GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Zhang, Bicheng; Huang, Jun; Li, Hong-Liang; Liu, Ting; Wang, Yan-Yi; Waterman, Paul; Mao, Ai-**; Xu, Liang-Guo; Zhai, Zhonghe; Liu, Depei; Marrack, Philippa; Shu, Hong-Bing

doi:10.1038/cr.2008.75

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

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Published: 01 July 2008

Volume 18, pages 900–910, (2008)
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GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

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Bicheng Zhang¹^na1,
Jun Huang²^na1,
Hong-Liang Li³^na1,
Ting Liu²,
Yan-Yi Wang⁴,
Paul Waterman¹,
Ai-** Mao⁴,
Liang-Guo Xu¹,
Zhonghe Zhai²,
Depei Liu³^na2,
Philippa Marrack¹^na2 &
…
Hong-Bing Shu⁴^na2

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Abstract

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the IAP family members and is required for GIDE's E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE-induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over- or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.

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Introduction

Apoptosis is required for normal development in all metazoans. Abnormal regulation of apoptosis leads to various diseases, such as cancer and autoimmune and neurodegenerative diseases. Apoptosis is mediated by activation of caspases, which cleave various cellular substrates and lead to characteristic apoptotic morphological changes ^{1, 2, 3}. Caspases are synthesized as cytoplasmic zymogen precursors and are activated in a hierarchical pattern in which downstream caspases are activated by upstream caspases ^{1, 2, 3}. The upstream caspases are activated through distinct mechanisms. For example, it has been proposed that the death receptor associated caspase-8 is activated by so-called “induced proximity” ⁴. In most cases, however, activation of the initial caspases involves mitochondria. Upon stimulation by various death signals, the outer membrane of the mitochondrion is permeabilized, resulting in the release of molecules, including cytochrome c, Smac/DIABLO, Omi/HtrA and GSPT1/eRF3, to the cytosol. Released cytochrome c causes cell death by binding to Apaf-1, an event that leads to recruitment and activation of pro-caspase-9 ⁵. Unlike cytochrome c, mitochondrial-released Smac/DIABLO, Omi/HtrA2 and GSPT1/eRF3 induce caspase activation and apoptosis through their interactions with proteins of the IAP family ^{6, 7, 8}.

IAP proteins are universally present in organisms from yeast to humans. All IAPs contain at least one baculovirus IAP repeat (BIR) domain and most also contain a C-terminal RING finger domain, which has E3 ubiquitin ligase activity. Although some IAP proteins have other activities, most IAPs inhibit apoptosis ⁹. Among the human IAPs, XIAP is the most extensively studied and the most potent inhibitor of apoptosis. XIAP binds to active caspase-9 with high affinity, and with lower affinity to caspase-3 and -7. Binding of XIAP to active caspases prevents their cleavage of cellular substrates, and thus inhibits apoptosis ¹⁰. XIAP may also target active caspase-3 for ubiquitination and degradation by the proteasome, thereby inhibiting apoptosis ¹¹. Binding of mitochondrial-released Smac/Diablo, Omi/HtrA2 and GSPT1/eRF3 to IAPs causes release of active caspases from inhibitory IAPs and/or inhibition of ubiquitination and degradation of caspases, and therefore promotes apoptosis ⁹. Recently, it has been shown that XIAP and the baculovirus Op-IAP can ubiquitinate Smac/Diablo and target it for degradation by the proteasome, suggesting that IAPs can also inhibit apoptosis through degradation of their antagonists, rather than by directly inhibiting caspases ¹². Whatever the mechanisms, the balance between mitochondrial-released death factors and cytosolic IAPs appears to be critically involved in the modulation of apoptosis.

Many extrinsic and intrinsic signals can trigger apoptosis. For example, JNK, a mitogen-activated protein kinase (MAPK) that is activated by stimuli that cause cell stress, such as DNA damaging reagents, UV light and proinflammatory cytokines, is pro-apoptotic under some circumstances in some cell types ^{13, 14}. In Drosophila, the JNK ortholog, DJNK, is required for apoptosis during embryonic patterning of the wing, eye and gut ¹⁵. DJNK phosphorylates DJun, which promotes transcription of Hid and Rpr, two proteins that bind to the Drosophila IAP ortholog DIAP1 and prevent DIAP1 from inhibiting the Drosophila caspase, DRONC, thus triggering apoptosis ¹⁶.

