Diagrams of wild-type p53 (p53wt) and of the variant p53 proteins (p53V1 and p53V2) that were used to study p53 stabilization after DNA damage. Tx, transactivation domain; DNA-B, sequence-specific DNA-binding domain; 4, native tetramerization domain; R, C-terminal regulatory region for the DNA-binding activity of p53; HA, hemagglutinin tag; IND, modified (independent) tetramerization domain that does not form heterotetramers with the native p53 tetramerization domain; H273, histidine substitution for Arg-273. Substitutions of Ser residues with Ala or Asp were performed in the context of p53V1 and p53V2.

Substitution of Ser-20 abrogates p53 protein stabilization after DNA damage in p53-nullizygous MEFs stably transfected with constructs expressing p53V1 (A–D) or p53V2 (E) proteins. The transfected cells were exposed to IR (A and C–E), UV light (B), or the proteasome inhibitor lactacystin (Lc) (C). p53 protein levels were determined by immunoblotting (IB) with antibody DO1 (A–C, E) and p53 mRNA levels by Northern blotting (D). The mutants are designated as in Fig. 2.

Substitution of Ser-20 abrogates p53 protein stabilization after DNA damage in U2-OS osteosarcoma cells. p53V1 proteins with Ser/Thr substitutions were expressed by transient transfection. The transfected cells were exposed to IR (A) or UV light (B) and p53 protein levels were determined by immunoblotting (IB) with an antibody that reacts with the HA tag in p53V1. Designations of the expressed p53 proteins correspond to the amino acid substitutions introduced in p53V1, indicating the position(s) of the replaced residue(s) and the type of residue introduced by using the single-letter code. p53V1wt has no Ser/Thr substitutions, p53V1QS has substitutions of Leu-22 → Gln and Trp-23 → Ser, and p53V1Δ1–39 has amino acids 1–39 deleted.

Effect of phosphorylation of Ser-20 on the interaction of p53 with Mdm2. (A) Capture of ³⁵S-labeled in vitro-translated full-length or N-terminally truncated (ΔN61) Mdm2 by phosphorylated and nonphosphorylated p53 peptides corresponding to residues 7–29 of human p53; -p, nonphosphorylated peptide; S15p, T18p, and S20p, peptides phosphorylated on Ser-15, Thr-18, and Ser-20, respectively. (B) Three-dimensional structure of the p53–Mdm2 complex (PDB ID code 1YCR). The p53 peptide shown (residues 17–29) is the entire p53 region that associates with Mdm2. Selected amino acid side chains of p53 and Mdm2 are shown and labeled by using the codon number and single-letter residue code: F, Phe; L, Leu; M, Met; Q, Gln; S, Ser; T, Thr; and W, Trp. The oxygen atoms of the hydroxyl groups of Thr-18 and Ser-20 are colored red. The figure was prepared by using molscript (58) and raster3d (59).

Phosphorylation of endogenous wild-type p53 on Ser-20 in response to DNA damage. (A) Specificity of antibodies DO1 and AbS20p for p53 not phosphorylated and phosphorylated on Ser-20, respectively. Biotinylated peptides corresponding to residues 7–29 of human p53 were coupled to avidin-agarose beads and incubated with antibodies DO1 or AbS20p. Antibodies bound to the beads were detected by fractionation on denaturing polyacrylamide gels and immunoblotting. -p, nonphosphorylated peptide; S15p, T18p, and S20p, peptides phosphorylated on Ser-15, Thr-18, and Ser-20, respectively. (B) Phosphorylation of p53 on Ser-20 in response to IR and UV light in U2-OS cells, as determined by immunoreactivity of p53 to antibodies DO1 and AbS20p. The fractions of p53 IP with antibody DO1 (not phosphorylated on Ser-20) and antibody AbS20p (phosphorylated on Ser-20) were determined by immunoblotting (IB) with antibody DO7. Because p53 protein levels increase in response to DNA damage, to facilitate comparisons between nonirradiated and irradiated cells, the amounts of cell extracts used were adjusted to have equal p53 protein levels in all reactions [as shown by immunoblotting (IB) with antibody DO7 control reactions not subjected to IP]. (C) Effect of protein phosphatase on the immunoreactivity of p53 to antibody DO1. Extracts from untreated or irradiated U2-OS cells were treated with protein phosphatase (PPase +) or mock-treated (PPase −) before IP with antibody DO1. The amounts of cell extracts used were adjusted to have equal p53 protein levels in all reactions.