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1.
Mol Cancer ; 9: 195, 2010 Jul 21.
Article in English | MEDLINE | ID: mdl-20663147

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/physiology , DNA-Binding Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Serine/metabolism , Trans-Activators/physiology , Transcription, Genetic/physiology , Tumor Suppressor Proteins/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Line, Transformed , DNA-Binding Proteins/genetics , Humans , Phosphorylation , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/genetics , Trans-Activators/chemistry , Transcription Factors , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
2.
Genome Biol ; 9(5): 222, 2008.
Article in English | MEDLINE | ID: mdl-18495049

ABSTRACT

A recent report identifies sites in the human genome that can associate with nucleoporin 93, a subunit of the nuclear pore complex. These associations are modulated by levels of global histone acetylation and highlight the dynamic nature of chromatin organization in the nucleus.


Subject(s)
Cell Nucleus/chemistry , Chromatin/chemistry , Porins/metabolism , Histone Deacetylases/metabolism , Humans , Nuclear Pore Complex Proteins/metabolism
3.
PLoS Genet ; 4(3): e1000039, 2008 Mar 21.
Article in English | MEDLINE | ID: mdl-18369458

ABSTRACT

The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. However, a crucial issue is whether this level of nuclear organisation directly affects gene function, rather than merely reflecting it. To directly investigate whether proximity to the nuclear periphery can influence gene expression in mammalian cells, here we relocate specific human chromosomes to the nuclear periphery by tethering them to a protein of the inner nuclear membrane. We show that this can reversibly suppress the expression of some endogenous human genes located near the tethering sites, and even genes further away. However, the expression of many other genes is not detectably reduced and we show that location at the nuclear periphery is not incompatible with active transcription. The dampening of gene expression around the nuclear periphery is dependent on the activity of histone deacetylases. Our data show that the radial position within the nucleus can influence the expression of some, but not all, genes. This is compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate the expression of selected genes during development and differentiation.


Subject(s)
Cell Nucleus/genetics , Base Sequence , Binding Sites/genetics , Cell Line , Cell Nucleus/metabolism , Chromosomes, Human/genetics , DNA Primers/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Histone Deacetylases/metabolism , Humans , In Situ Hybridization, Fluorescence , Lac Operon , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Envelope/genetics , Nuclear Envelope/metabolism , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Suppression, Genetic
4.
Cell Cycle ; 6(9): 1062-71, 2007 May 02.
Article in English | MEDLINE | ID: mdl-17426453

ABSTRACT

The last twenty years of research into p53 function has revealed some fascinating discoveries into the orchestration of tumor suppressor pathways with a multitude of putative drug targets being investigated. However, it was not until 1998 that the ancestral mother of p53 was documented. The eldest evolutionary conserved homolog of the p53 family is known today as p63. Originally, it was thought p63 was another tumor suppressor that could function in a similar capacity to p53. However, elegant demonstrations of the divergent roles that p63 plays as a key transcriptional regulator of the proliferation and differentiation cascade in stratified epithelia are documented. These data link deltaNp63alpha to adult tissue stem cell regulation and possibly "cancer stem cells". p63 lacks mutation in cancer development, which is in stark contrast to the classically high mutation status of p53 in a large compendium of cancer types. Perhaps suggesting a selective preference for p53 mutation. Why is p63 rarely mutated despite being part of the same gene family? Interestingly, p63 is often over-expressed and amplified in cancer, thus revealing a paradox. Is p63 required to provide cancer cell populations with a selective advantage as much as a loss of p53 function by mutation? Has p53 been masking a "phantom" with promising features as a target for drug development? Can we exploit the biochemical know how gained from the mass of p53 research to further elucidate deltaNp63alpha gene function? In this review, we will summarise the emerging advances that are elucidating deltaNp63alpha as a promising drug target.


