DeltaNp63 transcriptionally regulates ATM to control p53 Serine-15 phosphorylation.

BACKGROUND: DeltaNp63alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to DeltaNp63alpha-positive cells in the basal layer of human epithelium. RESULTS: We now report that phosphorylation of the p53 tumour suppressor is positively regulated by DeltaNp63alpha in immortalised human keratinocytes. DeltaNp63alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of DeltaNp63alpha in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct DeltaNp63alpha transcriptional target and that the DeltaNp63alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the DeltaNp63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. CONCLUSIONS: Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The DeltaNp63alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes.

The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members.

Genetic and biochemical studies have shown that Ser(20) phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser(20) phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser(20) kinases. Phosphorylation of a p53 transactivation domain fragment at Ser(20) by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser(20) kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser(20), (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.

The regulation of CHK2 in human cancer.

Exceptional progress has been made in the past two decades in mapping oncogenes and tumour suppressors, defining a function for these master switches, and identifying novel anti-cancer drug targets. The p53 tumour suppressor is a central component of a DNA-damage-inducible pathway controlled by the ataxia telangiectasia mutated (ATM) and CHK2 protein kinases that have a central role in cancer suppression. One limitation of current human cancer research is the difficulty in developing genetic models that reveal the post-translational regulation of a growth suppressor like CHK2 within the microenvironment of a human tumour. Gaining such insights is important since yeast models and human tissue culture cell lines do not necessarily predict how enzymes like CHK2 are regulated in vivo, and therefore what factors can affect CHK2 tumour suppressor function. Translational cancer research aims to link basic research methodologies and clinical biology by uncovering cancer-specific pathways not revealed by other approaches. This approach is exemplified by two studies in this edition of Oncogene: both use a set of well-characterized human cancers with the objective of identifying novel post-translational control of the tumour suppressor CHK2. The authors have revealed two unexpected epigenetic modifications of the CHK2 pathway in vivo: (1) constitutive phosphorylation of CHK2 at its ATM-activated site in the absence of exogenous DNA damage; and (2) the production of hyper-spliced and inactive isoforms of CHK2. These studies highlight the need to develop model systems to understand why CHK2-activating pathways are being triggered or suppressed in different human cancers and whether the splicing machinery can be manipulated to control the activity of CHK2 for therapeutic benefit.

Signaling to p53: the use of phospho-specific antibodies to probe for in vivo kinase activation.

Phospho-specific antibody technology has been recently adopted to study p53 phosphorylation both in vivo and in vitro. We have developed and carefully characterized p53 phosphospecific reagents directed to major amino- and carboxy-terminal regulatory sites. The specificities of both polyclonal and monoclonal reagents targeting the same phospho-epitope are discussed. We have defined the major chemical binding determinants for specific monoclonal reagents by determining the relative contribution of charge and sequence to epitope recognition. Remarkably, we have found that the utility of these reagents in different assay systems is not universal and depends both on epitope conformation and affinity. This is reflected in the striking differences in their ability to detect endogenous p53 and recombinant protein. Therefore, we conclude that this novel class of reagents is not generally applicable, but that the utility of each reagent must be determined empirically.

Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line.

In response to genotoxic insults, cells activate DNA damage response pathways that either stimulate transient cell cycle arrest and DNA repair or induce apoptosis. The Caenorhabditis elegans germ line is now well established as a model system to study these processes in a genetically tractable, multicellular organism. Upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late-pachytene stage undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to apoptosis of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptotic machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we briefly review the C. elegans DNA damage response field, discuss in detail methods currently used to assay DNA damage responses in C. elegans, and describe the development of new experimental tools that will facilitate a more comprehensive understanding of the DNA damage response.

A novel ataxia-telangiectasia mutated autoregulatory feedback mechanism in murine embryonic stem cells.

Ataxia-telangiectasia mutated (ATM) is known to play a central role in effecting the DNA damage response that protects somatic cells from potentially harmful mutations, and in this role it is a key anti-cancer agent. However, it also promotes repair of therapeutic damage (e.g. radiotherapy) and so frustrates the efficacy of some treatments. A better understanding of the mechanisms of ATM regulation is therefore important both in prevention and treatment of disease. While progress has been made in elucidating the key signal transduction pathways that mediate damage response in somatic cells, relatively little is known about whether these function similarly in pluripotent embryonic stem (ES) cells where ATM is also implicated in our understanding of adult stem cell ageing and in improvements in regenerative medicine. There is some evidence that different mechanisms may operate in ES cells and that our understanding of the mechanisms of ATM regulation is therefore incomplete. We investigated the behaviour of the damage response signalling pathway in mouse ES cells. We subjected the cells to the DNA-damaging agent doxorubicin, a drug that induces double-strand breaks, and measured ATM expression levels. We found that basal ATM gene expression was unaffected by doxorubicin treatment. However, following ATM kinase inhibition using a specific ATM inhibitor, we observed a significant increase in ATM and ataxia-telangiectasia and Rad3 related transcription. We demonstrate the use of a dynamical modelling approach to show that these results cannot be explained in terms of known mechanisms. Furthermore, we show that the modelling approach can be used to identify a novel feedback process that may underlie the anomalies in the data. The predictions of the model are consistent both with our in vitro experiments and with in vivo studies of ATM expression in somatic cells in mice, and we hypothesize that this feedback operates in both somatic and ES cells in vivo. The results point to a possible new target for ATM inhibition that overcomes the restorative potential of the proposed feedback.

The Barrett's antigen anterior gradient-2 silences the p53 transcriptional response to DNA damage.

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.