In mammals, there is evidence for both pro-apoptotic and anti-apoptotic roles of JNK. JNK activity is required for apoptosis of PC12 neuronal cells induced by NGF withdrawal, of MEFs in response to many stimuli and of thymocytes in response to T cell receptor ligation ^{Full size image}

Discussion

Here we report the identification of GIDE, an E3 ubiquitin ligase that, when overexpressed, induces apoptosis through novel caspase-9 and JNK activation-dependent pathways. When less overexpressed, GIDE slows cell growth rates and, when underexpressed, accelerates the cell cycle. This phenomenon may explain why, despite the fact that we have chimeric founders generated from GIDE^+/− ES cells, we have been unable to obtain heterozygous progeny from these mice (data not shown). The aberrant growth rate caused by heterozygous deletion of GIDE may be lethal at an early stage of embryonic development.

GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the IAP family members (Figure 1). The RING finger domain of GIDE is required for its E3 ligase activity and auto-ubiquitination (Figure 2). Aside from the RING finger domain, GIDE shows no conservation with known proteins, suggesting that GIDE may function through novel mechanisms. GIDE contains two transmembrane regions and localizes in the mitochondria. Overexpression of GIDE activates JNK, an event that precedes release of cytochrome c and Smac as well as apoptosis. These phenomena require GIDE's E3 ligase domain. In addition, caspase inhibitors crmA and z-VAD-fmk, as well as XIAP and an inactive mutant of caspase-9, block GIDE-induced apoptosis (Figure 4). These results suggest that GIDE induces caspase-dependent apoptotic pathways. Thus, our findings suggest that the E3 ubiquitin ligase GIDE induces apoptosis through activation of the protein kinase JNK and subsequent activation of caspases. The processes by which GIDE slows cell growth are currently unknown.

Other than itself, the physiological substrates for GIDE are unknown. We examined whether GIDE could ubiquitinate several candidate proteins, including XIAP; Survivin, an IAP family member that is highly expressed in cancer cells but not in normal cells and has an anti-apoptotic role; cFLIP, an anti-apoptotic protein that is also a substrate of the E3 ligase Itch and TAK1, a kinase-activating JNK. The results indicated that none of these proteins was a GIDE substrate (data not shown).

In co-immunoprecipitation experiments, GIDE interacted with the MAP3K family member TAK1 (Figure 5F). A kinase-inactive mutant of TAK1, but not mutants of MEKK1, MEKK3, ASK1 and TBK1, inhibited GIDE-induced JNK activation (Figure 5D). These results suggest that TAK1 is specifically involved in GIDE-induced JNK activation. GIDE-induced TAK1 and JNK activation requires GIDE's C-terminal RING finger domain, which confers its autoubiquitination activity (Figure 5). Although the exact mechanisms are unclear, GIDE-mediated TAK1 activation may be similar to the activation of TAK1 by TRAF6. TRAF6 is an E3 ubiquitin ligase that is critically involved in IL-1- and Toll-like receptor-induced NF-κB and JNK activation pathways ²⁵. In these pathways, TRAF6, which signals through TAK1 and the auto-ubiquitination of TRAF6, is sufficient to trigger the activation of TAK1 ²⁵. XIAP also interacts with TAK1, and may activate TAK1 through a similar mechanism.

