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1.
Virology ; 448: 33-42, 2014 Jan 05.
Article in English | MEDLINE | ID: mdl-24314634

ABSTRACT

In order to establish a successful infection, it is of crucial importance for invading viruses to alter the activities of the regulatory protein p53. Beta-herpesviruses stabilize p53 and likely direct its activities towards generation of a replication-friendly environment. We here study the mechanisms behind HHV-6B-induced stabilization and inactivation of p53. Stable transgene expression of the HHV-6B protein U19 was sufficient to achieve upregulation of p53. U19 bound directly to the p53-regulating protein HDM2 in vitro, co-precipitated together with HDM2 in lysates, and co-localized with HDM2 in the nucleus when overexpressed. U19 contained a sequence with a putative p53 BOX I-motif for HDM2 binding. Mutation of the two key amino acids within this motif was sufficient to inhibit all the described U19 functions. Our study provides further insight into p53-modulating strategies used by herpesviruses and elucidates a mechanism used by HHV-6B to circumvent the antiviral response.


Subject(s)
Herpesvirus 6, Human/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Roseolovirus Infections/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Tumor Suppressor Protein p53/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Motifs , Cell Line , Herpesvirus 6, Human/chemistry , Herpesvirus 6, Human/genetics , Humans , Protein Binding , Protein Stability , Protein Structure, Tertiary , Proto-Oncogene Proteins c-mdm2/genetics , Roseolovirus Infections/genetics , Roseolovirus Infections/virology , Trans-Activators/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Viral Proteins/genetics
2.
Exp Mol Pathol ; 94(1): 228-38, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22981919

ABSTRACT

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.


Subject(s)
Cell Transformation, Neoplastic , Proto-Oncogene Proteins c-mdm2/metabolism , T-Lymphocytes/parasitology , Theileria parva/pathogenicity , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Animals , Apoptosis , Cattle , Cell Line , Cisplatin/pharmacology , DNA Damage/drug effects , Enzyme Activation , Lymphocyte Activation , Molecular Sequence Data , NF-kappa B/metabolism , Naphthoquinones/pharmacology , Protein Isoforms , Proto-Oncogene Proteins c-mdm2/genetics , Signal Transduction , T-Lymphocytes/pathology , Theileria parva/immunology , bcl-2-Associated X Protein/biosynthesis
3.
Biochem J ; 450(3): 523-36, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23252402

ABSTRACT

The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.


Subject(s)
Proto-Oncogene Proteins c-mdm2/physiology , Receptor, Notch1/metabolism , Ubiquitination/physiology , Amino Acid Sequence , Catalytic Domain/genetics , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Biological , Neoplasms/genetics , Neoplasms/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , Proteolysis , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Receptor, Notch1/agonists , Receptor, Notch1/chemistry , Receptor, Notch1/genetics , Signal Transduction/genetics , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/physiology , Ubiquitination/genetics
4.
J Biol Chem ; 287(17): 14052-68, 2012 Apr 20.
Article in English | MEDLINE | ID: mdl-22337874

ABSTRACT

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.


Subject(s)
Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Proto-Oncogene Proteins c-mdm2/chemistry , Ubiquitin/chemistry , Apoptosis , Binding Sites , Cell Line, Tumor , Cell Lineage , Cell Proliferation , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Humans , Ligands , Protein Binding , Protein Structure, Tertiary , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitination
5.
Biochem J ; 418(3): 575-85, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19032150

ABSTRACT

Mdm2 (murine double minute 2)-mediated ubiquitination of the p53 tumour suppressor requires interaction of the ligase at two distinct binding sites that form general multiprotein-docking sites for the p53 protein. The first Mdm2-binding site resides in the transactivation domain of p53 and is an allosteric effector site for Mdm2-mediated p53 ubiquitination; the second site requires the acid domain of Mdm2 to recognize a 'ubiquitination signal' within p53's DNA-binding core. In order to expand on fundamental requirements for a protein to function as an Mdm2 substrate and the role of the acid domain in recognition, we have carried out a bioinformatics search for open reading frames that have homology with the Mdm2-docking sites in p53. IRF-2 [IFN (interferon) regulatory factor-2], an IFN-regulated transcription factor, has been identified as an Mdm2-binding protein and substrate requiring interactions with both the hydrophobic pocket and the acid domain of Mdm2. Mutation of either of the two Mdm2-binding sites on IRF-2 can attenuate substrate ubiquitination, confirming the requirement of a dual-site substrate interaction mechanism. Ligands that bind to the hydrophobic pocket are not sufficient to inhibit Mdm2 E3-ligase activity. Rather, acid domain-binding ligands act as E3-ligase inhibitors, lending additional support to the idea that the acid domain of Mdm2 is key to understanding its mechanism of action. The ability of Mdm2 and IRF-2 to form a complex in cells complements the biochemical assays and together establishes a novel substrate with which to develop insights into E3-ubiquitin ligase-substrate interactions in vitro and in cells.


