p31(comet)-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

Abstract

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31(comet) is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31(comet)-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31(comet), the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31(comet) overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31(comet). Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31(comet). Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wildtype Mad2 interacting with p31(comet), but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31(comet)-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31(comet) affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31(comet)-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.This study was supported by grants from National Research Foundation of Korea (2012M3A9B6055346), and nuclear R&D program of Korea Ministry of Sciences and Technology