Molecular regulation of death receptor- and DNA damage-induced apoptosis Maas, C.

UvADARE (Digital Academic Repository) Molecular regulation of death receptor and DNA damageinduced apoptosis Maas, C. Link to publication Citation for published version (APA): Maas, C. (2010). Molecular regulation of death receptor and DNA damageinduced apoptosis General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvADARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) Download date: 23 Jan 2018

Chapter 3 Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its Nterminal fragment Stephen W.G.Tait, Evert de Vries, Chiel Maas, Anna M. Keller, Clive S. D Santos and Jannie Borst The Journal of Cell Biology 2007: 179(7): 14531466

42 Chapter 3 Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its aminoterminal fragment Stephen W.G. Tait 1,2, Evert de Vries 1, Chiel Maas 1, Anna M. Keller 1, Clive S. D Santos 3,4 and Jannie Borst 1,5 1 Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 2 Current address: Department of Immunology, St Jude Children s Research Hospital, Memphis, TN 38105, USA 3 Department of Biomolecular Mass Spectrometry, Utrecht University, 3584 CA Utrecht, The Netherlands 4 Current address: Proteomic Unit Bergen, University of Bergen, N5009 Norway 5 Correspondence to Jannie Borst: j.borst@nki.nl Bcl2 family member Bid is subject to autoinhibition: in absence of stimuli, its Nterminal region sequesters the proapoptotic BH3 domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We report that upon Bid cleavage, the Nterminal fragment (tbidn) is ubiquitinated and degraded, thus freeing the BH3 domain in the Cterminal fragment (tbidc). Ubiquitination of tbidn is unconventional, since acceptor sites are neither lysines nor the aminoterminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly, but not exclusively in helix 1, which is required for ubiquitination and degradation of tbidn. Rescue of tbidn from degradation blocked Bid s ability to induce mitochondrial outer membrane permeability, but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasomedependent degradation of tbidn is required to unleash the proapoptotic activity of tbidc. Introduction In mammalian cells, mitochondria activate the apoptotic execution machinery. Apoptotic stimuli induce mitochondrial outer membrane permeability and release of proapoptotic proteins such as cytochrome c (Cyt c) into the cytosol. The collective action of these molecules results in effector caspase activation and apoptotic cell death [1]. Mitochondrial permeabilization is controlled by the Bcl2 protein family, which comprises the antiapoptotic Bcl2 subfamily and the proapoptotic Bax/Bak and BH3 domainonly subfamilies [2]. Their functional activity relies on homoand heterodimerization, which proceeds by interaction of the BH3 domain α helix with a groove, formed by BH1 and BH2 domains [3]. The BH3 domainonly subfamily has many members, which specialize in responsiveness to specific apoptotic stimuli [4]. BH3only proteins require Bax or Bak to induce cell death. They induce assembly of Bax/Bak into homomultimers in the mitochondrial outer membrane [5,6]. These multimers are postulated to either form transmembrane pores themselves, or to facilitate pore formation. Inhibitory Bcl2 family members sequester BH3only and/or Bax/Bak proteins, thereby preventing their participation in proapoptotic complexes [7]. The BH3 domainonly protein Bid conveys the

Unconventional ubiquitination allows Bid activity 43 apoptotic signal from death receptors, such as TNF receptor1, TRAIL receptors and Fas/CD95, to mitochondria [8,9]. Upon ligand binding, these death receptors induce formation of a complex between the intracellular adaptor FADD and procaspase8 or 10. This results in increased local concentrations of these inducer caspases, leading to their activation 10]. Active Caspase8/10 can cleave and activate Bid, as well as effector caspases. In certain cell types, the mitochondrial route is dispensable for death receptorinduced apoptosis, whereas in others it constitutes an essential amplification loop for effector caspase activation [11]. The three dimensional organisation of fulllength Bid has been resolved by NMR [12,13]. Residues 112 and 4377 form unstructured loops. The structured portion consists of eight α helices arranged in a compact fold (Supplementary Figure 1a). Of these, helix 3 constitutes the BH3 domain, which is fixed by hydrophobic contacts with helix 1 and 8. The sites for proteolytic cleavage by Caspase8 or Granzyme B lie in the second unstructured loop (D60 and D75, respectively). In solution, N and Cterminal fragments of Bid remain associated upon cleavage by Caspase8 and the complex undergoes no apparent conformational change [12,14,15]. Cleavage generates an Nterminal glycine in truncated Cterminal Bid (tbidc) that can be myristoylated. This lipid modification facilitates targeting of tbidc to mitochondria, but is not required for it [14]. Rather, helices 4, 5, and 6 appeared the critical elements for such targeting, specifically to cardiolipinenriched domains in the mitochondrial membrane [16]. Making the BH3 domain available for interactions with the BH1/BH2 groove of other Bcl2 family members is essential for Bid's proapoptotic function. The solution structure of Bid indicates that the BH3 domain must be freed from the structural constraint that is exerted by the tbidn fragment [12,13]. Indeed, a number of biochemical studies have shown that tbidn acts as an inhibitor of tbidc [15,17,18]. Specifically Tan et al. (1999) produced compelling evidence for autoinhibition of Bid, by interactions between helix 2 and the BH3 domain. A mutant full length Bid protein in which these interactions were abrogated was as effective as tbidc in inducing apoptosis and binding to BclxL [17]. The natural occurrence of an alternative splice form of Bid that lacks the BH3 domain, but has the inhibitory Nterminal sequences, lends further support to the idea that this is an important mode of tbidc regulation [18]. We have addressed the question how Bid cleavage can release tbidc from inhibition by tbidn. We reveal that this is accomplished by unconventional ubiquitination of the tbidn fragment, followed by its proteasomal degradation. Results tbidn disappears upon its generation To follow the fate of tbidn and tbidc, Bid was Nterminally Myctagged and Cterminally fused to GFP and expressed in MCF7 breast carcinoma cells, which were examined by confocal laser scanning microscopy (CLSM). In unstimulated cells, Myc and GFP signals indicated a cytoplasmic localization of full length Myc Bid GFP (Figure 1a, panels IIII). After stimulation with TNFα for 8 h, the GFP signal was punctate and colocalized with MitoTracker, indicating translocation of tbidc GFP to mitochondria (Figure 1a, panels V, VI). Surprisingly, Myc tbidn was undetectable by CLSM (Figure 1a, panel IV). Full length Myc Bid GFP was readily detectable by both antimyc and antigfp immunoblotting in unstimulated MCF7 cells and gradually decreased in amount after stimulation with TNFα (Figure 1b). While the tbidc GFP fragment could readily be visualized at 4 and 8 h time points, the Myc tbidn fragment (predicted molecular mass 7 kda) was undetectable (Figure 1b). Similar results were obtained with antibody that recognises both N and Cterminal epitopes on Bid (data not shown). After stimulation with the death receptor ligand TRAIL, a similar picture emerged. At the 8 h time point, fulllength Myc Bid GFP levels were decreased and tbidc GFP fragment had been generated. However, no Myc tbidn was detectable (Figure 1c). Collectively,

