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GIDE Is a Mitochondrial E3 Ubiquitin Ligase That Induces Apoptosis and Slows Growth

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GIDE Is a Mitochondrial E3 Ubiquitin Ligase That Induces Apoptosis and Slows Growth npg GIDE slows growth and induces apoptosis 900 Cell Research (2008) 18:900-910. © 2008 IBCB, SIBS, CAS All rights reserved 1001-0602/08 $ 30.00 npg ORIGINAL ARTICLE www.nature.com/cr GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth Bicheng Zhang1, *, Jun Huang2, *, Hong-Liang Li3, *, Ting Liu2, Yan-Yi Wang4, Paul Waterman1, Ai-Ping Mao4, Liang-Guo Xu1, Zhonghe Zhai2, Depei Liu3, §, Philippa Marrack1, §, Hong-Bing Shu4, § 1HHMI, National Jewish Medical and Research Center, Denver, CO 80206, USA; 2College of Life Sciences, Peking University, Bei- jing 100871, China; 3Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China; 4College of Life Sciences, Wuhan University, Wuhan 430072, China 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 cas- pases, 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. Keywords: GIDE, apoptosis, E3 ligase, mitochondria, caspase, JNK Cell Research (2008) 18:900-910. doi: 10.1038/cr.2008.75; published online 1 July 2008 Introduction In most cases, however, activation of the initial caspases involves mitochondria. Upon stimulation by various Apoptosis is required for normal development in all death signals, the outer membrane of the mitochondrion metazoans. Abnormal regulation of apoptosis leads to is permeabilized, resulting in the release of molecules, various diseases, such as cancer and autoimmune and including cytochrome c, Smac/DIABLO, Omi/HtrA neurodegenerative diseases. Apoptosis is mediated by and GSPT1/eRF3, to the cytosol. Released cytochrome c activation of caspases, which cleave various cellular sub- causes cell death by binding to Apaf-1, an event that leads strates and lead to characteristic apoptotic morphological to recruitment and activation of pro-caspase-9 [5]. Unlike changes [1-3]. Caspases are synthesized as cytoplasmic cytochrome c, mitochondrial-released Smac/DIABLO, zymogen precursors and are activated in a hierarchi- Omi/HtrA2 and GSPT1/eRF3 induce caspase activation cal pattern in which downstream caspases are activated and apoptosis through their interactions with proteins of the by upstream caspases [1-3]. The upstream caspases are IAP family [6-8]. activated through distinct mechanisms. For example, it IAP proteins are universally present in organisms from has been proposed that the death receptor associated cas- yeast to humans. All IAPs contain at least one baculo- pase-8 is activated by so-called “induced proximity” [4]. virus 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 *These three authors contributed equally to this work. activities, most IAPs inhibit apoptosis [9]. Among the §These three authors are co-corresponding authors. human IAPs, XIAP is the most extensively studied and Correspondence: Hong-Bing Shu the most potent inhibitor of apoptosis. XIAP binds to ac- Tel/Fax: +86-27-68753780 tive caspase-9 with high affinity, and with lower affinity E-mail: [email protected] Received 22 April 2008; revised 24 April 2008; accepted 24 April 2008; to caspase-3 and -7. Binding of XIAP to active caspases published online 1 July 2008 prevents their cleavage of cellular substrates, and thus Cell Research | Vol 18 No 9 | September 2008 Bicheng Zhang et al. npg 901 inhibits apoptosis [10]. XIAP may also target active nase (MAPK) that is activated by stimuli that cause cell caspase-3 for ubiquitination and degradation by the pro- stress, such as DNA damaging reagents, UV light and teasome, thereby inhibiting apoptosis [11]. Binding of proinflammatory cytokines, is pro-apoptotic under some mitochondrial-released Smac/Diablo, Omi/HtrA2 and circumstances in some cell types [13, 14]. In Drosophila, GSPT1/eRF3 to IAPs causes release of active caspases the JNK ortholog, DJNK, is required for apoptosis dur- from inhibitory IAPs and/or inhibition of ubiquitination ing embryonic patterning of the wing, eye and gut [15]. and degradation of caspases, and therefore promotes DJNK phosphorylates DJun, which promotes transcrip- apoptosis [9]. Recently, it has been shown that XIAP and tion of Hid and Rpr, two proteins that bind to the Dro- the baculovirus Op-IAP can ubiquitinate Smac/Diablo sophila IAP ortholog DIAP1 and prevent DIAP1 from and target it for degradation by the proteasome, suggest- inhibiting the Drosophila caspase, DRONC, thus trigger- ing that IAPs can also inhibit apoptosis through