scholarly journals Mapping of the p53 and mdm-2 interaction domains.

1993 ◽  
Vol 13 (7) ◽  
pp. 4107-4114 ◽  
Author(s):  
J Chen ◽  
V Marechal ◽  
A J Levine
Keyword(s):  
P53 Protein ◽  
Cellular Protein ◽  
Deletion Mutants ◽  
Cdna Clones ◽  
Transactivation Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Interaction Domains ◽  
General Transcription Machinery

The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.

Mapping of the p53 and mdm-2 interaction domains

1993 ◽  
Vol 13 (7) ◽  
pp. 4107-4114 ◽  
Author(s):  
J Chen ◽  
V Marechal ◽  
A J Levine
Keyword(s):  
P53 Protein ◽  
Cellular Protein ◽  
Deletion Mutants ◽  
Cdna Clones ◽  
Transactivation Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Interaction Domains ◽  
General Transcription Machinery

The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.

scholarly journals Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo

2005 ◽  
Vol 79 (16) ◽  
pp. 10528-10539 ◽  
Author(s):  
Reet Kurg ◽  
Kristiina Sild ◽  
Aigi Ilves ◽  
Mari Sepp ◽  
Mart Ustav
Keyword(s):  
Cellular Protein ◽  
Micrococcal Nuclease ◽  
Viral Gene ◽  
Genome Replication ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Proliferating Cells ◽  
E2 Protein ◽  
Nuclear Structures

ABSTRACT Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.

scholarly journals Characterization of the interaction domains of Ure2p, a prion-like protein of yeast

1999 ◽  
Vol 338 (2) ◽  
pp. 403-407 ◽  
Author(s):  
Eric FERNANDEZ-BELLOT ◽  
Elisabeth GUILLEMET ◽  
Agnès BAUDIN-BAILLIEU ◽  
Sébastien GAUMER ◽  
Anton A. KOMAR ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Deletion Mutants ◽  
Loss Of Function ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interaction Domains ◽  
Two Hybrid

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

scholarly journals Deletion of 5'-coding sequences of the cellular p53 gene in mouse erythroleukemia: a novel mechanism of oncogene regulation.

1987 ◽  
Vol 7 (2) ◽  
pp. 847-853 ◽  
Author(s):  
B Rovinski ◽  
D Munroe ◽  
J Peacock ◽  
M Mowat ◽  
A Bernstein ◽  
...  
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
Protein Molecule ◽  
Amino Acid Residues ◽  
Neoplastic Progression ◽  
Exon 2 ◽  
Coding Sequences ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The p53 gene is rearranged in an erythroleukemic cell line (DP15-2) transformed by Friend retrovirus. Here, we characterize the mutation and identify a deletion of approximately equal to 3.0 kilobases that removes exon 2 coding sequences. The gene is expressed in DP15-2 cells and results in synthesis of a 44,000-dalton protein that is missing the N-terminal amino acid residues of p53. The truncated protein is unusually stable and accumulates to high levels intracellularly. Moreover, it appears to have undergone a change in conformation as revealed by epitope mapping studies. This study represents the first description of an altered p53 gene product arising by mutation during neoplastic progression and identifies a region in the p53 protein molecule that plays a role in determining p53 stability in vivo.

PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials

1994 ◽  
Vol 14 (5) ◽  
pp. 3242-3252
Author(s):  
S C Bae ◽  
E Ogawa ◽  
M Maruyama ◽  
H Oka ◽  
M Satake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Myeloid Leukemia ◽  
Chromosome Mapping ◽  
Cdna Clones ◽  
Transactivation Domain ◽  
Amino Acid Residues ◽  
Runt Domain ◽  
Chromosome Translocations ◽  
Acid Protein ◽  
Amino Acid Protein

A murine transcription factor, PEBP2, is composed of two subunits, alpha and beta. There are two genes in the mouse genome, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. Two types of the alpha B cDNA clones, alpha B1 and alpha B2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8- and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described alpha B protein referred to as alpha B1, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed alpha B2, which is identical to alpha B1 except that it has an internal deletion of 64 amino acid residues. Both alpha B1 and alpha B2 associate with PEBP2 beta and form a heterodimer. The alpha B2/beta complex binds to the PEBP2 binding site two- to threefold more strongly than the alpha B1/beta complex does. alpha B1 stimulates transcription through the PEBP2 site about 40-fold, while alpha B2 is only about 25 to 45% as active as alpha B1. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that alpha A, alpha B, and beta genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AML1 on chromosome 21q22 and PEBP2 beta/CBF beta on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that previously described chimeric gene products, AML1/MTG8(ETO) and AML1-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AML1.

Immunologically distinct p53 molecules generated by alternative splicing

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain.

1990 ◽  
Vol 10 (10) ◽  
pp. 5473-5485 ◽  
Author(s):  
P Bull ◽  
K L Morley ◽  
M F Hoekstra ◽  
T Hunter ◽  
I M Verma
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Regulatory Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
In Cis ◽  
Transcriptional Transactivator

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

scholarly journals Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 1115-1124 ◽  
Author(s):  
Margaret A. Kenna ◽  
Carrie Baker Brachmann ◽  
Scott E. Devine ◽  
Jef D. Boeke
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Reverse Transcriptase Activity ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Localization Signal

ABSTRACT Retrotransposon Ty1 faces a formidable cell barrier during transposition—the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

scholarly journals Immunologically distinct p53 molecules generated by alternative splicing.

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239 ◽  
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals Different Regions of Hepatitis B Virus X Protein Are Required for Enhancement of bZip-Mediated Transactivation versus Transrepression

2000 ◽  
Vol 74 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Sangeeta Barnabas ◽  
Ourania M. Andrisani
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
X Protein ◽  
Amino Acid Region ◽  
B Virus

ABSTRACT The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIγ and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIγ interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX49–115 is as effective as the full-length pX in enhancing the ATF3- and ICERIIγ-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX49–140are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.

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