Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Linghu Nie,Mark Sasaki,Carl G Maki

doi:10.1074/jbc.m610515200

Abstract

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

Highlights

Mutant p53s Are Cytoplasmic and Dependent on Their C-terminal nuclear export signals (NES) and an Intact Ubiquitination Pathway—Cancerassociated mutations in p53 occur almost exclusively in the DNA-binding domain (DBD) and alter the conformation and localization of p53 to varying extents. p53 conformation can be monitored by assessing reactivity with the conformation-specific antibodies pAb1620 and pAb240 [23, 24]
The results suggest that confor- was mostly nuclear when expressed either alone or with mational change induced by the p53 mutation renders p53 sus- MDM2, demonstrating that it is resistant to MDM2-mediated ceptible to ubiquitination in both the DBD and C terminus. nuclear export
MDM2-mediated nuclear export of p53 is believed to require ubiquitination of one or more lysines located in a cluster of six lysines in the extreme C terminus of p53

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfections—Saos-2 (p53-null) and U2OS (p53 wild-type) are osteosarcoma cell lines. p53/MDM2 double knock-out (DKO) mouse embryo fibroblasts were from Rudy Alarcon (Stanford University). Cells were transfected with 0.5–1.0 ␮g of FLAG p53 DNA alone or with 1.0 ␮g of DNA encoding MDM2 and/or Myc-tagged ubiquitin (myc-Ub). Plasmid DNAs—FLAG-tagged wild-type p53 has been described previously [32] and was from Zhi-min Yuan (Harvard School of Public Health) This DNA contains wild-type p53 sequences cloned into BamHI and XbaI sites downstream of the FLAG epitope. P53 DNAs encoding a TEV protease cleavage site at different positions were generated by two-step PCR. The resulting supernatant was mixed for 1 h with 30 ␮l of protein A-agarose beads to pre-clear the lysate The supernatant from this pre-clear was immunoprecipitated for 12–15 h at 4 °C with 2 ␮l of anti-FLAG polyclonal antibody (F-7145, Sigma) or 1.75 ␮l of p53 wild-type conformation-specific antibody (pAb1620, Ab-5, Calbiochem) or mutant conformation-specific antibody (pAb240, Ab-3, Calbiochem). The staining pattern for p53 was scored for 100 –150 cells in two or three separate experiments

RESULTS

This involved immunoprecipitation

DISCUSSION