E2 Ubiquitin-conjugating Enzyme, UBE2C Gene, Is Reciprocally Regulated by Wild-type and Gain-of-Function Mutant p53

Swati Bajaj,Sk.Kayum Alam,Kumar Singha Roy,Arindam Datta,Somsubhra Nath,Susanta Roychoudhury

doi:10.1074/jbc.m116.731398

Abstract

Spindle assembly checkpoint governs proper chromosomal segregation during mitosis to ensure genomic stability. At the cellular level, this event is tightly regulated by UBE2C, an E2 ubiquitin-conjugating enzyme that donates ubiquitin to the anaphase-promoting complex/cyclosome. This, in turn, facilitates anaphase-onset by ubiquitin-mediated degradation of mitotic substrates. UBE2C is an important marker of chromosomal instability and has been associated with malignant growth. However, the mechanism of its regulation is largely unexplored. In this study, we report that UBE2C is transcriptionally activated by the gain-of-function (GOF) mutant p53, although it is transcriptionally repressed by wild-type p53. We showed that wild-type p53-mediated inhibition of UBE2C is p21-E2F4-dependent and GOF mutant p53-mediated transactivation of UBE2C is NF-Y-dependent. We further explored that DNA damage-induced wild-type p53 leads to spindle assembly checkpoint arrest by repressing UBE2C, whereas mutant p53 causes premature anaphase exit by increasing UBE2C expression in the presence of 5-fluorouracil. Identification of UBE2C as a target of wild-type and GOF mutant p53 further highlights the contribution of p53 in regulation of spindle assembly checkpoint.

Highlights

The tumor suppressor gene TP53 plays a pivotal role in the maintenance of cellular and genomic integrity by preventing accumulation of errors in the cell duplication process under stressed conditions [1]
It has been previously demonstrated that mutant p53 interacts with the CCAAT-binding factor NF-Y, and this complex serves to up-regulate NF-Y target genes such as CCNA, CCNB, CDK1, CDC25C, and EFNB2 to promote cell cycle progression and chemoresistance following drug treatment [4, 15]
Similar up-regulation of UBE2C was observed in two other mutant p53 cell lines, C33A (R237C p53 mutant) and SKBR3 (R175H GOF p53 mutant), suggesting that this is not a cell type-specific phenomenon

Summary

Introduction

The tumor suppressor gene TP53 plays a pivotal role in the maintenance of cellular and genomic integrity by preventing accumulation of errors in the cell duplication process under stressed conditions [1]. We observed a dose-dependent decrease in the activity of all three UBE2C promoter-luciferase constructs in HCT116p53Ϫ/Ϫ cells when wild-type p53 is ectopically expressed (Fig. 2B). We observed a significant increase of UBE2C promoter activity upon ectopic expression of wild-type NF-YA (NF-YA–WT) but not for the dominant-negative mutant alone or in combination (Fig. 4B).

Results

Conclusion