Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (

Mitochondrial proteins function as essential regulators in apoptosis. Here, we show that mitochondrial adenylate kinase 2 (AK2) mediates mitochondrial apoptosis through the formation of an AK2-FADD-caspase-10 (AFAC10) complex. Downregulation of AK2 attenuates etoposide- or staurosporine-induced apoptosis in human cells, but not that induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL). During intrinsic apoptosis, AK2 translocates to the cytoplasm, whereas this event is diminished in Apaf-1 knockdown cells and prevented by Bcl-2 or Bcl-X(L). Addition of purified AK2 protein to cell extracts first induces activation of caspase-10 via FADD and subsequently caspase-3 activation, but does not affect caspase-8. AFAC10 complexes are detected in cells undergoing intrinsic cell death and AK2 promotes the association of caspase-10 with FADD. In contrast, AFAC10 complexes are not detected in several etoposide-resistant human tumour cell lines. Taken together, these results suggest that, acting in concert with FADD and caspase-10, AK2 mediates a novel intrinsic apoptotic pathway that may be involved in tumorigenesis.

O steopontin (OPN) is a secreted glycosylated phosphoprotein that is involved in a number of physiological events including bone formation and remodeling (1), immune responses (2, 3), and tumor progression, such as cell proliferation, angiogenesis, metastasis, and anti-apoptosis (4). Especially, OPN is highly up-regulated in cancer patients' plasma, thus it is considered a candidate as a prognostic marker for human cancer diagnosis (4). Multiple cancer-related functions of OPN are mediated by its interaction with integrins or CD44 variants as a cytokine. Generally, secreted OPN acts as an intact protein or fragments cleaved by thrombin; Arg-Gly-Asp (RGD) motif in OPN interacts with integrins (␣ v ␤ 3 , ␣ v ␤ 5 ) and C-terminal region of OPN binds to CD44 variants, which subsequently activates a PI3K-AKT, NIK, or MEKK1 kinase cascade (4, 5). In addition, alternative isoform of OPN is found in cytosol (6) and OPN is detected as a CD44-ERM complex in the cytosolic side of CD44 (7). Further, OPN also associates with polo-like kinase-1 in the nucleus during cell cycle (8). These observations show diverse roles and subcellular localizations of OPN.OPN is also highly induced during hypoxia/reoxygenation (Hyp/ RO), which is closely related to pathological conditions including myocardial ischemia/reperfusion injury, stroke, inflammation, and solid tumors (9, 10). During Hyp/RO, cell death generally occurs after massive generation of reactive oxygen species (ROS) and caspases activation. Several caspases including caspases-8, -9, and -3 were reported to be activated during reoxygenation, which is required for Hyp/RO-induced cell death (11,12). Among these caspases, caspase-8 is a well known receptor-proximal caspase. However, accumulating evidence suggests atypical roles of caspase-8 in nonreceptor-mediated cell deaths (13, 14) and . In addition, caspase-8 deficiency is also detected in human cancers (16,17) and facilitates cellular transfomation (18), showing critical functions of caspase-8 in tumorigenesis and cell death. In the group of hundreds' cellular substrates of various caspases, only a few proteins, such as Bid, p28 Bap31, RIP-1, and plectin, are reported as caspase-8 substrates (19)(20)(21)(22).In this study, we performed genome-wide screening and isolated OPN as a caspase-8 substrate. OPN expression is rapidly increased during Hyp/RO and subsequently cleaved by caspase-8, leading to both inactivation of AKT survival signal and activation of cell death signal via its caspase cleavage fragment in tumor cells. Results OPN Is Cleaved by Caspase-8 in Vitro and in Apoptotic Cells DuringHyp/RO. To unearth caspase substrates, we undertook caspase substrate screening using human cDNA library. Small cDNA pools were transcribed and translated in vitro in the presence of [35 S]methionine and then incubated with recombinant caspases (23). From this analysis, we isolated OPN as a putative substrate of caspase-8. To characterize the cleavage, in vitro translated OPN was incubated with various recombinant active caspases (ca...

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