Subject(s)
DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Cell Cycle , Cell Proliferation , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Genes, Tumor Suppressor , Humans , Models, Biological , Molecular Sequence Data , Mutation , Neoplasms/drug therapy , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Trans-Activators/chemistry , Trans-Activators/genetics , Transcription Factors , Tumor Protein p73 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
5.
Cell Cycle ; 5(21): 2489-94, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17106255

ABSTRACT

The activity of the tumor suppressor protein p53 is controlled by a balance between E3-ligase mediated p53 protein degradation and protein kinase-mediated assembly of p53:p300 transcription machinery. Genetic studies in mice have shown that mutation of the CK2 phospho-acceptor site in p53 increases UV-induced skin cancer formation,(11) highlighting an unexpected role for p53 phosphorylation in mediating p53-dependent tumor suppression. However, it is not known in which cell types CK2-mediated phosphorylation of p53 occurs. Using human skin as a model to determine whether there is cell-selectivity in modulating p53 phosphorylation, we have found a selective induction of p53 phosphorylation at the CK2-site in the basal cells of UV irradiated human skin. Dual-immunofluorescence also revealed that Ser392 and Ser15 phosphorylation of p53 also occur in the same basal cells, although often within distinct regions of the nucleus. Given that p63alphaDeltaN is required for p53 activation after DNA damage, we examined and found a high proportion of cells co-express p63alphaDeltaN and CK2-phosphorylated p53 after UV-irradiation. As controls, the proliferation marker Ki67 and p63alphaDeltaN generally exhibit mutually exclusive expression. These data identify a physiological model with which to identify signaling pathways that mediate cross-talk between p63alphaDeltaN and activating p53 kinase pathways after DNA damage in basal cell populations.


Subject(s)
Casein Kinase II/physiology , Gene Expression Regulation , Skin/metabolism , Skin/radiation effects , Stem Cells/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , DNA Damage , DNA-Binding Proteins/metabolism , Disease Progression , Humans , Mice , Phosphorylation , Signal Transduction , Stem Cells/cytology , Trans-Activators/metabolism , Transcription Factors , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ultraviolet Rays
6.
Biochem J ; 397(2): 355-67, 2006 Jul 15.
Article in English | MEDLINE | ID: mdl-16579792

ABSTRACT

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53(F270A)) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53(R175H) or p53(F270A) also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53(R175H) or p53(F270A) unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53(F270A:6KR) chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53(F270A) and the p53(F270A:6KR) chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.


Subject(s)
Mutation, Missense , Tumor Suppressor Protein p53/genetics , Ubiquitin/metabolism , Amino Acid Motifs , Cell Line, Tumor , Cell-Free System , Humans , In Vitro Techniques , Models, Molecular , Mutation , Protein Conformation , Protein Structure, Secondary , Proto-Oncogene Proteins c-mdm2/metabolism , Transfection
7.
J Biol Chem ; 279(47): 49395-405, 2004 Nov 19.
Article in English | MEDLINE | ID: mdl-15337767

ABSTRACT

Two high affinity Ser-20-phospho-LXXLL p53-binding domains of p300 map to the C-terminal interferon-binding domain (IBiD) and N-terminal IBiD homology domain (IHD) regions. Purified fractions of a recombinant IHD miniprotein are active in a set of in vitro assays highlighting its affinity to the N-terminal LXXLL domain of p53 including (i) dose-dependent binding to Ser-20-phosphorylated p53 tetramers; (ii) DNA-stimulated binding to p53 tetramers; and (iii) inhibition of MDM2-mediated p53 ubiquitination. The active component of the IHD miniprotein was localized to a 75-amino-acid fragment corresponding to amino acids 401-475 on human p300. This minimal IHD miniprotein can function in vivo as a p53-binding polypeptide in assays including: (i) complex formation with VP16-LXXLL peptide motifs in the two-hybrid assay; (ii) action as a dominant negative inhibitor of p53 from p21 luciferase templates; and (iii) attenuation of endogenous p21 protein levels. Further, we show here that the IRF-1-dependent stabilization and reactivation of p53DeltaPRO protein (LXXLL+/PXXP-) can be neutralized by the minimal IHD miniprotein, suggesting that IHD can bind to the p53 LXXLL domain in vivo. Phage-peptide display to the IHD miniprotein gave rise to an LSQXTFSXLXXLL consensus binding site that displays significant homology to the LXXLL transactivation domain of p53. These data validate the IHD scaffold as an independent LXXLL peptide-binding domain within the p300 protein, complementing the known peptide-binding domains including IBiD, C/H1, and C/H3.


