Pro-apoptotic activity and mono-/diubiquitylation of Xenopus Bid in egg extracts

Saitoh, Takashi; Tsuchiya, Yosuke; Kinoshita, Toshihiko; Itoh, Masamichi; Yamashita, Shigeru

doi:10.1016/j.bbrc.2009.05.004

Cited by 4 publications

(25 citation statements)

References 26 publications

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“…Cytostatic factor (CSF) 2 -arrested metaphase egg extracts were prepared as described previously (17,33,36,39). Interphase egg extracts were prepared by the addition of 0.4 mM CaCl 2 and 0.1 mg/ml cycloheximide.…”

Section: Rt-pcr and Cdna Cloning Of Xenopus Bcl-2 Family Proteins-mentioning

confidence: 99%

“…We have been studying the functions and contributions of apoptosis regulators present in Xenopus egg extracts (17,33,36,39). We recently reported the pro-apoptotic activity of endogenous Xenopus Bid (xBid), a BH3-only member of the Bcl-2 family of proteins, in egg extracts (17). To further extend this finding, here we systematically examined the mRNA expression profiles and molecular functions of 13 Xenopus Bcl-2 family proteins.…”

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confidence: 99%

“…They are classified as either anti-apoptotic or pro-apoptotic, and the functional balance between these two groups controls the mitochondria-mediated intrinsic pathway of apoptosis. Although more than 10 members of the Bcl-2 family of proteins have been identified in Xenopus laevis (simply referred to as Xenopus hereafter), their functional characterizations are not yet complete (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”

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confidence: 99%

“…20 and 21). Egg extracts, as well as the mitochondria isolated from them, are frequently used for biochemical experiments of apoptosis (17,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Apoptosis in egg extracts is triggered solely by the cytoplasmic release of cytochrome c from mitochondria, and neither damage-inducing treatment nor death receptor-ligand signaling is required.…”

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confidence: 99%

“…For this reason, endogenous Bcl-2 family proteins present in Xenopus egg extracts or mitochondria are still poorly characterized. We have been studying the functions and contributions of apoptosis regulators present in Xenopus egg extracts (17,33,36,39). We recently reported the pro-apoptotic activity of endogenous Xenopus Bid (xBid), a BH3-only member of the Bcl-2 family of proteins, in egg extracts (17).…”

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confidence: 99%

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Anti-apoptotic Activity and Proteasome-mediated Degradation of Xenopus Mcl-1 Protein in Egg Extracts

Tsuchiya

Yamashita

2011

Journal of Biological Chemistry

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Xenopus egg extracts execute spontaneous apoptosis without the requirement of transcription and translation, and this intrinsic mechanism is supposed to be involved in the physiological elimination of aged eggs. Although apoptosis in this system is carried out by maternally stockpiled materials, the endogenous apoptosis regulators present in egg extracts are still poorly characterized. Here we examined the mRNA expression profiles and apoptosis-regulating functions of 13 Xenopus Bcl-2 family proteins in egg extracts. Among these, we found that endogenous Xenopus Mcl-1 (xMcl-1) physiologically inhibited apoptosis by counteracting the pro-apoptotic activity of endogenous Xenopus Bid in egg extracts. Exogenously added recombinant xMcl-1 was rapidly degraded by proteasome in egg extracts, and we identified the destabilizing region in the N terminus of xMcl-1. Our results suggest that the proteolytic decay of xMcl-1 may change the functional balance between pro-and anti-apoptotic activities of Bcl-2 family proteins, thereby regulating the timing of cytochrome c release in egg extracts.

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Section: Rt-pcr and Cdna Cloning Of Xenopus Bcl-2 Family Proteins-mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Anti-apoptotic Activity and Proteasome-mediated Degradation of Xenopus Mcl-1 Protein in Egg Extracts

Tsuchiya

Yamashita

2011

Journal of Biological Chemistry

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Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid

Yue

Messaoud

López

2015

Journal of Biological Chemistry

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Hyperosmotic shock induces early calpain activation, Smac/ DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-x L markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-x L co-expression. In these conditions a significant amount of Bid in the cytosol is mono-and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes (1). Recently, we have shown that hyperosmotic shock also induces rapid calpain activation and Smac/DIABLO (second mitochondrion-derived activator of caspases/direct IAP-binding protein with low PI Smac/DIABLO) release from the mitochondria, as well as p38 and JNK (c-Jun N-terminal kinase) activation. These four pathways, induced early by osmostress, converge on the mitochondria to trigger late cytochrome c release and caspase-3 activation (2). Moreover, we have found that caspase-3 activation induces rapid phosphorylation of p38, thus creating a positive feedback loop in osmostress-induced apoptosis (3). However, the role of Bcl-2 family members in osmostress-induced apoptosis was not addressed in previous studies. It is not clear which specific p38 and JNK isoforms are activated by osmostress and how they regulate the apoptotic program. Bid is a member of the BH3 2 -only proteins that plays a crucial role in regulating the permeability of the outer mitochondrial membrane. The BH3 region is required for interaction with both pro-apoptotic Bax or anti-apoptotic protein Bcl-x L (4). Bid also contains a large unstructured loop (amino acids 42-79) with a variety of sites that are subjected to post-translational modifications, regulating Bid localization and apoptotic function (5).Bid is a caspase-8 substrate, and the resulting tBid translocates to mitochondria and initiates mitochondrial protein release. The cleavage site of caspase-8 in human Bid is Asp-60 (6) and in Xenopus laevis is Asp-52 (7). Xenopus Bid can also be proteolyzed by caspase-10␤ (7). In addition, it has been reported that caspase-3 can cleave human Bid at Asp-60 (8), and cleavage sites for non-caspase proteases have been detected, such as Gly...

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Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts

2015

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When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G 1 or G 2 in a checkpoint-dependent manner, either to repair the damage or to die by apoptosis. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest, and it is not clear whether apoptosis in early embryonic cells occurs at a specific cell cycle stage or at random points. Here, we examined the functional molecular link between the embryonic cell cycle and apoptosis using Xenopus egg extracts. When apoptosis was induced in egg extracts by addition of exogenous cytochrome c during cellcycle progression, cyclin B accumulation was inhibited, Cdc2 was not activated, and the cell cycle arrested at interphase. However, addition of recombinant cyclin B failed to activate Cdc2 due to the strong inhibitory phosphorylation of Cdc2 Tyr15 in apoptotic egg extracts. We found that endogenous Cdc25C, which activates the Cdc2-cyclin B complex by dephosphorylating Cdc2 Tyr15, was inactivated by caspase-mediated cleavage at two sites in the N-terminal regulatory domain. When the hyperactive Cdc25A catalytic fragment was added together with recombinant cyclin B to artificially dephosphorylate Cdc2 Tyr15, M-phase induction was restored in apoptotic egg extracts, indicating that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibited Cdc2 activation. Apoptosis induction in cytostatic factor-arrested metaphase egg extracts resulted in inactivation of Cdc2 without cyclin B degradation. These results suggest that apoptotic inactivation of Cdc25C plays an important role in arresting the embryonic cell cycle at interphase during apoptosis.

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Pro-apoptotic activity and mono-/diubiquitylation of Xenopus Bid in egg extracts

Cited by 4 publications

References 26 publications

Anti-apoptotic Activity and Proteasome-mediated Degradation of Xenopus Mcl-1 Protein in Egg Extracts

Anti-apoptotic Activity and Proteasome-mediated Degradation of Xenopus Mcl-1 Protein in Egg Extracts

Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid

Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts

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