Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53

Abstract

The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53's transactivation activity and (ii) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm.

Figure 1

The nuclear localization sequence in Hdm2. A part of the Hdm2 primary structure that resembles the NLSs of various nuclear proteins is aligned with these known NLSs. The basic amino acid residues that are critical for NLS function are shown in bold. Numbers refer to the position of the amino acids in the proteins. A mutant Hdm2 (Hdm2NLS) was generated with the amino acid changes (R181T, R183L).

Figure 2

Cellular localization of Hdm2 mutants. 2KO and H1299 cells were (co)transfected with the expression plasmid(s) encoding Hdm2 mutants as indicated above the pictures. Cellular localization of Hdm2 mutants in 2KO (a–c) or H1299 (d–f) cells was determined by using immunofluorescence 24 hours after transfection as described in Materials and Methods.

Figure 3

Effect of wild-type Hdm2 and its mutants on p53 stability. 2KO cells were cotransfected with p53 expression plasmid and the plasmids encoding either wild-type Hdm2 or its mutants as indicated above the figure. At 24 hours after transfection, steady-state levels of p53 (A) and Hdm2 (B) were determined by immunoblotting as described in Materials and Methods. Two species of the Hdm2 protein with different molecular weights were detected (B). This may be a result of different levels of phosphorylation or some other posttranslational modification. The apparent molecular weight of Hdm2Δ150–230 is lower than that of the full-length Hdm2 proteins because of the deletion (B, lane 7).

Figure 4

Coexpression of either Hdm2NLS or Hdm2NES with wild-type Hdm2 has no effect on Hdm2’s ability to promote p53 degradation. 2KO cells were cotransfected with p53 expression plasmid and the Hdm2 plasmid alone or with the plasmid encoding its mutant (as indicated). At 24 hours after transfection, steady-state levels of p53 was determined by using immunoblotting as described in Materials and Methods.