The signaling mechanism by which the Rb/E2F pathway in general, and E2F1 specifically, communicates with p53 has been thought to involve the p19ARF/Mdm2 pathway. Indeed, we find that p19ARF and Mdm2 are required for the majority of p53 protein accumulation observed following E2F1 expression. However, contrary to the role of the p19ARF pathway in p53 accumulation, we find that E2F1 induces apoptosis in MEFs lacking p19ARF
(Fig. ). It is unlikely that E2F1 induction of p73
is directly responsible for the apoptosis observed in p19ARF−/−
MEFs since we show that E2F1 failed to induce apoptosis in MEFs lacking both p19ARF
. These results are in contrast to those of earlier studies which concluded that p19ARF
is important for E2F1-mediated apoptosis (16
). However, during revisions to the manuscript, reports published by Tsai et al. (70
) and Tolbert et al. (68
) showed that p19ARF
was not required for apoptosis in Rb
-deficient mouse embryos or following Rb inactivation by transgenic expression of a fragment of the simian virus 40 large-T antigen, respectively. Likewise, a report published by Russell et al.
showed that crossing an E2F1
transgenic mouse into a p19ARF−/−
background does not reduce apoptosis caused by expression of the E2F1
). Thus, studies that used several different models arrived at the conclusion that the apoptosis associated with deregulated E2F activity does not require p19ARF
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 (more ...)
If p19ARF is not required for E2F1 to activate p53, what role does p19ARF have in this context? Expression of p19ARF in p53−/−
MEFs causes growth arrest, and overexpression of E2F1 overcomes this arrest (8
), suggesting that p19ARF may act as a negative regulator of the Rb/E2F proliferation pathway. We speculate that induction of the p19ARF/Mdm2/p53 pathway by the E2F family functions primarily to attenuate the proliferation-promoting effects of E2F transcriptional activity (Fig. ). Indeed, human p14ARF binds E2F1 and inhibits its transcriptional activity (20
). Moreover, p19ARF physically interacts with E2F1, E2F2, and E2F3 and promotes their degradation in a proteasome-dependent manner (47
). Another possibility is that p19ARF/Mdm2 activates p53 and modulates proliferation by inducing the p21 cyclin-dependent kinase inhibitor, a known transcriptional target of p53 (18
). In either case, the p19ARF/Mdm2/p53 pathway would act as a sensor and attenuator of proliferation.
It is conceivable that forcing cells into S phase by ectopic E2F expression can induce the apoptotic response. However, it is not likely to simply be ectopic S-phase induction following E2F expression that triggers apoptosis, since both E2F1 and E2F2 can induce S phase at similar efficiencies (15
), and we find that E2F2 does not induce apoptosis in MEFs. Moreover, there does not appear to be a specific phase of the cell cycle in which E2F1 induces apoptosis (14
). Our findings that ectopic E2F1, but not E2F2, expression results in increased p53 phosphorylation in the absence of p19ARF
and that this covalent modification of p53 contributes to E2F1-mediated apoptosis suggest that E2F1 may activate a cellular response similar to DNA damage. Activation of p53 by covalent modification, specifically phosphorylation of serine 15 (serine 18 in the mouse) and serine 20 in response to DNA damage, has been a topic of great interest due to the location of these residues within the Mdm2 interaction domain of p53. Mutational analysis of p53 sites known to be covalently modified, including serine 15 and serine 20, have been inconsistent regarding the requirements for phosphorylation at any one of these sites for p53-dependent apoptosis in response to DNA damage (2
). While our study examined the phosphorylation status of two N-terminal residues on p53 known to be phosphorylated following DNA damage, we have not established that either of these modifications are causal in p53-mediated apoptosis, nor have we ruled out the involvement or importance of modification(s) to other residues on p53 following E2F1 expression. However, our finding that apoptosis is reduced when E2F1 is coexpressed with a p53 mutant lacking many of these phosphorylation sites demonstrates a contribution by potential p53 phosphorylation targets to E2F1-induced apoptosis. Additionally, the observation that caffeine inhibits E2F1-mediated apoptosis suggests that the action of one or more p53 kinases is likely to be important for E2F1 signaling. Signaling cascades that activate protein kinases responsible for phosphorylating N-terminal residues on p53 upon DNA damage are well documented (1
). Candidate kinases include ATM, ATR, CHK1, and CHK2. ATM and ATR phosphorylate p53 at Ser15
and activate the CHK kinases by phosphorylation (3
). Active CHK kinases can then phosphorylate p53 at Ser15
Recently, ATM/ATR has been shown to phosphorylate the N terminus of E2F1 but not that of E2F2 or E2F3 (43
). This observation, together with the data presented here, leads us to speculate that ectopic E2F1 expression or activation of endogenous E2F1 upon phosphorylation by ATM, leads to increases in the activity and, perhaps, levels of one or more of the p53 kinases, which then phosphorylate p53 and promote apoptosis. Given that the E2F1 DNA binding mutant E2F1e132
did not induce p53 phosphorylation or apoptosis, transcriptional activation of one or more p53 kinases might provide a mechanism for E2F1 activation of this pathway. However, overexpression of the ATM/ATR, CHK1, or CHK2 kinases has been found to be insufficient for their activation (45
). Therefore, an additional signal(s) may be necessary to stimulate their activity.
It is conceivable that E2F1, E2F2, or E2F3 could signal through the p19ARF/Mdm2 pathway to increase p53 protein levels and that the increased pools of p53 would provide more substrate for the p53 kinases activated by E2F1 (Fig. ). Thus, the p19ARF/Mdm2 pathway may act both as an attenuator of proliferation by targeting E2F family members for degradation and as an amplifier of a DNA damage signal by increasing pools of p53 available for phosphorylation. The decision to undergo growth arrest or apoptosis would then depend on the cellular context or extent of DNA damage.
Our data implicates p53 phosphorylation as a key step in E2F1-mediated p53-dependent apoptosis. These observations raise the possibility that E2F1 signaling and DNA damage response pathways may converge and involve the same or related kinases to activate p53. Alternatively, E2F1 may contribute to or be a component of the DNA damage pathway. Given these possibilities, it is possible that a role of E2F1 may be to amplify DNA damage signals, resulting in p53-mediated apoptosis. Future studies are needed to test this hypothesis and to define the kinase(s) involved in p53 phosphorylation and the mechanisms by which E2F1 activates them.