44 Chapter 3 a Myc (Nterminus) GFP (Cterminus) Overlay + MitoTracker b Control TNF 4 h TNF 8 h 60 III Control TNF 4 h TNF 8 h Myc Bid GFP 15 m I II III control 60 36 Myc Bid GFP tbidc GFP Overlay TNF 6 8 h antigfp blot antimyc blot IV V VI actin actin (43 kd) c 60 36 Control TRAIL antigfp blot Myc Bid GFP tbidc GFP actin 60 6 Control TRAIL antimyc blot Myc Bid GFP actin (43 kd) Figure 1. tbidn disappears upon its generation. (a) MCF7 cells expressing Myc Bid GFP were stimulated with TNFα for 8 h or not (control), stained to detect mitochondria (MitoTracker) and the Nterminal Myc tag and examined by CLSM. (b,c) MCF7 cells expressing Myc Bid GFP were stimulated with TNFα or TRAIL. Equal amounts of total lysate protein were separated by SDSPAGE, blotted to nitrocellulose, probed with antibodies to the Nterminal Myc and Cterminal GFP tags and reprobed with antiactin antibody as loading control. Molecular mass (kd) of marker proteins are indicated in this and all following figures. these results suggest that the tbidn fragment that is generated by Caspase8/10 is rapidly degraded. tbidn is degraded in a proteasomedependent manner To investigate whether tbidn was degraded by the proteasome, MCF7 cells were stimulated with TNFα in presence or absence of proteasome inhibitor MG132. Stimulation with TNFα for 8 h resulted in the appearance of the endogenous tbidc fragment, but the tbidn fragment was barely detectable (Figure 2a, left panel). When cells were stimulated in presence of MG132, the amount of endogenous tbidn was clearly increased. Similar results were obtained with transfected Myc Bid GFP: the Myc tbidn fragment was only detectable when cells were stimulated in presence of MG132 (Figure 2a, right panel). To determine whether targeting of tbidn for proteasomal degradation required a proapoptotic stimulus, the tbidn fragment was expressed in cells. The degradation pathway was constitutively active, since protein synthesis inhibition with cycloheximide (CHX) for 8 h resulted in loss of HAtagged tbidn (Figure 2b). Proteasome inhibition with MG132 abrogated this loss and increased the HA tbidn pool in cells that had not been treated with CHX (Fig. 2b). HA tbidn expressing cells were pulse labelled for 2 h with 35 Smethionine and cysteine and the fate of the labelled pool was followed during a 60 min chase period. The halflife of the labelled HA tbidn pool was about 5 min (Figure 2c). Untagged tbidn was even more unstable than the HAtagged version: it was only detectable when cells had been incubated with MG132 (Figure 2d). Collectively, these results indicate that tbidn, upon its generation by Caspase8/10, is targeted for proteasomedependent degradation by a pathway that is not dependent on apoptotic stimulation. tbidn is ubiquitinated in an unconventional manner Since tbidn was targeted to the proteasome, we investigated whether it was ubiquitinated. To this end, tbidn Nterminally tagged with the HA epitope (sequence YPDVPDYA)

Unconventional ubiquitination allows Bid activity 45 a 17 14 6 b Control TNF antibid blot antiha blot TNF + MG132 Bid tbidc tbidn actin + + CHX + + MG132 actin 49 38 28 17 14 6 6 HA tbidn (7 kd) Control TNF TNF + MG132 antibid blot antimyc blot c Myc Bid GFP tbidc GFP Myc tbidn Myc tbidn actin (43 kd) chase pulse15 30 45 60 min 15505 1781 490 673 433 IDV antiha IP Figure 2. tbidn is degraded by the proteasome. (a) MCF7 cells that were not transfected (left) or transfected with Myc Bid GFP (right) were stimulated with TNFα alone or in combination with MG132 for 8 h. Total lysates were probed for endogenous Bid (left) or for transfected Myc Bid GFP (right) and cleavage fragments thereof with antibid antibody. Reprobe was performed with antimyc antibody. (b) HeLa cells expressing HAtagged tbidn were treated for 8 h with CHX and proteasome inhibitor MG132 as indicated. Total cell lysates were probed for HA tbidn with antiha antibody. (c) HeLa cells expressing HA tbidn were labelled with [35S] methionine and cysteine for 2 h, followed by chase with nonradioactive amino acids for the indicated time periods. Bid protein was immunoprecipitated (IP) from detergent lysates with antiha mab and resolved by SDSPAGE. Radioactive signals were quantified by phosphorimaging (IDV = integrated density value). (d) HeLa cells transfected to express untagged tbidn were treated or not with MG132 for 16 h. Total lysates were probed for tbidn with anti Bid antibody. Blots were reprobed with antiactin antibody as indicated. d + MG132 HA tbidn (7 kd) antibid blot tbidn (6 kd) actin (43 kd) was expressed in HeLa cells with or without FLAGtagged ubiquitin. An antiha reactive smear, characteristic of polyubiquitination, was associated with immunoprecipitated HA tbidn (Figure 3a). In addition, we examined ubiquitination of tbidn as generated from fulllength Bid after death receptor stimulation. MCF7 cells expressing Myc Bid GFP were left untreated or stimulated with TNFα for 8 h in presence of MG132. Lysates were immunoprecipitated with antigfp and sequentially with antimyc antibody, to recover Myc tbidn fragment. Immunoblotting for Bid and ubiquitin revealed that ubiquitin was uniquely associated with cleaved, but not fulllength Bid and specified that Myc tbidn contained ubiquitin (Figure 3b). Ubiquitin was also found to be associated with tbidc, in accordance with published data [19]. Ubiquitination was surprising, since tbidn has no lysine residues that conventionally act as ubiquitin acceptor sites (Supplementary Figure 1b). Typically, Ubiquitin can either be linked via a peptide bond to the ε amino group of a lysine residue, or to the α amino group of an Nterminal residue [20]. Since linkage to a lysine was excluded, we investigated whether the Nterminus of tbidn acted as the Ubiquitin acceptor site. To this end, tbidn was fused Cterminally to a TAP tag, which contains a Calmodulin binding domain and Protein A sequence, separated by a tobacco etch virus (TEV) protease sensitive site (ENLYFQG). This allows for twostep purification: first on IgG beads and after cleavage by TEV on Calmodulin beads [21]. In one tbidn TAP construct (7 TEV tbidn TAP), sequences encoding the first 7 amino acids of tbidn and a TEV protease site were cloned upstream of the tbidn coding region (Figure 3c). If tbidn would indeed be ubiquitinated at its Nterminal residue, TEV cleavage should remove the associated ubiquitin. tbidn TAP and 7TEV tbidn TAP were expressed with HAubiquitin in HeLa cells.

46 Chapter 3 a 49 38 28 17 14 6 c HA tbidn Blot: antiha HA tbidn HA tbidn + FLAG Ub * * IP: antiha antiflag tbidn Cal TEV Prot A TEV tbidn Cal TEV Prot A HA Ubiquitin + tbidn TAP Blot: Isolation: HA tbidn + FLAG Ub ubiquitin HA tbidn tbidn TAP 7TEV tbidn TAP tbidn TAP 7TEV tbidn TAP I II III antibid antibid antiha IgG (50 kd) Myc Bid GFP (40 kd) tbidc GFP Calmodulin b 98 62 49 38 28 IP: antigfp antimyc (seq.) tbidn TAP Control 7TEV tbidn TAP 49 38 28 17 14 Control TNF MG132 antiubiquitin blot antibid blot TNF MG132 tbidn TAP 7TEV tbidn TAP Myc tbidn (7 kd) Figure 3. tbidn is ubiquitinated in an unconventional manner. (a) HeLa cells were transfected to express HA tbidn alone, or in combination with FLAGtagged ubiquitin. HA tbidn was immunoprecipitated with antiha mab and probed with antiha mab to detect Bid and with antiflag mab to detect ubiquitin. Asterisks indicate heavy and light chains of antiha mab. (b) MCF7 cells expressing Myc Bid GFP were stimulated with TNFα in presence of MG132 for 8 h or not stimulated (control). Lysates were immunoprecipitated with antigfp antibody and sequentially (seq) with antimyc mab and immunoprecipitates (IP) were probed with antibid antibody and with P4D1 antiubiquitin mab. (c) Assay to determine whether the Nterminus is ubiquitinated. In the drawing, the tbidn TAP proteins assessed for ubiquitination. CalTEV Prot A indicates the TAP tag with calmodulin and IgG binding sites, separated by a cleavage site for TEV protease (arrows). HeLa cells were transfected to express HA Ubiquitin and TAPtagged tbidn. TAPtagged tbidn proteins were isolated with IgG beads, digested with TEV protease and reisolated with Calmodulin beads. Isolates were probed for ubiquitin with antiha mab and for tbidn with antibid antibody. Note that in panel I, the Prot A sequence is still attached to tbidn TAP species and the antibid antibody binds aspecifically to it. Panel I in Figure 3c shows the position of tbidn TAP proteins prior to TEV cleavage, as isolated with IgG beads. TEV cleavage was successful as shown by the difference in migration of Bid species recovered on Calmodulin beads (Figure 3c, panel II). After TEV cleavage, Ubiquitin remained attached to digestion product of 7TEV tbidn TAP (Figure 3c, panel III). TEV protease had efficiently cleaved at the Nterminal site, since the difference in molecular mass between the two Bid species was smaller than prior to TEV cleavage (compare panels I and II). A very small difference remained, because TEV cleaves within the added protease recognition sequence. Ubiquitin was attached to Bid and not to the TAP tag, since the TAP tag alone did not carry ubiquitin upon coexpression with HA Ubiquitin in HeLa cells (Supplementary Figure 2). This experiment argues against the possibility that the Nterminal residue of tbidn is the Ubiquitin acceptor site. Moreover, analysis of the first tryptic peptide of tbidn TAP by MALDI ToF/ToF mass spectrometry determined that the Nterminus of Bid is acetylated and thereby not available for ubiquitin modification (results not shown). These data indicate that tbidn is ubiquitinated at a site that is neither a lysine nor the Nterminus.

