Characterization of Functional Domains Necessary for Mutant p53 Gain of Function

Wensheng Yan,Xinbin Chen

doi:10.1074/jbc.m109.097253

Abstract

Tumor cells, including SW480 carcinoma cells that carry a mutant p53, are addicted to the mutant for their survival and resistance to growth suppression by chemotherapeutic agents. Here, we investigated whether various classes of p53 mutants share a common property and functional domains necessary for mutant p53 gain of function. To test this, we generated SW480 cell lines in which endogenous mutant R273H/P309S can be inducibly or stably knocked down, whereas a small interfering RNA-resistant mutant p53 along with a mutated functional domain can be inducibly or stably expressed. We found that both contact-site (R248W and R273H) and conformation (G245S and R249S) mutants are able to maintain the transformed phenotypes of SW480 cells conferred by endogenous mutant p53. We also found that activation domains 1-2 and the proline-rich domain are required for mutant p53 gain of function. Interestingly, we showed that the C-terminal basic domain, which is required for wild-type p53 activity, is an inhibitory domain for mutant p53. Furthermore, we showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1-2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function. These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are also required for mutant p53 tumor promotion. Thus, the inhibitory basic domain and the common property for p53 mutants can be explored for targeting tumors with mutant p53.

Highlights

We showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1–2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function
These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are required for mutant p53 tumor promotion
The functional domains in wild-type p53 have been subject to extensive analysis [13,14,15]. p53 with a mutation in activation domain 1 (AD1) is deficient in transcriptional activity and subsequently unable to induce growth suppression and cell cycle arrest [16]

Summary

Introduction

We generated a series of SW480 cell lines in which endogenous mutant p53 can be inducibly knocked down, whereas an siRNA-resistant tumor-derived hot spot mutant, including contact-site mutants (R248W and R273H) and conformation mutants (G245S and R249S), was stably expressed. We showed that inducible expression of an exogenous mutant p53 was able to promote the colony-forming ability of SW480 cells along with reduced sensitivity to treatment with camptothecin when endogenous mutant p53 was stably knocked down (Fig. 3, B, D, F, and H).

Results

Conclusion