Journal: Journal of Biological Chemistry

Article Title: The Bradykinin Type 2 Receptor Is a Target for p53-mediated Transcriptional Activation

doi: 10.1074/jbc.m909810199

Figure Lengend Snippet: FIG. 1. The rat bradykinin B2 receptor promoter contains two p53-like binding sites. A, schematic showing the promoter-reporter BK2 (pBK2-CAT) construct and the relative positions of the p53-bind- ing sites, P1 and P2. B, sequence comparisons of the P1 and P2 sites with the consensus p53 binding sequence (14). Underlined bases denote sequence variation from the consensus p53 element. C, species conser- vation of P1 site.

Article Snippet: Western blots using the p53 antibodies, PAb421 (Oncogene Science), and FL-393 (Santa Cruz), showed that SVT-2 cells contain p53 (data not shown).

Techniques: Binding Assay, Construct, Sequencing

Journal: Journal of Biological Chemistry

Article Title: The Bradykinin Type 2 Receptor Is a Target for p53-mediated Transcriptional Activation

doi: 10.1074/jbc.m909810199

Figure Lengend Snippet: FIG. 2. p53 binds the p53-like motif (P1 site) in the BK2 pro- moter. A, EMSA using a 32P-labeled BK2-P1 oligoduplex (59-AGGGGG- GAGGTGCCCAGGAGAGTGATGACA-39). Lane 1, free probe; lanes 2–4, in the presence of SVT-2 nuclear extracts (210 mg). Lanes 3 and 4 contain 1 or 2 ml, respectively, of the p53 activating antibody, PAb421. B, EMSA using 32P-labeled oligoduplexes containing the p53 consensus sequence (59-TAGGCATGTCTAGGCATGTCTAAGCT-39) (lanes 1–7); mutant BK2-P1 oligoduplexes (59-AGGGGTGAGGCGCACAGGAGAT- TGATGACA-39, lane 8; 59-AGGGGTGAGGTTACCAGGAGAGTGAT- GACA-39, lane 9; 59-AGGGGGGAGGTAGCAAGGAGAGTGATGACA- 39, lane 10), or wild-type BK2-P1 oligoduplex (lanes 11 and 12). All lanes contain SVT-2 nuclear extracts. In lane 2, 100-fold excess of unlabeled consensus p53 sequence was added. Lanes 3–5 represent competition with increasing amounts (100–300-fold molar excess) of unlabeled oli- goduplex corresponding to the p53 DNA binding sequence in the p21waf-1 gene (59-ATCAGGAACATGTCCAACATGTTGGAACATGTCC- CAACATGTTGAGCTG-39). Lanes 6 and 7 represent competition with 100- or 200-fold excess of BK2-P1. Lanes 8–10 show lack of specific binding of mutant BK2-P1 site to nuclear extracts. Lanes 11 and 12 show that protein binding to the p53-like motif in the P1 site is com- peted by 100-fold excess unlabeled p53 consensus sequence. C, EMSA using recombinant p53 and either radiolabeled BK2-P1 oligoduplex (lanes 1–3) or the consensus p53 binding oligoduplex (lanes 4–7) as probes. Unlabeled competitors (100-fold excess) used included the p53 sequence in the p21waf-1 gene (lane 2 and 6), consensus p53 site (lanes 3 and 5), or BK2-P1 (lane 7). D, EMSA using recombinant p53 and BK2-P1 probe. Unlabeled competitors used included 200-fold excess of BK2-P1 (lane 2), consensus p53 sequence (lane 3), or the p53 site in the p21 gene (lane 4).

Article Snippet: Western blots using the p53 antibodies, PAb421 (Oncogene Science), and FL-393 (Santa Cruz), showed that SVT-2 cells contain p53 (data not shown).