Unconventional ubiquitination allows Bid activity 47 Helix 1 in Bid is critical for ubiquitination To identify regions in tbidn that were required for ubiquitination and concomitant proteasomemediated degradation, we made progressive Nterminal deletions. Deletion mutants of tbidn GFP were expressed in HeLa cells, which were treated or not with CHX for 8 h and stability was assessed by immunoblotting of total cell lysates. While tbidn GFP remained highly unstable upon deletion of up to 13 Nterminal residues, deletion of 15 or more (17, 23, 33) Nterminal amino acids dramatically increased its halflife (Figure 4a). To assess whether stabilization correlated with decreased ubiquitination, wildtype and 15 tbidn GFP were expressed in HeLa cells together with FLAGtagged Ubiquitin. AntiBid immunoblotting of total cell lysates showed that wildtype and 15 tbidn GFP were expressed to a similar extent (Figure 4b). Ubiquitinated tbidn GFP species were immunoprecipitated with antiflag antibody and identified by antibid immunoblotting. This analysis showed that wildtype tbidn GFP was polyubiquitinated, whereas the 15 tbidn GFP mutant bore virtually no Ubiquitin (Figure 4b). FLAG Ubiquitin expression was comparable in all transfectants (data not shown). The combined findings in the stability and ubiquitination assays lead to the conclusion that residues 14 and/or 15 contain information that is critical a tbidn GFP wt 7 13 15 17 23 33 Blot: 38 antibid 17 Actin (43 kd) b + + + + + + + CHX c tbidn GFP tbidn GFP GFP wt 15 GFP wt 15 + FLAG Ubiquitin 38 * ** 17 TCL antiflag IP * * HA tbidn TEV43 TEV30 TEV10 + FLAG ubiquitin + + + TEV 188 98 antiflag blot 62 49 38 28 17 14 6 antibid blot 6 3 antibid blot Figure 4. Helix 1 is critical for tbidn ubiquitination and degradation. (a) Wildtype (wt) tbidn GFP and deletion (Δ) mutants lacking 7, 13, 15, 17, 23 or 33 Nterminal amino acids were expressed in HeLa cells, which were treated (+) or not () with CHX for 8 h. The tbidn GFP proteins were detected in total lysates by immunoblotting with antibid antibody. (b) GFP, wt and Δ15 tbidn GFP were expressed in HeLa cells together with FLAGtagged ubiquitin. Total cell lysates (TCL) were probed with antibid antibody to assess tbidn expression levels. Ubiquitinated protein species were isolated with antiflag mab (IP) and probed with antibid antibody. Single asterisk indicates heavy and light chains of the antiflag mab, double asterisk endogenous full length Bid. (c) FLAGtagged ubiquitin was coexpressed in HeLa cells with HA tbidn versions with a TEV protease cleavage site at position 10, 30 or 43. HA tbidn molecules were isolated, incubated with (+) or without () TEV protease, run on gel and probed by immunoblotting for Bid and ubiquitin (antiflag). In case of HA tbidn TEV10, the HAtagged cleavage fragment repeatedly did not resolve on gel, but remaining undigested HA tbidn TEV10 and weakly ubiquitinated species of the cleavage fragment (arrows) are clearly visible. All experiments in this figure were performed three times with similar results.

48 Chapter 3 for ubiquitination and destabilization of tbidn. These are the first residues that take part in the α helix 1 of Bid (see Supplementary Figure 1b). Their deletion is expected to disrupt this structure and may therefore result in loss of ubiquitination site(s), and/or loss of a binding site for the Ubiquitin ligase. To ascertain whether helix 1 contained ubiquitination site(s), we performed mapping experiments. Cleavage sites for the TEV protease were introduced in HA tbidn after residues 10, 14, 30 or 43 and these recombinant proteins were expressed in presence of FLAG Ubiquitin. They were isolated with antiha antibody, digested with TEV protease and probed for the presence of Ubiquitin and Bid. In case of the HA tbidn TEV43 and 30 proteins, cleavage with TEV protease resulted in a shift in molecular mass of both Bid and the Ubiquitin signal. There was no apparent loss of the Ubiquitin signal (Figure 4c). This proves that tbidn is directly ubiquitinated and indicates that predominant Ubiquitin acceptor sites are amongst residues 130 of tbidn. The HA tbidn TEV14 protein did not cleave with TEV protease (data not shown). Upon TEV digestion of HA tbidn TEV10, the Ubiquitin signal was strongly reduced (Figure 4c). The HA tbidn 110 fragment did not resolve on gel, but weakly ubiquitinated species of this fragment are visible (arrows). The remaining Ubiquitin was mainly associated with the undigested HA tbidn TEV10. We conclude that tbidn is predominantly ubiquitinated on sites in between residues 10 to 30, but also to a certain extent on sites in between residues 1 to 10. The deletion and mapping experiments collectively indicate that helix 1 (residues 14 29, Supplementary Figure 1b) is critical for ubiquitination to occur. tbidn is likely ubiquitinated on cysteine, serine and/or threonine residues Lack of lysine residues is a common feature of tbidn in different species (human, chimpanzee, mouse, rat, cow, pig; Supplementary Figure 1c). We surmised that tbidn might be ubiquitinated on cysteine residues. Three such sites are available among residues 130 of human tbidn: C3, C15 and C28. Ubiquitin linkage to cysteine is not uncommon, since the E2 and E3 enzymes are charged with Ubiquitin in this manner. In such case, the carboxyl group of the Cterminal glycine in Ubiquitin forms a thioester bond with the sulfhydryl group in cysteine, which is easily reducible. To address this possibility, ubiquitinated HA tbidn was isolated and subjected to reduction with dithiothreitol (DTT) under denaturing conditions. HA tbidn was reprecipitated with antiha antibody to separate it from possibly dissociated Ubiquitin moieties. As a positive control, we used in vitro ubiquitinated E225K, an E2 that is monoubiquitincharged through a thioester bond in its active site [22]. Indeed, DTT treatment resulted in loss of Ubiquitin from E225K species (Figure 5a). Importantly, the same treatment diminished the amount of Ubiquitin appended to HA tbidn (Figure 5a). This experiment indicates that cysteine(s) is/ are among the Ubiquitin acceptor sites in tbidn. However, we found highly reproducibly that Ubiquitin was partially retained on tbidn upon reduction, arguing that other residues play a role as acceptor sites as well. Point mutation of C15 or C28 either alone, or in conjunction (Table 1) did not stabilize tbidn (Supplementary Figure 3). It also did not reproducibly reduce ubiquitination of the 130 fragment of HA tbidn TEV 30 (Fig. 5b). Additional mutation of C3 made HA tbidn TEV30 very unstable, such that it could only be expressed at lower levels than the wildtype protein (Figure 5b and Supplementary Figure 3). However, it could clearly be demonstrated that the cysteineless (C3;C15;C28) mutant of HA tbidn TEV30 still carried Ubiquitin after TEV digestion (Figure 5b). This ties in with the partial reducibility of the ubiquitination and leads to the important conclusion that residues 130 of tbidn harbour residues other than cysteine to which Ubiquitin can be conjugated. The potential alternative ubiquitination sites in tbidn (130) are serine and threonine residues, since the carboxyl group of the Cterminal glycine in Ubiquitin can theoretically form an ester bond with the hydroxyl group in the side

