PHAPI/pp32 Suppresses Tumorigenesis by Stimulating Apoptosis

Abstract

PHAPI/pp32 is a tumor suppressor whose expression is altered in various human cancers. Although PHAPI possesses multiple biochemical activities, the molecular basis for its tumor-suppressive function has remained obscure. Recently we identified PHAPI as an apoptotic enhancer that stimulates apoptosome-mediated caspase activation. In this study, we defined the structural requirement for its activity to stimulate caspase activation using a series of truncation mutants of PHAPI. Further, utilizing these mutants, we provide evidence to support the model that the apoptotic activity of PHAPI is required for its tumor-suppressive capability. Consistently, pp32R1, a close homolog of PHAPI and yet an oncoprotein, is not able to stimulate caspase activation. A highly discrete region between these two proteins localizes to an essential caspase activation motif of PHAPI. Additionally, PHAPI is predominantly a nuclear protein, and it can translocate to the cytoplasm during apoptosis. Disruption of the nuclear localization signal of PHAPI caused a modest decrease of its tumor-suppressive function, indicating that nuclear localization of PHAPI contributes to, but is not essential for, tumor suppression.Apoptosis plays a crucial role in multiple physiological events, and its deregulation can lead to various human diseases, including cancer, neurodegeneration, and autoimmune disorders (1, 2). Apoptosis is executed by a family of proteases known as caspases (3,4). In mammals, a major caspase activation pathway is the mitochondrial cytochrome c-mediated caspase activation pathway, also known as the intrinsic apoptotic pathway (5). In this pathway, a variety of apoptotic stimuli induce release of cytochrome c from mitochondria to the cytoplasm, a process controlled by the Bcl-2 family members (6 -8). The released cytochrome c binds to Apaf-1, the essential mediator of the pathway, and triggers its nucleotide binding/exchanging activity (9, 10). Subsequently, the activated Apaf-1 protein forms a multimeric protein complex called the apoptosome, which in turn recruits and activates the initiator caspase, caspase-9. The active apoptosome-caspase-9 holoenzyme (9, 11) then cleaves and activates the effector caspases, including caspase-3 and caspase-7, and therefore causes apoptotic cell death.The cytochrome c-mediated caspase activation pathway is under precise regulation. For example, in the upstream of mitochondria, there are both pro-apoptotic and anti-apoptotic Bcl-2 family proteins that dictate mitochondrial cytochrome c release (6 -8). In the downstream of mitochondria, inhibitor of apoptosis proteins negatively regulate apoptosis by inhibiting caspase activity (12-15). The function of inhibitor of apoptosis proteins can be antagonized by Smac/Diablo (16,17) and Omi/ Htr2A (18,19), which are also released from mitochondria during apoptosis. Further, nucleotide binding/exchanging of Apaf-1, a key biochemical event of the pathway, is also closely regulated. Recently, we found that the Apaf-1-dependent caspase activation can be enha...

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