JNK may promote apoptosis by engaging the mitochondrial-dependent intrinsic pathway. It has been shown that UV-induced Bax activation and cytochrome c release are absent in JNK1/JNK2-deficient MEFs, and Bax and Bak double-deficient cells are resistant to apoptosis induced by UV and JNK activation ^{26, 27, 28}. It has also been shown that JNK-dependent phosphorylation of the BH3 proteins Bim and Bmf causes their dissociation from dynein and myosin motor complexes, and, subsequently, these dissociated proteins induce apoptosis through Bax and Bak ²⁸. Recently, it has been shown that JNK activation leads to phosphorylation of 14-3-3 proteins, which causes dissociation of 14-3-3 proteins with Bax. The released Bax is translocated to mitochondria, where it triggers the intrinsic apoptotic pathway ²⁹. Moreover, in RelA-deficient MEFs or human HeLa cells expressing an IκBα mutant that blocks NF-κB activation, activation of JNK causes processing of Bid into tBid by an unknown mechanism. The processed tBid is translocated to the mitochondria, where it selectively triggers release of Smac, but not of cytochrome c. The released Smac causes activation of caspase-8 and promotes apoptosis ¹³. Based on these previous studies, it is possible that GIDE-induced apoptosis is also mediated by the 14-3-3 and Bcl2 family members. However, when overexpressed in Bax and Bak double knockout fibroblasts, GIDE could still induce apoptosis (data not shown), which is consistent with the observation that Bcl-XL and 14-3-3 could not block GIDE-induced apoptosis (Figure 3). In addition, although XIAP could block GIDE-induced apoptosis, GIDE could not ubiquitinate XIAP or cause XIAP degradation (data not shown), indicating that XIAP is not a direct target of GIDE. Thus, our findings suggest that GIDE induces a novel, non-classic apoptotic pathway.

Northern blot and western blot analysis suggests that GIDE is expressed in most normal human tissues at relatively high levels (Figure 2). However, we failed to detect GIDE expression in eight examined cancer cell lines, including five lung-derived (H549, H441, H290, H157, and 16HBE), one each of liver- (Hep3B), colon- (H1299) and kidney- (293) derived cancer cell lines (data not shown). Together with our observations that GIDE induces apoptosis or inhibits cell growth when ectopically expressed in immortal cell lines, these observations suggest the possibility that GIDE is a tumor suppressor gene that is down-regulated in cancer cells.

Materials and Methods

Reagents

Antibodies against Flag and HA epitopes (Sigma); ubiquitin, PARP, cyclin A, cyclin D1, cyclin E, CDK2,4 and β-actin (Santa Cruz Biotechnology); cytochrome c, Smac, caspase-7,8,9 and p53β (Cell Signaling Technology); Cox4 (Molecular Probes); z-VAD-fmk (R&D systems); Rh-123, SP600125 and SB203580 (Sigma); and human embryonic kidney 293 cells (ATCC, Manassas, VA) were purchased from the indicated manufacturers. NIH3T3 Tet-on cell line was provided by Dr James Hagman (National Jewish Medical and Research Center).

Constructs

Mammalian expression plasmids for IκBα(SS/AA), FADD-DN, crmA, TAK1(K/W), TBK1(K/W), ASK1(K/W), MEKK1(K/W), MEKK3, MEKK3(K/W), HA-TAK1, HA-TAB1, cIAP1, p53-DN, Bcl-XL, Caspase 8(c360s) and Caspase 9(c276s) were described previously ^{30, 31, 32}. Mammalian expression plasmids for HA- or FLAG-tagged GIDE, GIDEΔR, GIDE(H319A), XIAP, 14-3-3σ-DN and Caspase-9(c276s) were constructed by PCR amplification of the corresponding cDNA fragments and subsequently cloned into a CMV promoter-based vector containing an N-terminal HA or FLAG tag.

Northern blot

Human multiple tissue mRNA blots were purchased from Clontech (Palo Alto, CA). The blots were hybridized with ³²P-labeled GIDE cDNA in Rapid Hybridization Buffer (Clontech, Palo Alto, CA) under high-stringency conditions.

Transfection, co-immunoprecipitation, reporter gene, immunofluorescence, in vitro kinase, apoptosis assays and production of heterozygous ES cells

The methods used were as described previously ^{30, 31, 32}.

DNA fragmentation assays

Transfected cells were washed twice with cold PBS and lysed with 2% NP-40 containing 0.2 mg/ml Proteinase K. DNA in the lysate was precipitated with 2 volumes of ethanol. The pellets were dissolved in H₂O and analyzed by electrophoresis in a 2% gel.