Unconventional ubiquitination allows Bid activity 49 a HA tbidn + FLAG Ubiquitin E225K in vitro ubiquitinated + + + + reduction 188 98 62 49 38 28 28 17 17 14 14 6 6 IDV: 220 173 Blot: antiha antiflag antie225k antiubiquitin * c tbidn TAP + HA Ubiquitin ph 49 38 28 17 14 14 7.4 12 Blot: antiha antibid b Blot: antiflag 188 98 62 49 38 28 HA tbidn TEV30 + FLAG Ubiquitin + + + + + + + + + TEV antiha 6 3 wt S27;C28; T17; wt C3;C15; C3;C15; wt S9;S10 S9;S10; S29 S27;C28; C28 T17;S27; C15;T17; Figure 5. Ubiquitin is partially reducible, hydrolysable and affected by combined mutation of S, T and C residues. (a) HA tbidn was coexpressed with FLAG Ubiquitin in HeLa cells. AntiHA immunoprecipitates were reduced (+) or not () as described in Experimental Procedures and reprecipitated with antiha mab. Reprecipitates were probed for Bid (antiha) and ubiquitin (antiflag). Recombinant E225K as a positive control for cysteine ubiquitination was ubiquitinated in vitro, reduced or not, and probed with mab to E225K or ubiquitin. Asterisk indicates free recombinant ubiquitin. IDV = integrated density values, showing comparable tbidn loading. (b) Alanine substitution mutations were generated in HA tbidn TEV30. All mutants listed in Table I were tested for ubiquitination and part of the results are shown here. Wildtype and mutant proteins were expressed in HeLa cells together with FLAG Ubiquitin, isolated with antiha mab and treated (+) or not () with TEV protease. AntiHA immunoblotting was performed to assess Bid expression and cleavage and antiflag immunoblotting to examine ubiquitination. Arrows indicate positions of the ubiquitinated TEV cleavage fragment. (c) tbidn TAP was coexpressed with HA Ubiquitin in HeLa cells. Cell lysates were treated at ph 7.4 or ph 12 as indicated in Experimental Procedures and tbidn TAP was purified on the TAP tag. Calmodulinbound protein was separated by SDSPAGE and probed for Bid (antibid) and ubiquitin (antiha). All experiments in this figure were performed three times with similar results. chain of these amino acids. There is recent biological precedent for this type of Ubiquitin conjugation by viral Ubiquitin ligases [23]. To test this possibility, we investigated whether the Ubiquitin linkage was susceptible to alkaline hydrolysis. HeLa cells expressing tbidn TAP and HA Ubiquitin were lysed at either ph 7.4 or ph 12, heated for 10 min at 70 o C, diluted out in buffer with neutral ph and subjected to TAP purification. Isolates were probed for Bid and Ubiquitin. Equal amounts of tbidn TAP were recovered from both lysates, but after treatment at ph 12, the amount of Ubiquitin associated with tbidn was greatly diminished (Figure 5c). Thioester bonds are very easily hydrolysable, but the dramatic loss of ubiquitination after

50 Chapter 3 hydrolysis was compatible with the notion that hydroxyl ester bonds were involved as well. To verify that the hydrolysis procedure left conventional lysinelinked Ubiquitin moieties unaffected, we tested its effect on a complex of purified, in vitro ubiquitinated Ring1b and Bmi1 [23]. After the ubiquitination reaction, the complex was incubated at ph 7.4 or at ph12, exactly as done for tbidn TAP. In addition, it was heated in presence of βmercaptoethanol to test the effect of reduction. The conditions were neutralized, the complex was recovered by virtue of His tags, and examined for extent of ubiquitination by immunoblotting. None of the treatments affected the ubiquitination status of Ring1b (Supplementary Figure 4). For mutation analysis of serines and threonines, we focused on the residues in helix 1. Among these, T17 is completely conserved between species (Supplementary Figure 1c). However, alanine substitution of T17 alone did not stabilize tbidn, or affect ubiquitination of the 130 fragment of tbidn (not shown). Additional mutation of S27, C28 and S29, or mutation of all serines, threonines and cysteines in helix 1 did not stabilize tbidn either (Supplementary Figure 3). It reproducibly reduced ubiquitination of the 130 fragment to a certain extent, but did not abrogate it (Figure 5b). Additional mutation of C3 rendered HA tbidn TEV30 highly unstable (Supplementary Figure 3), but we could reveal ubiquitination on the 130 fragment, supporting the conclusion that other residues than cysteines are acceptor sites (Figure 5b). This result pointed to a possible role for S9 and/or S10. However, alanine substitution of these residues either alone, or in conjunction with the potential acceptor sites in helix 1 did not stabilize tbidn (Supplementary Figure 3). Importantly, ubiquitination of the 130 fragment was virtually abrogated when alanine substitution of S9 and S10 was combined with alanine substitution of cysteine, threonine and serine residues in helix 1 (Figure 5b). This result indicates that one or more of these residues is a ubiquitination site. We conclude from the combined biochemical and mutation analyses that tbidn is ubiquitinated on cysteine residues, but not exclusively so. There are ubiquitination sites other than cysteine amongst residues 130. We postulate that these are serine and/or threonine residues, given the hydrolysability of the ubiquitination and the results of mutation analysis. Unambiguously pinpointing the ubiquitination sites by mutation analysis is hampered by effects of mutation on protein stability and by the fact that the ubiquitination machinery is promiscuous and ubiquitinates multiple sites, including alternative residues when the primary target sites are not available due to mutation. Proteasomedependent degradation of tbidn is critical for its membranepermeabilizing activity To investigate whether degradation of tbidn was required for tbidc function, we determined the impact of proteasome inhibition on tbidc translocation. MCF7 cells expressing Myc Bid GFP were stimulated with TNFα for 8 h in presence or absence of MG132 and examined by CLSM for the distribution of Myc tbidn and tbidc GFP cleavage fragments. In accordance with the biochemical data (Figure 2a), Myc tbidn was rescued from degradation by MG132 (Figure 6a, panel IV). Despite this, tbidc GFP translocated to mitochondria (panel V), where it partially colocalized with Myc tbidn (panel VI). This result indicates that degradation of tbidn is not required for tbidc translocation. In line with a previous study [14], it also suggests that the cleaved, but still associated tbidn/tbidc complex can translocate to mitochondria. Next, we investigated whether proteasome inhibition affected the capacity of tbidc to permeabilize the mitochondrial outer membrane. Untransfected MCF7 cells were stimulated with TNFα in the presence or absence of MG132 and analyzed by flow cytometry for Cyt C content. The dramatic release of Cyt C induced by TNFα stimulation was greatly reduced upon coincubation with MG132 (Figure 6b), suggesting that degradation of tbidn is required for tbidc function. Since the proteasome may have other targets in this pathway, we aimed to test a Bid

Unconventional ubiquitination allows Bid activity 51 a b Myc (Nterminus)) GFP (Cterminus) Overlay Overlay 90 * 80 I II III TNF TNF + MG132 Cytochrome c release (%) 70 60 50 40 30 20 10 IV 15 m V VI 0 C TNF TNF + MG 132 Figure 6. Proteasome inhibition prohibits tbidc function. (a) MCF7 cells expressing Myc Bid GFP were stimulated with TNFα with or without MG132 for 8 h. Stimulation resulted in translocation of tbidc GFP (green) to mitochondria (panels II, III). In the presence of MG132, Myc tbidn (red) remained detectable (panel IV), indicating escape from proteasomal degradation. N and C terminal epitopes partially colocalized at mitochondria (panel VI). (b) TNFαinduced Cyt c release in presence or absence of MG132 was tested by flow cytometric analysis in untransfected MCF7 cells after 8 h of stimulation. Results are means plus SD from three independent experiments. Values are corrected for Cyt c release in absence of stimulus (15 + 5%). Asterisk indicates statistically significant difference (p<0.05 according to Student s t test). molecule harbouring tbidn that could escape from proteasomal degradation. Unfortunately, the stabilizing Nterminal deletion mutation 15 unleashed constitutive mitochondrion localisation and permeabilizing activity in full length Bid (data not shown) and was therefore not useful. In the course of our work, though, we had noted that addition of a bulky group such as GFP or a repetitive Myc tag to the Nterminus of tbidn greatly increased its stability. Therefore, we compared the functional activity of GFP Bid VSV, containing a stabilized GFP tbidn, with that of Myc Bid GFP, containing the wildtype, unstable Myc tbidn. To create a cell line that exclusively expressed stabilized tbidn, endogenous Bid was constitutively downregulated in MCF7 cells by RNA interference (RNAi) (Figure 7a). To express stabilized GFP Bid VSV and control (unstable) Myc Bid GFP into these cells, a silent mutation was introduced into the Bid sequence that was not targeted by the sirna. This allowed appropriate expression of the Bid constructs upon transient transfection (Figure 7a,b). Processing of Myc Bid GFP and GFP Bid VSV by Caspase8/10, as well as the halflife of the Bid fragments was followed kinetically after stimulation of the reconstituted MCF7 cells with TNFα. In case of Myc Bid GFP, the tbidc GFP cleavage product was visible from the 2 h time point onward and its signal increased in time. The Myc tbidn fragment was detected at the 2 h and 4 h time points, but not at the 8 h time point (Figure 7b). Processing of GFP Bid VSV was also visible at 2 h, but in this case not only the tbidc VSV fragment remained detectable in the 8 h time frame, but also the GFP tbidn fragment (Figure 7b). We conclude that the GFP Bid VSV protein is properly processed by Caspase8/10 and that the GFP tbidn fragment is longlived, whereas the Myc tbidn fragment has a short halflife. The fate of stabilized GFP tbidn was also followed by CLSM. In unstimulated cells, GFP and VSV signals were consistent with a cytoplasmic localization of full length GFP Bid VSV (Figure 7c, panels I, II). At 8 h after stimulation, the VSV signal was punctate and colocalized with MitoTracker, indicating mitochondrial localization of tbidc VSV (Panel

