Regulation of p53 function and stability by phosphorylation

Abstract

The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21(Waf1/Cip1)-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

FIG. 1

(A) Schematic representation of the human p53 protein indicating the important functional domains (transactivation, proline rich, sequence-specific DNA binding, oligomerization, and regulation of DNA binding), conserved box regions I to V, and potential sites of phosphorylation within the N- and C-terminal regions. To generate N-terminal phosphorylation mutant p53 (N-term), Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, and Ser37 were mutated to Ala in combination. To generate C-terminal phosphorylation mutant p53 (C-term), Ser315, Ser371, Ser376, Ser378, and Ser392 were mutated to Ala in combination. N- and C-terminal phosphorylation mutant p53 (N/C-term) was generated by a combination of the above. (B) Western blot analysis (using PAb1801) of Saos-2 cells transiently expressing wild-type p53 and the p53N-term, p53C-term, and p53N/C-term mutant proteins after transient transfection with 5 μg of the respective plasmid. Cells were harvested at 24 h.

FIG. 2

Relative phosphorylation of p53 mutant proteins in vivo. Phosphorylation site mutant p53 proteins (5 μg) were transiently transfected into Saos-2 cells. At approximately 4 h prior to harvesting (at 24 h), the cells were washed and incubated in 5 ml of phosphate-free medium (supplemented with 10% dialyzed FCS) containing 1 mCi of ³²P-labeled sodium orthophosphate (per 10-cm² dish). Cells were lysed with radioimmunoprecipitation assay lysis buffer, and p53 protein was immunoprecipitated with PAb1801-protein A Sepharose beads. (A) Phosphorylated protein was assessed by SDS–10% PAGE (top), and blots were reprobed with PAb1801 to assess immunoprecipitated p53 levels (bottom). (B) Phosphorylated protein was quantified by PhosphorImager analysis and represented graphically by using absolute integrated optical density units (I.O.D.). The graph represents the results of four independent experiments.

FIG. 3

Transcriptional activation by p53 phosphorylation mutant proteins assessed by using a luciferase reporter assay. p53 phosphorylation mutant proteins (50 ng) were cotransfected into Saos-2 cells with 5 μg of either a human p21^Waf1/Cip1, Mdm2, or bax luciferase reporter construct. Five micrograms of pCB6+βgal was included to assess transfection efficiency. Cells were harvested at 24 h, lysed, and assessed for luciferase and β-galactosidase activities. Each graph shows fold activation relative to that of the pCB6+ control harvested at 24 h.

FIG. 4

Wild-type p53 and p53 phosphorylation mutant proteins (5 μg) were transiently transfected into Saos-2 cells and the cells were harvested at 24 h for Western blot analysis to assess p21^Waf1/Cip1 protein levels by using an anti-p21 antibody.

FIG. 5

(A) In vitro association assay using in vitro-translated, unlabeled, human flag-Mdm2 (cold) and ³⁵S-labeled, in vitro-translated wild-type p53 and p53N-term, p53C-term, and p53N/C-term mutant proteins. Complexes were immunoprecipitated (IP) with anti-flag antibody in the presence of protein G Sepharose beads, and radioactively labeled p53 proteins were assessed by SDS–10% PAGE. (B) Western blot analysis (using PAb1801) of Saos-2 cells after transient cotransfection of the phosphorylation mutant p53 (3 μg), mouse Mdm2 (9 μg), and pEGFP-N1 (1 μg). Cells were harvested at 24 h. The ΔI mutant protein (deletion of conserved box I, the region containing the Mdm2 binding site), which is resistant to Mdm2-mediated degradation, and the Δ370 mutant protein (C-terminal truncation mutant protein), which is partially resistant to Mdm2-mediated degradation, were used as controls. Relative transfection efficiency was monitored by expression of cotransfected GFP. (C) Western blot analysis (using PAb1801) of Saos-2 cells after transient cotransfection of the p53-15A/37A, -15A/37D, -15D/37A, and -15D/37D combination mutant proteins (3 μg), mouse Mdm2 (9 μg), and pEGFP-N1 (1 μg). Cells were harvested at 24 h. Relative transfection efficiency was monitored by expression of cotransfected GFP. (D) Western blot analysis (using PAb1801) of Saos-2 cells after transient cotransfection of the p53-20A, -20DD, -18, and -18D combination mutant proteins (3 μg), mouse Mdm2 (9 μg), and pEGFP-N1 (1 μg). Cells were harvested at 24 h. Relative transfection efficiency was monitored by expression of cotransfected GFP.

FIG. 6

(A) Schematic representation of the conserved box II deletion mutant proteins. (B) Western blot analysis (using PAb1801) of Saos-2 cells transiently expressing p53ΔII and the p53ΔII 15A/37A, p53ΔII N-term, and p53ΔII C-term mutant proteins after transient transfection with 5 μg of the respective plasmid. Cells were harvested at 24 h.

FIG. 7

Stable expression of p53ΔII combination mutant proteins in MCF-7 cells which express wild-type (wt) p53. Stable clones were isolated, and p53 protein levels were assessed by Western blot analysis using PAb1801 or D01 after treatment with either 5 nM actinomycin D (Act; harvested at 10 h) or 50-J/m² UV (harvested at 6 h). (A) Western blot analysis of MCF-7 stable cell lines expressing either the pCB6+ control (leftmost blot), p53ΔII (middle blot), or p53ΔI-ΔII (rightmost blot). Equal protein loading was confirmed by actin expression. endo., endogenous; exo, exogenous. (B) Western blot analysis of two independent MCF-7 stable cell lines (clone 1, left; clone 2, right) expressing either p53ΔII15A/37A (top), p53ΔIIN-term (middle), or p53ΔIIC-term (bottom). Equal protein loading was confirmed by actin expression. (C) Western blot time course analysis of actinomycin D (ActD) treatment (0, 3, and 6 h) of MCF-7 cells stably expressing either pCB6+ (control), p53ΔII (clone 1), p53ΔII15A/37A (clone 1), and p53ΔIIN-term (clone 1). All blots were reprobed with an antiactin monoclonal antibody to assess protein levels. Equal protein loading was confirmed by actin expression.

FIG. 8

Stable and transient expression of p53 proteins in p53-null MEFs. (A) Western blot analysis of MEFs stably expressing either a vector control (pCB6+) or p53ΔII with or without treatment with 5 nM actinomycin (ActD). (B) Western blot analysis of MEFs transiently expressing a vector control (pCB6+) or the indicated p53 proteins without DNA damage or following treatment with actinomycin D (Act; 5 nM) or UV irradiation (50 J/m²). (C) Western blot analysis of MEFs transiently expressing a vector control (pCB6+), wild-type p53, or p53N-term without DNA damage or following treatment with actinomycin D (ActD; 5 nM) or UV irradiation (50 J/m²).