53BP1 and hPTIP required for protein-protein interaction and focus formation. Data characterizing the cellular roles of hPTIP are also presented. Small interfering RNA was used to show that hPTIP is required for ATM-mediated phosphorylation of p53 at Ser(15) and for IR-induced up-regulation of the cyclin-dependent kinase inhibitor p21. Lowering hPTIP levels also increased cellular sensitivity to IR, suggesting that this protein plays a critical role in maintaining genome stability.