52 Chapter 3 c a Myc Bid GFP (50 kd) (43 kd) Actin Myc Bid GFP Myc Bid GFP + mutant + control 49 tbidc GFP 28 14 Myc tbidn 6 3 GFP (Nterminus) VSV (Cterminus) Overlay + MitoTracker 15 m Bid RNAi control antigfp blot Bid RNAi I II III b Myc Bid GFP 0 2 4 8 0 2 4 8 h TNF control TNF 8 h antibid blot d Cytochrome C release (%) 70 60 50 40 30 20 10 GFP Bid VSV full length Bid GFP tbidn tbidc VSV * IV V VI 0 MCF7 MCF7 Bid RNAi Bid GFP GFP Bid MCF7 Bid RNAi + rescue mutants Figure 7. Stabilization of tbidn prohibits tbidc function. (a) MCF7 cells were depleted of endogenous Bid by stable expression of a Bid sirna. MCF7 Bid RNAi cells were reconstituted by transient transfection with Myc Bid GFP or GFP Bid VSV with silent mutations to allow escape from RNAi. The result for reconstitution with Myc Bid GFP wildtype and RNAi escape mutant is shown by immunoblotting for GFP in total cell lysates. (b) Myc Bid GFP and GFP Bid VSV were expressed in MCF7 Bid RNAi cells and cells were stimulated for the indicated time periods with TNFα. Full length Bid and cleavage fragments were detected by antibid immunoblotting of total cell lysates. Fragments were identified additionally by immunoblotting with antibodies to Myc, GFP and VSV (not shown). ECL signals originate from the same blotted gel, but the lower panel represents a longer exposure than the upper panel to allow detection of Myc tbidn. (c) MCF7 cells expressing GFP Bid VSV were stimulated with TNFα for 8 h or left untreated (control). Signals diagnostic for stabilized GFP tbidn are shown individually and in combination with the signals for tbidc (VSV) and MitoTracker. The zooms allow assessment of colocalization of tbidn and tbidc at mitochondria. (d) TNFαinduced Cyt c release was tested after 8 h of stimulation by flow cytometric analysis in parental MCF7 cells, in cells lacking Bid (MCF7 Bid RNAi) and the same cells reconstituted with either Myc Bid GFP (unstable tbidn) or GFP Bid VSV (stabilized GFP tbidn). MCF7 and MCF7 Bid RNAi cells were additionally transfected to express HistoneGFP. Cyt c content was monitored in cells gated for equal GFP expression to standardize for transfected Bid levels. Results are means plus SD from three independent experiments. Asterisk indicates statistically significant difference (p<0.05 according to Student s t test). V). The GFP signal was still present at this time point (Panel IV) and partially colocalized with VSV and MitoTracker signals (Panel VI). This indicated longterm stabilisation of GFP tbidn (compare with disappearance of Myc tbidn, Figure 1a). As in the case of proteasome inhibition (Figure 6a), tbidn stabilization did not interfere with mitochondrial translocation of tbidc and both fragments partially colocalized at mitochondria. This suggests that tbidn and tbidc may remain associated after cleavage of fulllength Bid and translocate to mitochondria as a complex. Next, Cyt C release upon TNFα stimulation was compared in wildtype MCF7 and Bid RNAi cells, before and after reconstitution with unstable or stabilized Bid versions. Bid RNAi effectively blocked TNFαinduced Cyt C release (Figure 7d). Importantly, introduction of GFP Bid VSV, bearing stabilized tbidn, did not restore the ability of TNFα to induce Cyt C release, whereas introduction of Myc Bid GFP,

Unconventional ubiquitination allows Bid activity 53 bearing unstable tbidn did (Figure 7d). Taken together, these results indicate that degradation of tbidn regulates the proapoptotic function of tbidc. Based on these data, we propose a model in which selective removal of tbidn by proteasomemediate degradation liberates tbidc to induce mitochondrial membrane permeabilization. Discussion Sequences in Bid s Nterminal fragment sequester the proapoptotic BH3 domain. Through this autoinhibitory mechanism, full length Bid is kept in an inactive state [12,17]. Bid can be converted to its active form by cleavage in its second unstructured loop, such as at D60 by Caspase8/10 and at D75 by Granzyme B [8,25,26]. Mechanistically, it was unclear why cleavage should lead to Bid activation. Upon Bid cleavage in vitro, the Nterminal and Cterminal fragments remain associated according to both NMR and biochemical analyses [6,12,14]. Moreover, several investigators have demonstrated that the tbidnfragment, when expressed independently, can block the proapoptotic activity of the tbidc fragment [15,17,18]. Collectively, these data suggest a critical inhibitory effect of the Bid Nterminus upon the Cterminal proapoptotic function both before and after Bid cleavage. Based on our results, we propose that cleavage of Bid acts as a proapoptotic activation signal by allowing ubiquitination and subsequent proteasomal destruction of the inhibitory Nterminal fragment. While originally recognized as a signal for the steady state destruction of aged and misfolded proteins, ubiquitination is now known to play an essential regulatory role in cellular signaling. Various apoptosis signaling molecules are regulated by proteasomal destruction [2729]. These include inhibitory Bcl2 family members Bcl2 [30], Mcl1 [31,32] and A1 [33], proapoptotic Bax [25], as well as BH3only proteins Bim [34,35] and Bik [36] and the Cterminal fragment of Bid [30]. Polyubiquitination and degradation generally negatively regulate the function of the target protein by its complete elimination. The case of tbidn ubiquitination is different, since it represents a form of positive regulation: it is required for the proapoptotic function of Bid. In the noncanonical NFκB pathway, polyubiquitination and partial protein degradation of p105/p100 also acts as an activating principle [29]. Both in case of the NFκB precursor and in the case of Bid, a portion of the molecule escapes from degradation and is released to perform its function. Mutation analysis specifically implicated 5 out of 13 lysines in Mcl1 in its destruction [32], while mutation of all 4 lysine residues in tbidc was shown to increase its halflife [19]. In case of the other Bcl2 family members, no specific target sites have been identified. In case of Bid, ubiquitination occurred with certainty on the Bid fragment encompassing residues 130. Indeed, the TEV protease mapping experiments indicate that tbidn can be ubiquitinated on sites among amino acid 1130, as well as to a lesser extent on sites among amino acids 110. This classifies the ubiquitination as unconventional, since it did not concern a peptide bond to an ε amino group of lysine or an α amino group at the Nterminus of the Bid protein. Following reduction, ubiquitination was partially, but not completely lost. This indicates that cysteine residues are among the Ubiquitin acceptor sites. A thioester linkage is not unusual for Ubiquitin, since the ubiquitin conjugating (E2) and Ubiquitin ligase (E3) enzymes bear Ubiquitin in such a manner. However, to our knowledge, cysteine ubiquitination of a target other than E2 or E3 proteins has only one precedent in the existing literature. This concerns the cytoplasmic tail of MHC class I that contains no lysine residues, but was ubiquitinated in a reducible manner by an E3 encoded by a herpesvirus [36]. Ubiquitination of tbidn was to a significant degree maintained upon reduction (Figure 5b) consistent with the possibility that threonine and serine residues are primary targets of the ubiquitination machinery. The fact that a tbidn mutant lacking all three cysteine residues was still ubiquitinated on fragment 130 proves

