Protease activity of procaspase-8 is essential for cell survival by inhibiting both apoptotic and nonapoptotic cell death dependent on receptor-interacting protein kinase 1 (RIP1) and RIP3.

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.

Identification of mechanism that couples multisite phosphorylation of Yes-associated protein (YAP) with transcriptional coactivation and regulation of apoptosis.

The transcriptional coactivator Yes-associated protein (YAP) has been implicated in tumorigenesis by regulating cell proliferation and apoptosis. YAP interacts with the transcription factor TEAD and is essential in mediating TEAD-dependent gene expression. Here we show that YAP is hyperphosphorylated and activated in response to genotoxic stress such as UV irradiation and cisplatin treatment. Using high resolution mobility shift assay for phosphorylated proteins, we identified multiple sites of phosphorylation induced by UV irradiation. Pretreatment with p38 and JNK inhibitors completely suppressed the mobility retardation of phosphorylated YAP in UV-irradiated cells. Co-immunoprecipitation experiments showed that the physical interaction of YAP with TEAD was markedly enhanced by UV irradiation or CDDP treatment but suppressed by pretreatment with p38 and JNK inhibitors. Similarly, pretreatment with p38 and JNK inhibitors suppressed the expression of YAP/TEAD target genes, which were elevated on exposure to genotoxic stress. Using phosphomimetic and phosphorylation-deficient YAP mutants, we showed that the coactivator activity of YAP correlated with its state of phosphorylation and sensitivity to cisplatin-induced apoptosis. Our results demonstrate that multisite phosphorylation of YAP induces YAP/TEAD-dependent gene expression and provides a mechanism by which YAP regulates apoptosis differently depending on cellular context.

Role of the tumor suppressor RASSF1A in Mst1-mediated apoptosis.

Mammalian sterile 20-like kinase 1 (Mst1) is activated by both caspase-mediated cleavage and phosphorylation in response to apoptotic stimuli, including Fas ligation. Here, we examined the possible role of the tumor suppressor RASSF1A in Mst1 activation and Mst1-mediated apoptosis induced by death receptor signaling. Immunoprecipitation and immunofluorescence analyses revealed that Mst1 was associated with RASSF1A in cultured mammalian cells, with both proteins colocalizing to microtubules throughout the cell cycle. Whereas purified recombinant RASSF1A inhibited the kinase activity of purified recombinant Mst1 in vitro, overexpression of RASSF1A increased the kinase activity of Mst1 in intact cells, suggesting that regulation of Mst1 by RASSF1A in vivo involves more than the simple association of the two proteins. Both the activation of Mst1 and the incidence of apoptosis induced by Fas ligation were markedly reduced in cells depleted of RASSF1A by RNA interference and were increased by restoration of RASSF1A expression in RASSF1A-deficient cells. Moreover, the stimulatory effect of RASSF1A overexpression on Fas-induced apoptosis was inhibited by depletion of Mst1. These findings indicate that RASSF1A facilitates Mst1 activation and thereby promotes apoptosis induced by death receptor signaling.

Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) inhibits Fas-induced apoptosis of neutrophils. However, the exact step in the apoptotic pathway blocked by GM-CSF remained unclear. Here, we found that pretreatment of neutrophils with GM-CSF inhibits the recruitment of Fas-associated protein with death domain (FADD) to Fas, abolishing the formation of the death-inducing signaling complex required for Fas-induced apoptosis. Two-dimensional electrophoresis revealed that GM-CSF modifies the ratio of FADD subspecies. These GM-CSF-triggered changes were abrogated, and Fas-induced apoptosis was restored by an inhibitor of classical protein kinase C (PKC), Go6976, and by the combination of a phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, and an inhibitor of mitogen-activated protein kinase kinase (MEK)1, PD98059. Go6976 blocked GM-CSF-elicited phosphorylation of Akt/PKB and extracellular signal-regulated kinase (ERK)1/2. These results indicated that GM-CSF suppresses Fas-induced neutrophil apoptosis by inhibiting FADD binding to Fas, through redundant actions of PI-3K and MEK1-ERK1/2 pathways downstream of classical PKC.

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes.

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.

Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).

Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.