The C terminus of p53 binds the N-terminal domain of MDM2

Abstract

The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former’s N-terminal region and core domain. Yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2–p53 association. We show that, the Mdm2–p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C-terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches we demonstrate that a peptide from p53 C-terminus directly binds Mdm2 N-terminus in vitro. We also show that p300-acetylated p53 binds inefficiently to Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro.

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

“…The IVT proteins (5 ml of the IVT reaction) were preincubated with the indicated amounts of Nutlin-3A in 500 ml PLB (20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet-P40, 1 mM PMSF plus complete protease inhibitors (Roche, Milan, Italy) for 2 h at 4 1C, and then allowed to react with 4 mg of either GST/hTP53 or GST recombinant proteins in the presence of 40 ml of 50% slurry glutathione sepharose 4B resin (GE Healthcare, Milan, Italy) for 2 h at 4 1C. The amount of Nutlin-3A used in these experiments (0, 80, and 400 mM) was established experimentally according to Hu et al 17 and Poyurovsky et al 25 Efficient inhibition of the interaction between full-length TP53 and full-length MDM2 was used as a positive control. After extensive washings, the bound proteins were separated by means of SDS-PAGE, after which the gels were stained with Coomassie Brilliant Blue, dried and exposed to X-ray film (Kodak, Cinisello Balsamo, Italy).…”

Interaction between p53 and estradiol pathways in transcriptional responses to chemotherapeutics

“…p53 activators can vary in their impact on p53 post-translational modifications and alter transcriptome responses. 58,59 It is important to note that, while p53 has been implicated, there may be other reasons for the genotoxic stress/ER synergistic responses.…”

Induction of ASAP (MAP9) contributes to p53 stabilization in response to DNA damage

“…Indeed, ASAP binds to both N-ter p53 and N-ter MDM2, thus not only disrupting p53-MDM2 interaction, but probably also facilitating C-ter p53 modifications as described. 16 However, since various proteins affect the p53-MDM2-p300 pathway, we cannot exclude that ASAP interacts or competes with other p53-interacting proteins that modify the outcome of the p53 response, as shown for other co-factors, 29 or with different drug combination treatments. 30 It is noteworthy that even polymorphism in the MDM2 promoter can alter its function and, consequently, and/or spindles and have mitotic-specific roles independent of their functions during interphase.…”