54 Chapter 3 that residues other than cysteine are ubiquitin acceptors. This is consistent with the fact that cysteines are not fully conserved in tbidn. In fact, rat tbidn does not contain any cysteines (Supplementary Figure 1c). Our extensive mutation analysis has not pinpointed the ubiquitination sites. These experiments are hampered by the fact that Ubiquitin is likely appended to other candidate sites when the original sites are not available, including residues outside region 130. Mutation of all cysteines, threonines and serines in tbidn made it much more unstable than the wildtype protein, such that it was barely detectable even in the presence of proteasome inhibitor (not shown). In this case, the protein likely bears little structural resemblance to the wildtype tbidn and results are difficult to interpret. Thus far, attempts to map the ubiquitination sites by mass spectrometry have not been successful. Lability of the (thio)ester bonds is the most likely limiting factor in this analysis, particularly after tryptic digestion which converts the polyubiquitin chain into a reactive Gly Gly amine. Theoretically, Ubiquitin can link to serine and threonine by the formation of an ester bond between the carboxyl group of the Cterminal glycine and the hydroxyl group in the side chain of these amino acids. The first evidence for such linkage in a biological system has been reported when this manuscript was in final stage of preparation. It again concerned the cytoplasmic tail of MHC class I, which was ubiquitinated by another herpesvirusderived E3 than the one responsible for cysteine ubiquitination [23]. Our work provides additional evidence for such linkage and adds that it can occur in a mammalian system. While 13 tbidn was as unstable as the wildtype version, the 15 mutant was fully stabilized. C15 is a good candidate Ubiquitin acceptor site, since it lies at the border of helix 1 and is accessible in the Bid structure [12,13]. However, point mutation of C15 did not affect ubiquitination. In stark contrast, ubiquitination was almost completely lost from tbidn GFP upon deletion of residues 115. This suggests that these residues, and in particular residues 14 and 15 (given the instability of the 13 mutant) are critical for binding of the (undefined) ubiquitination machinery to tbidn. In our CLSM experiments, we found tbidn together with tbidc at the mitochondrial membrane after TNFα stimulation, when tbidn degradation was blocked by proteasome inhibition (Figure 6a) or GFP fusion (Figure 7c). It is therefore clear that tbidn degradation is not required for mitochondrial translocation of tbidc. We suggest that the stabilized tbidn fragment inhibits the function of tbidc at the mitochondria. In time course experiments with wildtype Myc Bid GFP, we also found a degree of colocalization of Myc and GFP signals at mitochondria at 2 h after TNFα stimulation (Supplementary Figure 5). These findings tie in with the in vitro observation that the cleaved complex stays intact [12] and a previous study showing association of the cleaved Bid complex with mitochondria [14]. These data suggest that tbidn and tbidc may remain associated during mitochondrial translocation. Further experiments are required to resolve whether tbidn ubiquitination takes place at the mitochondrial membrane and whether this event determines its dissociation from tbidc. We have shown here that Bid s proapoptotic function is activated through cleavageinducible ubiquitination and proteasomal degradation of tbidn. Freeing of the BH3 domain by this mechanism may apply to other Bcl2 family members as well. For instance, Bim EL is cleaved by caspases at an early stage during apoptosis induction, rendering it more proapoptotic [37]. Moreover, Bcl2, Bfl1 and Bclx L can be converted into proapoptotic proteins following cleavage by caspases or Calpain [7,33,38]. Perhaps in these cases a similar mechanism operates as we have shown here for Bid activation. Acknowledgements We are grateful to Gretel Buchwald, Puck Knipscheer and Titia Sixma for help with the in vitro ubiquitination experiments and

Unconventional ubiquitination allows Bid activity 55 information on Bid structure. We thank Martina O Flaherty, Albert J. Heck, Clive Slaughter and Andrew High for help with mass spectrometry, Douglas R. Green for support and Huib Ovaa for sharing his chemical expertise and commenting on the manuscript. This work was funded by the Dutch Cancer Society. Materials and methods Cells and reagents Human cervix carcinoma line HeLa, breast carcinoma line MCF7 and 293 embryonic kidney cellderived packaging line ΦNXAmpho were grown in DMEM with fetal calf serum. Human TNFα was used at 10 ng/ml in combination with CHX at 10 µg/ml (both from SigmaAldrich), soluble human FLAGtagged TRAIL in combination with enhancer (Alexis Biochemicals) at 100 ng/ml., MG132 (Calbiochem) at 50 µm. Monoclonal antibodies used were: antiha 12CA5, antimyc 9E10, anti VSV P5D4 (purified Ig prepared from available hybridomas), antiflag M2 (SigmaAldrich), antiactin C4 (Chemicon), anti Cyt c 6H2.B4 (BDBiosciences), antiubiquitin P4D1 (Santa Cruz Biotechnology). Polyclonal rabbit sera used were: anti Bid raised against a GST fusion protein of full length human Bid [39], available from BDBiosciences, antigfp (Clontech), antie225k UG9520 (Biomol). Horse radish peroxidase (HRP) conjugated rabbit antimouse Ig and swine antirabbit Ig were from DAKO A/S (Glostrup, Denmark); HRPconjugated anti HA and antiflag mab from Sigma and goat antimouse Ig conjugated to Alexa Fluor 594 from Molecular Probes. Plasmids For the construction of cdna encoding full length human Bid fused Cterminally to egfp (Bid GFP), we made use of Clontech vector egfpn1. The construct encoding full length Bid GFP with an Nterminal Myc tag was created by placing a polylinker encoding a Myc epitope 5 in frame with the Bid intiation codon. cdna encoding the tbid Nterminal residues 160 (tbidn) was cloned into egfpn1, egfpc1 (Clontech), pmt2sm, pmt2sm VSV and pmt2smha [39] by PCR/restriction enzyme based methods. The plasmid encoding histone 2BGFP has been described [40]. Plasmid pcdna3.1 encoding FLAGtagged human ubiquitin was a kind gift of Dr. Simon Cook (Babraham Institute, Cambridge, UK). Plasmid pmt2sm encoding HAtagged human ubiquitin has been described [41]. pcdna3.1 TAP was made by restriction enzyme based subcloning of the TAP tag from pzomec1 (CellZome) into pcdna3.1. tbidn cdna and deletion mutants were cloned into pcdna3.1tap by restriction enzyme based subcloning producing constructs that were Cterminally fused to the TAP tag [21]. Where used, polylinkers encoding TEV protease recognition sequences (encoding ENLYFQG) were inserted following the appropriate restriction digest. Point mutants and TEV insertion constructs were generated using QuikChange site directed mutagenesis kit according to the manufacturers instructions (Stratagene). Western blotting, metabolic labelling and immunoprecipitation For immunoprecipitation, all cells (adherent and floating) were harvested, washed twice in PBS and lysed in Nonidet P40 (NP 40) buffer, consisting of 50 mm TrisHCl, ph 7.4, 1% NP40, 150 mm NaCl, 1 mm PMSF and Complete EDTAfree Protease Inhibitors (Roche Molecular Biochemicals). Cell lysates were clarified by centrifugation for 15 min at 13.000 g and protein content was measured by BioRad protein assay. All SDSPAGE was done on precast 412% NuPAGE minigels, according the manufacturer s protocol (Invitrogen). In this procedure, samples are heated for 10 min at 70 o C in reducing (100 mm DTT) sample buffer prior to electrophoresis. Equal amounts of protein per lane (20 µg) were separated by SDSPAGE and transferred to nitrocellulose sheets. Immunoblotting and enhanced chemiluminescence were performed as described [40]. For metabolic labelling, cells (1 x 10 6 cells in a 10 cm dish) were starved in methionine and cysteinefree DMEM for 2 h, radiolabelled with [ 35 S]methionine and cysteine (13.25 MBq per dish, Amersham) for 2 h in the same medium. For the chase, cells were incubated for the indicated time in complete DMEM medium, supplemented with unlabelled methionine and cysteine (2.5 mm). Immunoprecipitated proteins were resolved by SDSPAGE, detected by autoradiography and quantified by phosphorimaging (Fuji Film FLA3000 and AIDA software). Tandem affinity purification Cells (1x10 6 ) were lysed in NP40 buffer. Lysates were clarified by centrifugation and incubated with rabbit IgG agarose (Sigma) for 2 h at 4 o C. Agarose beads were washed in NP40 buffer and in TEV cleavage buffer (10 mm Tris ph 8, 0.1 % NP40, 150 mm NaCl, 0.5 mm EDTA, 1 mm DTT) and incubated with 10 units TEV protease (Invitrogen) in cleavage buffer overnight at 4 o C. Next, supernatant was diluted 3fold in calmodulinbinding buffer (10 mm TrisHCl ph 8, 0.1% NP40, 150 mm NaCl, 1 mm Mg acetate, 1 mm imidazole, 2 mm CaCl 2 ) and incubated with calmodulin sepharose beads (Stratagene) at 4 o C for 1 h. Calmodulinbound proteins were separated by SDSPAGE and detected by Western blotting. For alkaline hydrolysis experiments, cells were lysed in PBS ph 7.4 or ph 12, 1% NP40 and Complete EDTAfree Protease Inhibitors. Lysates were clarified by centrifugation, heated at 70 o C for 10 min, diluted out 100fold in NP40 buffer and subjected to TAP purification. Treatment of immunoprecipitates For TEV protease treatment, HA Bid immunoprecipitates were resuspended in 300 µl TEV cleavage buffer and incubated with or without 2 µl TEV protease overnight at 4 o C. Beads were spun down, washed three times and taken up in sample buffer for SDSPAGE. For reduction, HA Bid immunoprecipitates were suspended in 10 µl NP40 buffer. To this, 15 µl of 100 mm TrisHCl ph 6.8, 4% SDS, 8 M urea, 20% glycerol was added, with or without 600 mm fresh DTT. Control samples without DTT were kept on ice, while samples with DTT were heated at 95 o C for 10 min. Next, samples were diluted out 80 times in NP40 buffer with 10 mm iodoacetamide and 50 µg/ml RNAse A (as carrier protein). Beads were spun down and supernatants