Subject(s)
Interferon Regulatory Factor-2/metabolism , Proto-Oncogene Proteins c-mdm2/physiology , Ubiquitination/physiology , Animals , Computational Biology , Interferon Regulatory Factor-2/genetics , Mice , Protein Structure, Tertiary , Proto-Oncogene Proteins c-mdm2/chemistry , Ubiquitin-Protein Ligases/antagonists & inhibitors
6.
FEBS J ; 275(19): 4875-86, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18754770

ABSTRACT

Murine double minute 2 (MDM2) protein exhibits many diverse biochemical functions on the tumour suppressor protein p53, including transcriptional suppression and E3 ubiquitin ligase activity. However, more recent data have shown that MDM2 can exhibit ATP-dependent molecular chaperone activity and directly mediate folding of the p53 tetramer. Analysing the ATP-dependent function of MDM2 will provide novel insights into the evolution and function of the protein. We have established a system to analyse the molecular chaperone function of MDM2 on another of its target proteins, the transcription factor E2F1. In the absence of ATP, MDM2 was able to catalyse inhibition of the DNA-binding function of E2F1. However, the inhibition of E2F1 by MDM2 was stimulated by ATP, and mutation of the ATP-binding domain of MDM2 (K454A) prevented the ATP-stimulated inhibition of E2F1. Further, ATP stabilized the binding of E2F1 to MDM2 using conditions under which ATP destabilized the MDM2:p53 complex. However, the ATP-binding mutant of MDM2 was as active as an E3 ubiquitin ligase on E2F1 and p53, highlighting a specific function for the ATP-binding domain of MDM2 in altering substrate protein folding. Antibodies to three distinct domains of MDM2 neutralized its activity, showing that inhibition of E2F1 is MDM2-dependent and that multiple domains of MDM2 are involved in E2F1 inhibition. Dimethylsulfoxide, which reduces protein unfolding, also prevented E2F1 inhibition by MDM2. These data support a role for the ATP-binding domain in altering the protein-protein interaction function of MDM2.


Subject(s)
Adenosine Triphosphate/metabolism , E2F1 Transcription Factor/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Animals , DNA-Binding Proteins/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Humans , Mice , Molecular Chaperones/metabolism , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/immunology , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
7.
J Biol Chem ; 282(45): 32603-12, 2007 Nov 09.
Article in English | MEDLINE | ID: mdl-17848574

ABSTRACT

The murine double minute (mdm2) gene encodes an E3 ubiquitin ligase that plays a key role in the degradation of p53 tumor suppressor protein. Nevertheless recent data highlight other p53-independent functions of MDM2. Given that MDM2 protein binds ATP, can interact with the Hsp90 chaperone, plays a role in the modulation of transcription factors and protection and activation of DNA polymerases, and is involved in ribosome assembly and nascent p53 protein biosynthesis, we have evaluated and found MDM2 protein to possess an intrinsic molecular chaperone activity. MDM2 can substitute for the Hsp90 molecular chaperone in promoting binding of p53 to the p21-derived promoter sequence. This reaction is driven by recycling of MDM2 from the p53 complex, triggered by binding of ATP to MDM2. The ATP binding mutant MDM2 protein (K454A) lacks the chaperone activity both in vivo and in vitro. Mdm2 cotransfected in the H1299 cell line with wild-type p53 stimulates efficient p53 folding in vivo but at the same time accelerates the degradation of p53. MDM2 in which one of the Zn(2+) coordinating residues is mutated (C478S or C464A) blocks degradation but enhances folding of p53. This is the first demonstration that MDM2 possesses an intrinsic molecular chaperone activity, indicating that the ATP binding function of MDM2 can mediate its chaperone function toward the p53 tumor suppressor.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Adenosine Triphosphate/metabolism , Cell Line, Tumor , Humans , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Protein Folding , Proto-Oncogene Proteins c-mdm2/genetics , Tumor Suppressor Protein p53/genetics
8.
Mol Cell ; 23(2): 251-63, 2006 Jul 21.
Article in English | MEDLINE | ID: mdl-16857591

ABSTRACT

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.