Mitochondrial membrane potential assays

293 cells (∼2 × 10⁵) were transfected with 1 μg of expression plasmids for the indicated time. The transfected cells were harvested and washed with PBS(+) three times and stained with Rh123 (10 μg/ml) at 37 °C for 30 min, then washed three times with PBS (+). The mitochondrial membrane potential was analyzed by flow cytometry.

Induced expression of GIDE in NIH3T3 tet-on cells

NIH3T3 tet-on cells were transfected with pTRE-HA-GIDE (or pTRE) and pTK-Hyg in the ratio 10:1 by Lipofectamine 2000 following the manufacturer's instructions (Invitrogen, Carlsbad, CA). One day after transfection, cells were selected with hygromycin (500 μg/ml) for 15 days. Individual clones were induced with doxcycline (1 μg/ml), and induction of GIDE expression was confirmed by western blot.

Growth curves

The growth rate of stable clones of transfected NIH3T3 tet-on cells was measured by counting the number of cells. Cells were subcultured into six-well plates at a density of 1.5 × 10⁴ cells/well in medium with or without doxcycline (1 μg/ml). The medium was replaced with fresh medium every 2 days. The cells were trypsinized and counted every 24 h for 5 days. Similar assays were performed for GIDE^+/+ and GIDE^+/− ES cells, except that doxycyclin was not added, and the cells were grown on irradiated feeder cells.

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Acknowledgements

The authors thank Saiphone Webb for her help in production and analysis of the GIDE^+/− ES cells. This work was supported by Hi-Tech Research and Development Program of China (863 program, Grant No. 2006AA02A306), the Chinese 111 project (Grant No. B06018), and National Institutes of Health (Grant No. CA108771).

Author information

Bicheng Zhang, Jun Huang and Hong-Liang Li: These three authors contributed equally to this work.
Depei Liu, Philippa Marrack and Hong-Bing Shu: These three authors are co-corresponding authors.

Authors and Affiliations

HHMI, National Jewish Medical and Research Center, Denver, 80206, CO, USA
Bicheng Zhang, Paul Waterman, Liang-Guo Xu & Philippa Marrack
College of Life Sciences, Peking University, Bei**g, 100871, China
Jun Huang, Ting Liu & Zhonghe Zhai
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Bei**g, 100005, China
Hong-Liang Li & Depei Liu
College of Life Sciences, Wuhan University, Wuhan, 430072, China
Yan-Yi Wang, Ai-** Mao & Hong-Bing Shu

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Bicheng Zhang
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Jun Huang
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Hong-Liang Li
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Ting Liu
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Yan-Yi Wang
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Paul Waterman
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Ai-** Mao
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Liang-Guo Xu
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Zhonghe Zhai
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Depei Liu
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Philippa Marrack
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Hong-Bing Shu
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Correspondence to Hong-Bing Shu.

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Zhang, B., Huang, J., Li, HL. et al. GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth. Cell Res 18, 900–910 (2008). https://doi.org/10.1038/cr.2008.75

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Received: 22 April 2008
Revised: 24 April 2008
Accepted: 24 April 2008
Published: 01 July 2008
Issue Date: September 2008
DOI: https://doi.org/10.1038/cr.2008.75
Springer Nature Singapore Pte Ltd.

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Abstract

Similar content being viewed by others

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly

Introduction

Discussion

Materials and Methods

Reagents

Constructs

Northern blot

Transfection, co-immunoprecipitation, reporter gene, immunofluorescence, in vitro kinase, apoptosis assays and production of heterozygous ES cells

DNA fragmentation assays

Mitochondrial membrane potential assays

Induced expression of GIDE in NIH3T3 tet-on cells

Growth curves

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding author

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About this article

Cite this article

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GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Abstract

Similar content being viewed by others

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly

Introduction

Discussion

Materials and Methods

Reagents

Constructs

Northern blot

Transfection, co-immunoprecipitation, reporter gene, immunofluorescence, in vitro kinase, apoptosis assays and production of heterozygous ES cells

DNA fragmentation assays

Mitochondrial membrane potential assays

Induced expression of GIDE in NIH3T3 tet-on cells

Growth curves

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding author

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About this article

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