CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.     

Blocking Cytochrome c Activity within Intact Neurons Inhibits Apoptosis

Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal–induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal–induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.     

The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event

During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.     

Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis

During apoptosis, a key event is the release of Smac/DIABLO (an inhibitor of XIAP) and cytochrome c (Cyt-c, an activator of caspase-9) from mitochondria to cytosol. It was not clear, however, whether the releasing mechanisms of these two proteins are the same. Using a combination of single living-cell analysis and immunostaining techniques, we investigated the dynamic process of Smac and Cyt-c release during UV-induced apoptosis in HeLa cells. We found that YFP-labeled Smac and GFP-labeled Cyt-c were released from mitochondria in the same time window, which coincided with the mitochondrial membrane potential depolarization. Furthermore, using immunostaining, we found that the endogenous Smac and Cyt-c were always released together within an individual cell. Finally, when cells were pre-treated with caspase inhibitor (z-VAD-fmk) to block caspase activation, the process of Smac release, like that of Cyt-c, was not affected. This was true for both YFP-labeled Smac and endogenous Smac. These results suggest that in HeLa cells, both Smac and Cyt-c are released from mitochondria during UV-induced apoptosis through the same permeability transition mechanism, which we believe is triggered by the aggregation of Bax in the outer mitochondrial membrane to form lipid-protein complex.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants

Bax, a proapoptotic member of the Bcl-2 family of proteins, resides in the cytosol and translocates to the mitochondrial membrane upon induction of apoptosis. It has been proposed that Bax does not translocate to mitochondria under normal physiological conditions, due to interaction between amino (ART) and carboxy (TM) terminal domains. Here, we report the physiological consequences of introducing a matrix targeting mitochondrial signal sequence (Su9) at the amino terminus of Bax and its mutants lacking ART, TM, or both segments. In vitro mitochondrial protein import assays of the fusion proteins suggests localization to the mitochondrial matrix. When expressed in Cos-1 cells, Su9 could target Bax to mitochondria in the absence of an apoptotic stimulus. However, mitochondrial localization did not result in apoptosis. When ART, TM, or both segments of Bax were deleted, expression of fusion proteins containing Su9 resulted in apoptosis via cytochrome c release. Cell death was inhibited by the pan-caspase inhibitor zVAD-fmk. We thus demonstrate that an effective mitochondrial matrix targeting signal can override the inhibition of import of Bax to the organelle, presumed to arise as a result of interaction between ART and TM segments, in the absence of apoptotic stimulus. We also demonstrate the ability of truncated variants of Bax to cause apoptosis when targeted to mitochondria by cytochrome c release from an ectopic environment.     

Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.     

Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency

Blockade of ionotropic glutamate receptors induces neuronal cell apoptosis. We investigated if mitochondria-mediated death signals would contribute to neuronal apoptosis following administration of glutamate antagonists. The administration of MK-801 and CNQX (MK-801/CNQX), the selective antagonists of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, produced widespread neuronal death in neonatal rat brain and cortical cell cultures. MK-801/CNQX-induced neuronal apoptosis was prevented by zVAD-fmk, a broad inhibitor of caspases, but insensitive to inhibitors of calpain or cathepsin D. Activation of caspase-3 was observed within 6-12 h and sustained over 36 h after exposure to MK-801/CNQX, which cleaved PHF-1 tau, the substrate for caspase-3. Activation of caspase-3 was blocked by high K+ and mimicked by BAPTA-AM, a selective Ca2+ chelator. Reducing extracellular Ca2+, but not Na+, activated caspase-3, suggesting an essential role of Ca2+ deficiency in MK-801/CNQX-induced activation of caspases. Cortical neurons treated with MK-801/CNQX triggered activation of caspase-9, release of cytochrome c from mitochondria, and translocation of Bax into mitochondria. The present study suggests that blockade of ionotropic glutamate receptors causes caspase-3-mediated neuronal apoptosis due to Ca2+ deficiency that is coupled to the sequential mitochondrial death pathway.