56 Chapter 3 were subjected to reprecipitation with antiha antibody. For the sequential immunoprecipitation shown in Fig. 3b, following immunoprecipitation with antigfp antibody, the lysate was incubated 3 times with Protein G sepharose beads followed by immunoprecipitation with antimyc antibody. In vitro ubiquitination To ubiquitinate the recombinant E2 core domain of E225K [22], recombinant human ubiquitin activating enzyme (E1) (120 ng; 0.2 µm), E225K 1155 (1.5 µg; 17 µm) and ubiquitin (2 µg; 47 µm) were coincubated in 20 mm TrisHCl ph 8.0, 100 mm NaCl, 5 mm βmercaptoethanol with 5 mm ATP and 5 mm MgCl 2 for 2 min at 23 C. Reactions were terminated by 1:1 dilution in sample buffer (100 mm Tris ph 6.8, 8 M urea, 4% SDS, 20% glycerol, with or without 600 mm DTT) and samples were kept on ice or heated at 95 o C as described above prior to electrophoresis. A complex of Histagged Ring1b (residues 1331) and fulllength Histagged Bmi1 were coexpressed in Sf9 insect cells by simultaneous infection with separate recombinant baculoviruses. The complex was purified on Niagarose (Hitrap column, Pharmacia) and further purified as described [24]. Ring1b/Bmi1 was incubated at 6 µm, together with human E1 (220 nm), human UbcH5c (2 µm), ubiquitin (20 µm) and ATP (3 mm) in a total volume of 50 µl ligase buffer, consisting of 50 mm TrisHCl ph 7.5, 100 mm NaCl, 10 mm MgCl 2, 1 µm ZnCl 2, 1 mm DTT at 30 o C for 1 h. For the control sample, the ubiquitination reaction was stopped by addition of LDS sample buffer (NuPAGE, Invitrogen). Samples for immunoprecipitation (IP) were treated as follows: 1) incubated with 12.5fold excess of ligase buffer as control, 2) heated for 8 min at 95 o C in presence of 5% βmercaptoethanol, after which 12.5fold excess ligase buffer was added, 3) incubated with 12.5fold excess PBS ph 7.4 and heated for 10 min at 70 o C, 4) incubated with 12.5fold excess PBS ph 12 and heated for 10 min at 70 o C. Next, samples were diluted with NP40 buffer (see above) to a total volume of 10 ml and subjected to immunoprecipitation with Niagarose beads (Qiagen). Reaction products were separated by NuPAGE in 412% BisTris precast gels (Invitrogen), blotted to nitrocellulose and probed independently with P4D1 antibody directed to ubiquitin and antiring1b (H. Koseki, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan). RNA interference A short interfering (si) oligonucleotide targeting human Bid (nt 238256, 5 gaagacatcatccggaata 3 ) was cloned into psuper and pretrosuper [42]. Stable expression of Bid sirna was achieved by retroviral transduction of cell lines of interest with the pretrosuper construct. For rescue experiments, two silent mutations were introduced in the appropriate Bid sequence (gaagacatcataaggaata), using the QuikChange sitedirected mutagenesis kit, in order to avoid sirnamediated silencing. Transfection and retroviral transduction Cells were transfected with FuGENE 6 according to manufacturer s instructions (Roche Molecular Biochemicals). Unless otherwise indicated, cells were manipulated 20 h after transfection. Production of amphotropic retrovirus was done in ΦNXAmpho cells as described [39]. For transduction, viruscontaining packaging cell supernatant was incubated on ice for 10 min with 10 µg/ml DOTAP (Roche) and subsequently incubated with target cells overnight. Cells were selected with puromycin (1 µg/ml) 48 h after transduction. CLSM Cells (1 x 10 5 per sample) were grown and transfected on glass cover slips in 6well plates, washed in PBS and fixed for 30 min with 4% paraformaldehyde in PBS. If applicable, cells were incubated for 30 min with MitoTracker Deep Red (100 nm, Invitrogen) prior to fixation. Fixed cells were washed in PBS, permeabilized by incubation in PBS with 1% Triton X100 for 10 min, followed by blocking with 0.5% BSA in TBSTween for 1 h. Cells were incubated in the same buffer with the appropriate primary antibody and subsequently washed three times in TBSTween and incubated with AlexaFluor 594conjugated goat antimouse IgG (Invitrogen). Next, cells were washed in TBSTween and mounted on slides using Vectashield (Vector Laboratories). All treatments were done at room temperature. CLSM was performed using a Leica TCS SP2 system (Leica Microsystems, Heidelberg, Germany) using a Leica 63x 1.32 NA oil immersion objective and Leica Confocal Software. Images were processed (cropping, level adjustment) using Adobe Photoshop software. To minimise spectral leakthrough, images were obtained by sequential scanning. Cyt c release assay Cells were analyzed for Cyt c release essentially as described [43]. All cells were collected, washed in PBS and permeabilized with 200 µg/ml digitonin in PBS, 150 mm KCl, 1 mm EDTA for 10 min on ice. Cells were washed once in PBS, fixed in 4% paraformaldehyde, incubated in blocking buffer (2% BSA in PBS) for 30 min at room temperature and incubated with anticyt c mab 6H2.B4 at 1:200 dilution in blocking buffer overnight at 4 o C. Cells were washed in blocking buffer and then incubated with a Cy5conjugated goat antimouse Ig antibody (Molecular Probes) at 1:200 dilution in blocking buffer for 1 h at room temperature. After washing, cells were analyzed using a FACSCalibur and CellQuest software (BDBiosciences). Cyt c content was analyzed specifically in transfected cells by gating the GFPpositive population. References [1] Wang, X. (2001) The expanding role of mitochondria in apoptosis. Genes Dev 15, 29222933. [2] Adams, J.M., and S. Cory. (1998) The Bcl2 protein family: arbiters of cell survival. Science 281, 1322 1326. [3] Sattler, M., H. Liang, D. Nettesheim, et al. (1997) Structure of BclxLBak peptide complex: recognition between regulators of apoptosis. Science 275, 983 986. [4] Puthalakath, H., and A. Strasser. (2002) Keeping killers on a tight leash: transcriptional and posttranslational

