Identification of an additional negative regulatory region for p53 sequence-specific DNA binding

Abstract

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393. The C-terminal residues 364-393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

Figure 1

Affinity precipitation of recombinant p53 deletion constructs with peptides, analyzed by Western blot. Bacterial lysates containing 1.5 μg of the indicated p53 constructs were precipitated with different peptide affinity matrices. p53 in eluates was determined by Western blot analysis with PAb240. Lanes 1 show lysate samples containing 7.5 ng of the respective input p53 construct. The cysteine-blocked matrix was used for the control analyses in lanes 2. The indicated peptide-matrices were used for precipitation in lanes 3–5. Position of molecular mass standards is indicated.

Figure 2

Recombinant p53 constructs. Schematic overview of the recombinant p53 constructs that were generated and bacterially expressed.

Figure 3

Effects of C-terminal peptide and PAb421 on DNA binding of recombinant p53 deletion constructs. EMSAs of indicated p53 constructs with radiolabeled Ep21 oligonucleotide. Lanes 1 represent basal uninduced DNA binding activity of the respective proteins. In lanes 2–5 the indicated agents were added to the DNA binding reactions at following concentrations: 3 mM control peptide (lanes 2); 300 μM peptide 369–383 (lanes 3); 3 mM peptide 363–373 (lanes 4); and 10 nM PAb421 (lanes 5).

Figure 4

Activation of p53 by synthetic polyproline peptides spanning residues 80–93. Sequences of the two peptides in single letter code. PXXP motifs, termed “A–E,” are depicted in boldface letters (A). EMSAs of full-length p53 with radiolabeled Ep21 oligonucleotide (B and C). Lanes 1 represent basal uninduced p53 DNA binding activity. In lanes 2–7 increasing concentrations of the indicated polyproline peptide had been added to the DNA binding reactions:0.1 mM (lanes 2); 0.25 mM (lanes 3); 0.5 mM (lanes 4); 1 mM (lanes 5); 2.5 mM (lanes 6); 7.5 mM (lanes 7). Lane 8: 0.75 mM of control peptide had been added to the reaction mixture.

Figure 5

Affinity precipitation of R vs. T state full-length p53 with C-terminal peptide analyzed by Western blot. p53aa1–393 (lysate containing 750 ng) was titrated into R state conformation with increasing amounts of PAb421. p53 was subsequently analyzed for peptide binding by affinity precipitation with peptide 363–373. p53 in eluates was analyzed by Western blotting with PAb240 (A). In parallel, same epitope-tagged p53aa1–393 lysate was preincubated with an anti-peptide-tag mAb as control (B). Lanes 1 in both A and B represent p53aa1–393 bound to the peptide-matrix without addition of any antibody. The following increasing amounts of PAb421 (A) and anti-peptide-tag antibody (B) were used for preincubation with p53: 1 μg (lanes 2), 5 μg (lanes 3), and 10 μg (lanes 4). Position of molecular mass standards is indicated.

Figure 6

Correlation of peptide binding with the T state conformation of p53. Columns 1 and 2 summarize DNA binding data. Column 1 scores basal DNA binding activity of the p53 deletion constructs as determined in EMSAs. −, Low-affinity DNA binding activity of p53; +, high-affinity DNA binding of p53. Column 2 scores the ability of the respective p53 deletion constructs to be activated by the C-terminal peptides. +, Significant activation of p53 DNA binding activity; −, the nonsusceptibility of the respective protein to further activation by the peptides. *, Data not shown. Column 3 scores the results of the peptide affinity precipitation analyses of the various deletion constructs and therefore summarizes the data presented in Fig. 1. +, Specific interaction of the respective protein with the C-terminal peptides; −, significantly reduced affinity of the protein for the peptides.