Techniques: Labeling, Sequencing, Mutagenesis, Binding Assay, Protein Binding, Recombinant

Journal: Journal of Biological Chemistry

Article Title: The Bradykinin Type 2 Receptor Is a Target for p53-mediated Transcriptional Activation

doi: 10.1074/jbc.m909810199

Figure Lengend Snippet: FIG. 3. Wild-type p53 activates BK2 promoter-driven transcription in HeLa cells. A, representative CAT assay result. Using the LipofectAMINE rea- gent, 1.2 mg of each reporter construct was co-transfected with 50 ng of the plas- mid expressing wild-type p53. Cells were harvested 24 h after transfection and an- alyzed for CAT activity. Transfection and CAT assays were performed in duplicate in at least three independent experi- ments, and consistent results were ob- tained. B, dose-response effect of wild- type p53 (10, 50, and 75 ng) on CAT activity of the BK2 promoter constructs, pBK2(21184) and pBK2(294). C and D, co-transfection of pBK2(21184) construct with a fixed amount, 50 ng, of pCMV- p53(wt), and increasing amounts (0, 50, 100, and 500 ng) of pCMV-p53(mutant) showing that the dominant negative mu- tant p53 suppresses the transcriptional activation of the BK2 promoter by wild- type p53.

Article Snippet: Western blots using the p53 antibodies, PAb421 (Oncogene Science), and FL-393 (Santa Cruz), showed that SVT-2 cells contain p53 (data not shown).

Techniques: Construct, Transfection, Expressing, Activity Assay, Cotransfection, Mutagenesis, Dominant Negative Mutation, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: The Bradykinin Type 2 Receptor Is a Target for p53-mediated Transcriptional Activation

doi: 10.1074/jbc.m909810199

Figure Lengend Snippet: FIG. 4. The transcriptional co-activators, CBP/p300, augment p53-mediated transactivation of the BK2 promoter. HeLa cells were co-transfected with pBK2(21184) (A and B) or pBK2-(294) (C) constructs (1.2 mg) with or without expression plasmids for wild-type p53 (50 ng) and increasing amounts of CBP (A) or p300 (B) (0, 10, and 50 ng of DNA). FIG. 5. The upstream BK2-P2 sequence modulates p53-medi- ated transactivation. A, EMSA gels. 32P-Labeled oligoduplexes cor- responding to the BK2-P2 sequence (59-ACTCTTGCCTGGTCTTCCCT- 39) were incubated with SVT-2 nuclear extracts (210 mg protein/ sample) in the presence or absence of the p53-activating antibody, PAb421 (1 ml). The position of the band shifts is indicated by an asterisk (*). Addition of PAb421 enhanced the DNA binding activity, while addition of unlabeled p53 sequence from the p21 gene attenuated the binding. B, HeLa cells were co-transfected with pBK2 reporter con- structs either containing (21184) or lacking (2635) the P2 site with expression plasmids for wild-type (wt) or mutant (mut)p53. CAT activ- ity is 2-fold higher in pBK2(2635) than pBK2(21184) (n 5 3 assays in duplicates). A DNA-binding mutant p53 fails to activate the BK2 pro- moter. C, representative CAT assay showing the effect of deletion of the BK2-P2 site on p53-mediated activation of the BK2 promoter. HeLa cells were transfected with 1.2 mg each of the promoter-reporter plas- mids. BK2 promoter activity is increased in a dose-dependent manner by wild-type p53. Removal of 3/4 or 4/4 pentamers of the P2 site amplifies the p53-mediated activation. D, quantitative analysis of the CAT assay in C; white bar, pBK-2(21184); gray bar, pBK2-(P2D15bp); black bar, pBK2-(P2D20bp). Similar results were obtained in three inde- pendent experiments.

Article Snippet: Western blots using the p53 antibodies, PAb421 (Oncogene Science), and FL-393 (Santa Cruz), showed that SVT-2 cells contain p53 (data not shown).

Techniques: Transfection, Construct, Expressing, Sequencing, Labeling, Incubation, Binding Assay, Activity Assay, Mutagenesis, Activation Assay