Article Figures & Data
Figures
- Fig. 1.
p53 from HTLV-1-transformed cells binds to DNA in a sequence-specific manner. (A) ML-1 and C81 cell lysates were incubated with biotinylated oligonucleotides containing either the wild-type (WT) or mutant (MT) p53 binding sites from WAF1 (lanes 3 to 6) and GADD45 (lanes 7 to 10) promoters. Immunoblot analysis was performed to detect DNA-bound p53. One-fourth of the input amount of p53 is shown in lanes 1 (ML-1) and 2 (C81). Sizes are indicated in kilodaltons. (B) The ability of endogenous p53 from two additional HTLV-1-transformed cell lines (MT-2 and HUT102) to bind to theWAF1 promoter was tested. (C) EMSAs of a32P-end-labeled WAF1 oligonucleotide probe were performed with nuclear lysates of ML-1 cells untreated (lane 1) or induced with 6 Gy of ionizing radiation (lane 2) and with nuclear extracts of C81 cells (lane 3). Specificity of binding was confirmed by competition with a 100-fold excess of either cold wild-type (WT; lane 4) or mutated (MT; lane 5) WAF1 probe. The specific bound complex is indicated by the lower arrow. PAb421 supershifted this bound complex (lane 6), indicated by the upper arrow.
- Fig. 2.
HTLV-1 p53 DNA complex does not contain TBP or MDM2. DNA-bound p53 complexes isolated as described for Fig. 1 were analyzed by immunoblotting for the presence of p53, Tax, TBP, MDM2, and E2F. One-fourth of the input amount is shown. WT, wild type; MT, mutant.
- Fig. 3.
Phosphopeptide map of HTLV-1 p53. Tryptic/chymotryptic digestion of purified in vivo 32P-labeled p53 from C81 (A) and dexamethasone-induced GM47.23 (B) cells were performed as described previously (47) and separated by electrophoresis and chromatography. (C) Diagram showing the migration of synthetic peptides where individual phosphopeptides were assigned numbers 1 to 8 (47). Ori, origin.
- Fig. 4.
p53 phosphorylation-specific antibodies confirm increased phosphorylation of Ser15 and Ser392 in HTLV-1-transformed cells. Antibodies specific for P-Ser15 (A) and P-Ser392 (B) and antibody DO-1, which reacts with phosphorylated or unphosphorylated p53 (C), were used in immunoblot analysis of lysates from HTLV-1-transformed cell lines C81 (lane 1), MT-2 (lane 2), and HUT102 (lane 3) or from untransformed dexamethasone-induced GM47.23 cells (lane 4), serum-starved ML-1 cells (lane 5), and peripheral blood leukocytes (PBL; lane 6). (D and E) Characterization of P-Ser15- and P-Ser392-specific antibodies. The graphs of absorbance at 490 nm represent the reactivities of the sera against the synthetic peptides indicated at the right.
- Fig. 5.
Western blot analysis of chymotryptic digestion of p53 from serum-starved ML-1 and C81 cells stained with either antibody PAb421, a C-terminal epitope (lanes 3 and 4), or DO-1, an N-terminal epitope (lanes 5 and 6). Lanes 1 and 2 show the amount of p53 in each extract prior to digestion. Sizes are indicated in kilodaltons.
- Fig. 6.
Phosphorylation at p53 Ser15 inhibits TFIID binding. (A) Diagram of biotinylated peptides which correspond to the activation domain (amino acids 1 through 39) of human p53. Biotinylated peptides were incubated with the 1 M phosphocellulose TFIID fraction from HeLa cell extracts. Bound complexes were captured with magnetic streptavidin beads and analyzed by Western blotting for the presence of TBP, using an anti-TFIID antibody (Santa Cruz). (B) Lane 1, one-fourth of input TFIID fraction; lane 2, no peptide; lane 3, unphosphorylated 1-39 peptide; lane 4, 1-39 peptide phosphorylated at Ser15; lane 5, 1-39 peptide phosphorylated at Ser20. Sizes are indicated in kilodaltons. (C) Peptides were either mock treated (lanes 2 to 5) or treated with DNA PK. The reaction was stopped by addition of 0.5 μM wortmannin. Lane 1, one-fourth of input TFIID fraction; lane 2, unphosphorylated 1-39 peptide; lane 3, 1-39 peptide phosphorylated at Ser15; lane 4, 1-39 peptide phosphorylated at Ser20; lane 5, 1-39 peptide phosphorylated at Ser37; lane 6, DNA PK-treated peptide 1-39; lane 7, DNA PK-treated peptide 1-39P15; lane 8, DNA PK-treated peptide 1-39P20; lane 9, DNA PK-treated peptide 1-39P37. Below lanes 2 to 9 are captured peptides stained with Coomassie blue to determine the amount of peptide recovered in the binding assays.
- Fig. 7.
Phosphorylation of p53 by DNA PK abolishes MDM2 binding. (A) Western blot analysis of MDM2 from whole-cell extracts bound to peptides as indicated at the top. (B) MDM2 binding to peptides 1-39 (lane 1), 1-39 P15 (lane 2), 1-39 treated with DNA PK (lane 3), and 1-39 P15 treated with DNA PK (lane 4). The lane M indicates positions of molecular weight standard in kilodaltons.
Tables
- Table 1.
32P radioactivity in phosphopeptides from C81 and GM47.23 (wild-type) cells
Phosphopeptidea % of total labeled peptideb Peptide no.a Sequence No. of sites phosphorylated (position) GM47.23 (wild type) C81 1 p53-(25–53) 1 (S37) 18.4 ± 5.4 9.5 ± 2.9 4 Ac-p53-(1–19) 1 (S9) 36.9 ± 6.3 17.7 ± 0.8 5 Ac-p53-(1–19) 2 (S9 + S15) 12.4 ± 3.3 22.0 ± 1.7 7 p53-(307–319) 1 (S315) 11.8 ± 1.8 15.7 ± 1.3 8 p53-(387–393) 1 (S392) 8.2 ± 2.1 28.5 ± 1.6 ↵a Numbered as indicated in Fig. 3. Synthetic peptide standards corresponding to phosphopeptides 1, 4, 5, 7, and 8 were used.
↵b The amount of 32P associated with each phosphopeptide was determined by using a PhosphorImager and the ImageQuant program (Molecular Dynamics) and is expressed as the percentage of total radioactivity recovered after digestion and performic acid treatment. Values are given as the mean ± standard deviation of three independent experiments.