degrada- ing apoptosis [16]. tion of their antagonists, rather than by directly inhibiting In mammals, there is evidence for both pro-apoptotic caspases [12]. Whatever the mechanisms, the balance be- and anti-apoptotic roles of JNK. JNK activity is required tween mitochondrial-released death factors and cytosolic for apoptosis of PC12 neuronal cells induced by NGF IAPs appears to be critically involved in the modulation withdrawal, of MEFs in response to many stimuli and of of apoptosis. thymocytes in response to T cell receptor ligation [17-19]. Many extrinsic and intrinsic signals can trigger apop- However, knockout of JNK1 and JNK2 in mice can in- tosis. For example, JNK, a mitogen-activated protein ki- crease apoptosis in the brain during embryonic develop- A TM1 hGIDE 1 45 mGIDE 1 45 hGIDE 46 90 mGIDE 46 90 hGIDE 91 135 mGIDE 91 135 hGIDE 136 180 mGIDE 136 180 hGIDE 181 225 mGIDE 181 225 TM1 hGIDE 226 270 mGIDE 226 270 hGIDE 271 315 mGIDE 271 315 hGIDE 316 352 mGIDE 316 352 B GIDE 301 343 XIAP 449 488 clAP1 569 609 clAP2 556 595 Livin 251 289 Figure 1 Sequence and structural analysis of GIDE. (A) Alignment of human and mouse GIDE amino acid sequences. The putative transmembrane regions (TM) are indicated. (B) Alignment of conserved RING-fingers of human GIDE, XIAP, c-IAP1, c-IAP2 and Livin. www.cell-research.com | Cell Research npg GIDE slows growth and induces apoptosis 902 ment [20]. Identification and tissue distribution of GIDE In this study, we identified a mitochondrial protein, Due to our interest in the anti-apoptotic functions of GIDE, that contains a RING finger domain homologous the RING finger domains of IAPs, we searched the EST to those of the IAP family members. GIDE slows the databases for novel proteins that contained C-terminal growth of cells and, when overexpressed, induces apop- RING finger domains like those of the IAPs. We thus tosis. GIDE has E3 ubiquitin ligase activity, which is identified the novel protein GIDE (for Growth Inhibition required for its ability to induce apoptosis. and Death E3 Ligase); through alignment of the available cDNA and EST sequences, we obtained an ~2.5 kb human Results GIDE cDNA sequence that encodes a 352 aa protein. Hu- man GIDE displays an approximately 90% amino acid A B Brain Heart Skeletal muscle Colon Thymus Spleen Kidney Liver Small intestine Placenta Lung Leukocyte Brain Heart Liver Lung Kidney Placenta Pancreas Ovary Testis 4.4- 250- 148- 2.4- 60- 42- 30- 1.4- C D ΔR Flag-GIDE + Flag-GIDEΔR + Vector Flag-GIDEFlag-GIDEFlag-GIDE(H319A) HA-GIDE + + IP Abs: IgG αFlag lgG αFlag IP: α-Flag -Ubiquitinated WB: αHA -HA-GIDE WB: α-Ubiquitin -Flag-GIDE α IP: α-Flag WB: -Flag -Flag-GIDEΔR WB: α-Flag E F αFlag Mito tracker Overlay – Flag-GIDE Cyt. Mito Cyt. Mito kDa Flag-GIDE 82- -Ubiquitinated 64- 49- GIDE -TOM20 Flag-GIDE -CPP32 (H319A) Figure 2 Expression, ubiquitination and localization of GIDE. (A) Northern blot analysis of human GIDE mRNA expression in various tissues. (B) Western blot analysis of human GIDE protein expression in various tissues. (C) GIDE is auto-ubiquitinat- ed. 293 cells were transfected with the indicated Flag-tagged plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody, the immunoprecipitates were analyzed by western blot with anti-ubiquitin (upper panel) and anti-Flag (lower panel) antibodies. (D) GIDE forms oligomers. 293 cells were transfected with the indicated GIDE or its mutant plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody or control mouse IgG; the immunoprecipitates were analyzed by western blot with anti-HA antibody. (E) Biochemical study of GIDE localization. 293 cells were transfected with Flag-GIDE or left un- transfected for 12 h. Mitochondrial and cytosolic fractions were isolated and analyzed by western blots with the indicated anti- bodies. (F) Immunofluorescent staining of GIDE and its mutant GIDE(H319A). HeLa cells were transfected with the indicated plasmids and stained with the indicated antibodies. Cell Research | Vol 18 No 9 | September 2008 Bicheng Zhang et al. npg 903 A GIDE B C Vector GIDE GIDEΔR (H319A) GIDE Vector 120 100 300 GIDE GIDEΔR 80 250 Vector 200 60 GIDE 150 40 Rh-123 FD(%) 100 20 50 0 Survival cell numbers 0 Time (h) 0 3 6 9 12 14 16 Time (h) 0 6 12 14 16 GIDE D Vector GIDE E Time (h) 0 6 12 16 24 kDa Time (h) 0 6 12 16 24 0 6 12 16 24 -32 Caspase-3 -Cyto.c -20 -17 -Smac -35 Caspase-7 -COX4 -20 Cytosol β -55 - -actin Caspase-8 -43 Caspase-9 -46 -Cyto.c -37 -Smac -116 PARP -85 -COX4 Mitochondria b-actin -42 5 Figure 3 GIDE induces apoptosis in 293 cells.
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