Subject(s)
Proto-Oncogene Proteins c-mdm2/metabolism , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Cells, Cultured , Ligands , Models, Biological , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Retinoblastoma Protein/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/chemistry
9.
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
10.
J Mol Biol ; 357(2): 442-56, 2006 Mar 24.
Article in English | MEDLINE | ID: mdl-16438982

ABSTRACT

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.


Subject(s)
DNA/metabolism , Protein Conformation , Protein Isoforms/chemistry , Tumor Suppressor Protein p53/chemistry , Acetylation , Animals , Antibodies, Monoclonal/metabolism , Base Sequence , Biological Assay , Epitopes , Humans , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Denaturation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
11.
Mol Cell Biol ; 24(22): 10083-98, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15509808

ABSTRACT

Interferon regulatory factor 1 (IRF-1) and p53 control distinct sets of downstream genes; however, these two antioncogenic transcription factors converge to regulate p21 gene expression and to inhibit tumor formation. Here we investigate the mechanism by which IRF-1 and p53 synergize at the p21 promoter and show that stimulation of p21 transcription by IRF-1 does not require its DNA-binding activity but relies on the ability of IRF-1 to bind the coactivator p300 and to stimulate p53-dependent transcription by an allosteric mechanism. Deletion of the p300-binding sites in IRF-1 eliminates the ability of IRF-1 to stimulate p53 acetylation and associated p53 activity. Complementing this, small peptides derived from the IRF-1-p300 interface can bind to p300, stabilize the binding of p300 to DNA-bound p53, stimulate p53 acetylation in trans, and up-regulate p53-dependent activity from the p21 promoter. The nonacetylatable p53 mutant (p53-6KR) cannot be stimulated by IRF-1, further suggesting that p53 acetylation is the mechanism whereby IRF-1 modifies p53 activity. These data expand the core p300-p53 protein LXXLL and PXXP interface by including an IRF-1-p300 interface as an allosteric modifier of DNA-dependent acetylation of p53 at the p21 promoter.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor Protein p53/metabolism , Acetylation , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , DNA/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , E1A-Associated p300 Protein , Humans , Interferon Regulatory Factor-1 , Mice , Models, Biological , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Promoter Regions, Genetic , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trans-Activators/genetics , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics
12.
Mol Cell Biol ; 24(12): 5606-19, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15169919

ABSTRACT

Phosphorylation of target proteins by cyclin D1-Cdk4 requires both substrate docking and kinase activity. In addition to the ability of cyclin D1-Cdk4 to catalyze the phosphorylation of consensus sites within the primary amino acid sequence of a substrate, maximum catalytic activity requires the enzyme complex to anchor at a site remote from the phospho-acceptor site. A novel Cdk4 docking motif has been defined within a stretch of 19 amino acids from the C-terminal domain of the Rb protein that are essential for Cdk4 binding. Mutation or deletion of the docking motif prevents Cdk4-dependent phosphorylation of full-length Rb protein or C-terminal Rb fragments in vitro and in cells, while a peptide encompassing the Cdk4 docking motif specifically inhibits Cdk4-dependent phosphorylation of Rb. Cyclin D1-Cdk4 can overcome the growth-suppressive activity of Rb in both cell cycle progression and colony formation assays; however, while mutants of Rb in which the Cdk4 docking site has been either deleted or mutated retain growth suppressor activity, they are resistant to inactivation by cyclin D1-Cdk4. Finally, binding of Cdk4 to its docking site can inhibit cleavage of exogenous and endogenous Rb in response to distinct apoptotic signals. The Cdk4 docking motif in Rb gives insight into the mechanism by which enzyme specificity is ensured and highlights a role for Cdk4 docking in maintaining the Rb protein in a form that favors cell survival rather than apoptosis.


Subject(s)
Cyclin-Dependent Kinases/metabolism , Proto-Oncogene Proteins , Retinoblastoma Protein/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites/genetics , Caspase 3 , Caspases/metabolism , Cell Division , Cell Line, Tumor , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases/genetics , Humans , In Vitro Techniques , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinoblastoma Protein/genetics , Substrate Specificity
13.
J Biol Chem ; 277(32): 28446-58, 2002 Aug 09.
Article in English | MEDLINE | ID: mdl-11925449

ABSTRACT

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.


Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/chemistry , Tumor Suppressor Protein p53/chemistry , Ubiquitin/metabolism , Alleles , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Dose-Response Relationship, Drug , Genes, p53 , Humans , Immunohistochemistry , Leucine/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Mutation , Peptide Library , Precipitin Tests , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Sequence Homology, Amino Acid , Serine/chemistry , Transcriptional Activation , Transfection , Tumor Cells, Cultured
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