E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis

Abstract

It has been proposed that the E2F1 transcription factor serves as a link between the Rb/E2F proliferation pathway and the p53 apoptosis pathway by inducing the expression of p19ARF, a protein that regulates p53 stability. We find that although p19ARF contributes to p53 accumulation in response to E2F expression, p19ARF is not required for E2F1-mediated apoptosis. E2F1 can signal p53 phosphorylation in the absence of p19ARF, similar to the observed modifications to p53 in response to DNA damage. These modifications are not observed in the absence of p19ARF following expression of E2F2, an E2F family member that does not induce apoptosis in mouse embryo fibroblasts but can induce p19ARF and p53 protein expression. p53 modification is found to be crucial for E2F1-mediated apoptosis, and this apoptosis is compromised when E2F1 is coexpressed with a p53 mutant lacking many N- and C-terminal phosphorylation sites. Additionally, E2F1-mediated apoptosis is abolished in the presence of caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases that phosphorylate p53. These findings suggest that p53 phosphorylation is a key step in E2F1-mediated apoptosis and that this modification can occur in the absence of p19ARF.

FIG. 1.

E2F1 induces apoptosis in the absence of p19ARF. (A) Immunoblot analysis of p19ARF levels in lysates of wild-type (wt) and p19ARF^−/− MEFs. Cells were infected with recombinant adenoviruses encoding E2F1, E2F1_e132, or a control (Con) virus. Samples were harvested, and lysates were generated at 24 hpi. The actin blot is shown as a protein loading control. ns, nonspecific band. (B) Analysis of apoptosis in MEFs. Wild-type and p19ARF^−/− MEFs were infected at a MOI of 100, 300, or 500 with virus encoding the marked cDNA. Cells were harvested and fixed at 72 hpi, stained with PI, and analyzed by flow cytometry. Apoptotic cells were scored as the percentage of cells with a sub-2N DNA content (marked by the bar in each plot). (C) Analysis of apoptosis in p19ARF^−/− and p19ARF^−/−/p53^−/− MEFs. Infection and harvest conditions were the same as described for panel B. Percentages represent cells with sub-2N DNA content.

FIG. 2.

Contribution of p19ARF and Mdm2 to E2F1-mediated accumulation of p53. (A) Immunohistochemical staining for endogenous p53 protein in wild-type (WT) and p19ARF^−/− MEFs. Cells were fixed for staining at 48 hpi. (B) Immunoblot analysis for p53 in lysates of wild-type, p19ARF^−/−, p53^−/−, and Mdm2^−/−/p53^−/− MEFs. Cells were coinfected with Adp53 and AdCon, AdE2F1, or AdE2F1_e132. Samples were harvested at 24 hpi. Actin blots are shown as protein loading controls.

FIG. 3.

E2F1 and E2F2 induce endogenous p53 accumulation and phosphorylation of the serine 18 residue of p53 in wild-type MEFs. Immunofluorescent detection of total p53 protein (rhodamine red X) and phospho-Ser¹⁸ p53 (FITC) following E2F1 or E2F2 expression in wild-type MEFs. DAPI (4′,6′-diamidino-2-phenylindole) staining is shown in blue. Merged images are also shown. Con, control virus.

FIG. 4.

E2F1 specifically induces endogenous p53 accumulation and phosphorylation of the serine 18 residue of p53 in p19ARF^−/− MEFs. Immunofluorescent detection of total p53 protein (Rhodamine Red-X) and phospho-Ser¹⁸ p53 (FITC) following E2F1 or E2F2 expression in p19ARF^−/− MEFs. DAPI (4′,6′-diamidino-2-phenylindole) staining is shown in blue. Merged images are also shown. Con, control virus.

FIG. 5.

Induction of apoptosis is specific to E2F1. (A) Apoptosis analysis in wild-type (wt) MEFs infected with AdCon, AdE2F1, or AdE2F2. At 72 hpi, cells were harvested and processed for PI staining and flow cytometry. Percentages represent cells with sub-2N DNA content. (B) Immunoblot analysis for p19ARF in lysates of wild-type and p19ARF^−/− MEFs. Cells were coinfected with Adp53 and either AdCon, AdE2F1, AdE2F1_e132, or AdE2F2. Cells were harvested, and lysates were generated at 24 hpi. The actin blot is shown as a protein loading control. ns, nonspecific band. (C) Immunoblot analysis for endogenous p53 and the phospho-Ser¹⁸ form of p53 in lysates of wild-type and p19ARF^−/− MEFs. Cells were infected with AdCon, AdE2F1, AdE2F2, or AdE2F1_e132. Cells were harvested, and lysates were generated at 24 hpi. The actin blot is shown as a protein loading control.

FIG. 6.

Induction of p53 phosphorylation by E2F1 and E2F2. Immunoblot analysis for phospho-Ser¹⁵ (A) and phospho-Ser²⁰ (B) forms of p53 or total p53 in lysates (C) of wild-type (wt) and p19ARF^−/− MEFs coinfected with Adp53 and either AdCon, AdE2F1, or AdE2F2. Cells were harvested, and lysates were generated at 24 hpi. The blot was sequentially stripped and reprobed. Actin blots are shown as protein loading controls.

FIG. 7.

Covalent modification of p53 contributes to E2F1-mediated apoptosis. (A) Immunoblot analysis of wild-type (wt) p53 and p53N/C in lysates of p53^−/− MEFs infected with Adp53 or Adp53N/C at an MOI of 10, 20, or 50. An actin blot is also shown. (B) Apoptosis analysis in p53^−/− MEFs. Cells were infected with Adp53 or Adp53N/C at an MOI of 20 and AdCon, AdE2F1, or AdE2F2 at an MOI of 500. At 72 hpi, cells were harvested and processed for PI staining and flow cytometry. Percentages represent cells with sub-2N DNA content.

FIG. 8.

Caffeine inhibits E2F1-mediated apoptosis. Analysis of apoptosis in wild-type MEFs. MEFs were infected with AdE2F1 at an MOI of 500, and caffeine was added at the indicated doses to the culture medium following infection. Cells were harvested at 72 hpi. Apoptosis was measured by detection of cytoplasmic DNA-histone complexes by ELISA. Error bars represent standard deviations calculated from three separate experiments. Con, control virus.

FIG. 9.

Model of E2F-mediated p53 accumulation and apoptosis. E2F1, E2F2, and to a lesser extent, E2F3 can signal p53 accumulation through the p19ARF/Mdm2 pathway. p53 phosphorylation contributes to E2F1-mediated apoptosis and can occur in the absence of p19ARF. p19ARF may function to attenuate E2F proliferation-promoting activity as shown by the dashed lines. Activation of the p19ARF/Mdm2 pathway may increase levels of p53, contributing substrate to the p53 kinases induced by E2F1 as shown by the dashed arrow.