Subject(s)
Nuclear Proteins/chemistry , Peptides/chemistry , Trans-Activators/chemistry , Transcriptional Activation , Tumor Suppressor Protein p53/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Gene Deletion , Genes, Reporter , Humans , Immunohistochemistry , Insecta , Luciferases/metabolism , Molecular Sequence Data , Peptide Library , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Serine/chemistry , Two-Hybrid System Techniques , Ubiquitin/chemistry
8.
Cell Cycle ; 3(1): 80-9, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14657672

ABSTRACT

Cyclin-dependent protein kinases play important roles in cell cycle progression and are attractive targets for the design of anti-proliferative drugs. Two distinct synthetic CDK1/2 inhibitors, Roscovitine and NU2058, are pharmacologically distinct in their ability to modify p53-dependent transcription and perturb cell cycle progression. Although such active-site CDK1/2 inhibitors comprise the most standard type of enzyme inhibitor, many protein kinases are proving to harbour high affinity docking sites that may provide a potentially novel interface for the design of kinase-inhibitors. We examined whether CDK2 has a docking site for its oligomeric substrate p53, whether small-peptide leads can be developed that inhibit CDK2 function, and whether such peptide-inhibitors are pharmacologically distinct from Roscovitine or NU2058. A docking site for CDK2 was identified in the tetramerization domain of p53 at a site that is distinct from the phospho-acceptor site. Peptides derived from the tetramerization domain of p53 block CDK2 phosphorylation and identification of critical CDK2 contacts in the tetramerization domain of p53 suggest that kinase docking does not require tetramerization of the substrate. Transient transfection assays were developed to show that the GFP-CDK2 docking site fusion protein (GFP-CIP) attenuates p53 activity in vivo and suppresses p21WAF1 induction which is similar to NU2058 but distinct from Roscovitine. A stable cell line with an inducible GFP-CIP gene attenuates p53 activity and induces significant cell death in a drug-resistant melanoma cell line, sensitizes cells to death induced by Doxorubicin, and suppresses cell growth in a colony formation assay. These data indicate that CDK2, in addition to cyclin A, can have a high affinity docking site for a substrate and highlights the possibility that CDK2 docking sites may represent effective targets for inhibitor design.


Subject(s)
CDC2-CDC28 Kinases/metabolism , Cell Death/physiology , Guanine/analogs & derivatives , Peptide Fragments/metabolism , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/pharmacology , Binding Sites/physiology , CDC2-CDC28 Kinases/antagonists & inhibitors , Cyclin A/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinases , Cyclins/metabolism , Enzyme Inhibitors/pharmacology , Guanine/pharmacology , Humans , Mutation , Peptide Fragments/pharmacology , Phosphorylation , Protein Binding , Purines/pharmacology , Roscovitine , Tumor Cells, Cultured , Tumor Suppressor Protein p53/antagonists & inhibitors
9.
Methods Mol Biol ; 234: 171-202, 2003.
Article in English | MEDLINE | ID: mdl-12824532

ABSTRACT

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.


Subject(s)
Antibodies, Phospho-Specific/metabolism , Phosphotransferases/metabolism , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Amino Acids/metabolism , Animals , Enzyme Activation , Enzyme-Linked Immunosorbent Assay/methods , Epitope Mapping , Epitopes , Humans , Mice , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Substrate Specificity
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