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.