MDM2 mediates p300/CREB-binding protein-associated factor ubiquitination and degradation

We recently reported that MDM2, a negative feedback regulator of the tumor suppressor p53, inhibits p300/CREB-binding protein-associated factor (PCAF)-mediated p53 acetylation. Our further study showed that MDM2 also regulates the stability of PCAF. MDM2 ubiquitinated PCAF in vitro and in cells. PCAF ubiquitination occurred at the N terminus and in the nucleus, as the nuclear localization signal sequence-deletion mutant of MDM2, which localized in the cytoplasm and degraded p53, was unable to degrade nuclear PCAF. Restriction of PCAF in the nucleus by leptomycin B did not affect MDM2-mediated PCAF degradation. Consistently, overexpression of MDM2 in p53 null cells caused the reduction of the protein level of PCAF, but not the mRNA level. Conversely, PCAF levels were higher in MDM2-deficient mouse p53(-/-)/mdm2(-/-) embryonic fibroblast (MEF) cells than that in MDM2-containing MEF cells. Furthermore, MDM2 reduced the half-life of PCAF by 50%. These results demonstrate that MDM2 regulates the stability of PCAF by ubiquitinating and degrading this protein.     

Acetyltransferase p300 regulates NBS1-mediated DNA damage response

The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1–p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

Structured summary

MINT-8058074, MINT-8058083: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti bait coimmunoprecipitation (MI:0006)MINT-8058111: p300 (uniprotkb:Q09472) and NBS1 (uniprotkb:O60934) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8058657: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by two hybrid (MI:0018)MINT-8058093: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti tag coimmunoprecipitation (MI:0007)     

Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response

Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21(WAF1) and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21(WAF1) and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.     

E2F1 plays a direct role in Rb stabilization and p53-independent tumor suppression

To better understand the role of E2F1 in tumor formation, we analyzed spontaneous tumorigenesis in p53-/-E2F1+/+ and p53-/-E2F1-/- mice. We show that the combined loss of p53 and E2F1 leads to an increased incidence of sarcomas and carcinomas compared to the loss of p53 alone. E2F1-deficient tumors show wide chromosomal variation, indicative of genomic instability. Consistent with this, p53-/-E2F1-/- primary fibroblasts have a reduced capacity to maintain genomic stability when exposed to S-phase inhibitors or genotoxic drugs. A major mechanism of E2F1’s contribution to genomic integrity lies in mediating stabilization and engagement of the Rb protein.     

A role for 14-3-3 tau in E2F1 stabilization and DNA damage-induced apoptosis

Genotoxic stress triggers apoptosis through multiple signaling pathways. Recent studies have demonstrated a specific induction of E2F1 accumulation and a role for E2F1 in apoptosis upon DNA damage. Induction of E2F1 is mediated by phosphorylation events that are dependent on DNA damage-responsive protein kinases, such as ATM. How ATM phosphorylation leads to E2F1 stabilization is unknown. We now show that 14-3-3 tau, a phosphoserine-binding protein, mediates E2F1 stabilization. 14-3-3 tau interacts with ATM-phosphorylated E2F1 during DNA damage and inhibits E2F1 ubiquitination. Depletion of 14-3-3 tau or E2F1, but not E2F2 or E2F3, blocks adriamycin-induced apoptosis. 14-3-3 tau is also required for expression and induction of E2F1 apoptotic targets, such as p73, Apaf-1, and caspases, during DNA damage. Together, these data demonstrate a novel function for 14-3-3 tau in the regulation of E2F1 protein stability and apoptosis during DNA damage.     

14-3-3 proteins integrate E2F activity with the DNA damage response

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.