Unconventional ubiquitination allows Bid activity 57 control of the proapoptotic activity of BH3only proteins. Cell Death Diff. 9, 505512. [5] Wei, M.C., T. Lindsten, V.K. Mootha, et al. (2000) tbid, a membranetargeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 14, 20602071. [6] Kuwana, T., M.R. Mackey, G. Perkins et al. (2002) Bid, Bax and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111, 331342. [7] Cheng, E.H., M.C. Wei, S. Weiler et al. (2001) BCL2, BCLXL sequester BH3 domainonly molecules preventing BAK and BAKmediated mitochondrial apoptosis. Mol. Cell 8, 705711. [8] Luo, X., I. Budihardjo, H. Zou, et al. (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481490. [9] Li, H., H. Zhu, C. Xu, and J. Yuan. (1998) Cleavage of BID by Caspase 8 mediates the mitochondrial damage in the fas pathway of apoptosis. Cell 94, 491501. [10] Boatright, K.M., and G.S. Salvesen. (2003) Mechanisms of caspase activation. Curr. Op. Cell Biol. 15, 725731. [11] Yin, X.M., K. Wang, A. Gross, et al. (1999) Biddeficient mice are resistant to Fasinduced hepatocellular apoptosis. Nature 400, 886891. [12] Chou, J.J., H. Li, G.S. Salvesen, et al. (1999). Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96, 615624. [13] McDonnell, J.M., D. Fushman, C.L. Miliman, et al. (1999) Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. Cell 96, 625634. [14] Zha, J., S. Weiler, K.J. Oh, et al. (2000) Posttranslational Nmyristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 17611765. [15] Kudla, G., S. Montessuit, R. Eskes, et al. (2000). The destabilization of lipid membranes induced by the Cterminal fragment of Caspase 8cleaved Bid is inhibited by the Nterminal fragment. J. Biol. Chem. 275, 22713 22718. [16] Lutter, M., M. Fang, X. Luo, et al. (2000). Cardiolipin provides specificity for targeting of tbid to mitochondria. Nat. Cell Biol. 2, 754756. [17] Tan, K.O., K.M.L. Tan, and V.C. Yu. (1999) A novel BH3like domain in BID is required for intramolecular interaction and autoinhibition of proapoptotic activity. J. Biol. Chem. 274, 2368723690. [18] Renshaw, S.A., C.E. Dempsey, F.A. Barnes, et al. (2004). Three novel Bid proteins generated by alternative splicing of the human Bid gene. J. Biol. Chem. 279, 28462855. [19] Breitschopf, K., A.M. Zeiher, and S. Dimmerler. (2000 Ubiquitinmediated degradation of the proapoptotic active form of Bid. J. Biol. Chem. 275, 2164821652. [20] Ciechanover, A., and R. BenSaadon. (2004) Nterminal ubiquitination: more protein substrates join in. Trends Cell Biol. 14, 103106. [21] Rigaut, G., A. Shevchenko, B. Rutz, et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotech. 17, 10301032. [22] Pichler, A., P. Knipscheer, E. Oberhofer, et al. (2005) SUMO modification of the ubiquitinconjugating enzyme E225K. Nat. Struct. Mol. Biol. 12, 264269. [23] Wang, X., R.A. Herr, W.J. Chua, et al. (2007) Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHCI by viral E3 ligase mk3. J. Cell Biol. 177, 613624. [24] Buchwald, G., P. van der Stoop, O. Weichenrieder, et al. (2006). Structure and E3ligase activity of the RingRing complex of Polycomb proteins Bmi1 and Ring1b. EMBO J. 25, 24652474. [25] Li, B., and Q.P. Dou. (2000) Bax degradation by the ubiquitin/proteasomedependent pathway: involvement in tumor survival and progression. Proc. Natl. Acad. Sci. U.S.A. 97, 38503855. [26] Sutton, V.R., M.E. Wowk, M. Cancilla, and J.A. Trappani. (2003) Caspase activation by Granzyme B is indirect and caspase autoproessing requires the release of proapoptotic mitochondrial factors. Immunity 18, 319 329. [27] Weissman, AM. (2001) Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2, 169178. [28] Jesenberger, V., and S. Jentsch. (2002) Deadly encounter: ubiquitin meets apoptosis. Nat. Rev. Mol. Cel.Biol. 3, 112121. [29] Chen, Z.J. (2005) Ubiquitin signalling in the NFκB pathway. Nat. Cell Biol. 7, 758765. [30] Breitschopf, K., J. Haendeler, P. Malchow, et al. (2000) Posttranslational modification of Bcl2 facilitates its proteasomedependent degradation: molecular characterization of the involved signaling pathway. Mol. Cell Biol. 20, 18861896. [31] Liu, H., H.S. Peng, Y.S. Cheng, et al. (2005). Stabilization and enhancement of the antiapoptotic activity of Mcl1 by TCTP. Mol. Cell. Biol. 25, 31173126. [32] Zhong, Q., W. Gao, F. Du, and X. Wang. (2005) Mule/ ARFBP1, a BH3only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl1 and regulates apoptosis. Cell 121, 10851095. [33] Kucharczak, J.F., M.J. Simmons, C.S. Duckett, and C. Gelinas. (2005) Constitutive proteasomemediated turnover of Bfl1/A1 and its processing in response to TNF receptor activation in FL5.12 prob cells convert it into a prodeath factor. Cell Death Differ. 12, 1225 1239. [34] Akiyama, T., P. Bouillet, T. Miyazaki, et al. (2003) Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3only Bcl2 family member Bim. EMBO J. 22, 66536664. [35] Ley, R., K. Balmanno, K. Hadfield, et al. (2003). Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasomedependent degradation of the BH3only protein, Bim. J. Biol. Chem. 278, 18811 18816. [36] Marshanksy, V., X. Wang, R. Bertrand, et al. (2001).

58 Chapter 3 Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl2 family members and compromise functioning of the electron transport chain in leukemic cells. J. Immunol. 166, 31303142. [36] Cadwell, K., and L. Coscoy. (2005) Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Science 309, 127130. [37] Chen, D., and Q. Zhou. (2004) Caspase cleavage of Bim EL triggers a positive feedback amplification of apoptotic signaling. Proc. Natl. Acad. Sci. U.S.A. 101, 12351240. [38] Clem R.J., E.H. Cheng, C.L. Karp, et al. (1998). Modulation of cell death by BclxL through caspase interaction. Proc. Natl. Acad. Sci. U.S.A. 95, 554559. [39] Werner, A.B., E. de Vries, S.W.G. Tait et al. (2002). Bcl2 family member Bfl1/A1 sequesters truncated Bid to inhibit its collaboration with proapoptotic Bak or Bax. J. Biol. Chem. 277, 2278122788. [40] Tait, S.W.G., A.B. Werner, E. de Vries, and J. Borst. (2004) Mechanism of action of Drosophila Reaper in human cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability. Cell Death Differ. 11, 800811. [41] de Melker, A.A., G. van der Horst, and J. Borst. (2001) ccbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route. J. Cell Science 114, 21672178. [42] Brummelkamp, T.R., R. Bernards, and R. Agami. (2002) Stable suppression of tumorigenicity by virusmediated RNA interference. Cancer Cell 2, 243247. [43] Waterhouse N.J., and J.A. Trappani. (2003) A new quantitative assay for cytochrome c release in apoptotic cells. Cell Death Differ. 10, 853855.

Unconventional ubiquitination allows Bid activity 59 Supplementary Information Table S1. Wildtype and Bid mutants tested for stabilization and ubiquitination HA tbidn TEV 30 Wild type T17A S9A;S10A S27A;C28A;S29A T17A;S27A;C28A;S29A C15A C28A C15A;C28A C15A;T17A;S27A;C28A;S29A S9A;S10A;C15A;T17A;S27A;C28A;S29A C3A;C15A;C28A C3A;C15A;T17A;S27A;C28A;S29A a Caspase8 cleavage site D60 Helix 2 Helix 1 Helix 3 BH3 domain NH 2 b 1 10 20 30 40 50 60 MDCEVNNGSSLRDECITNLLVFGGLQSCSDNSFRRELDALGHELPVLAPQWEGYDELQTD Helix 1 Helix 2 GNRSSHSRLGRIEADSESQEDIIRNIARHLAQVGDSMDRSIPPGLVNGLALQLRNTSR Helix 3, BH3 domain Helix 4 SEEDRNRDLATALEQLLQAYPRDMEKEKTMLVLALLAKKVASHTPSLLRDVFHTTVNFINQNLRTYVRSLARNGMD Helix 5 Helix 6 Helix 7 Helix 8 c Supplementary Figure 1. Bid structure and sequence. (a) The three dimensional structure of full length human Bid, as determined by NMR (Chou et al., 1999; McDonnell et al., 1999]. Cleavage by Caspase8 at D60 results in the tbidn fragment (residues 160) indicated in blue and the tbidc fragment indicated in red, which in vitro remain associated in a complex with the same structure as uncleaved Bid [Chou et al., 1999]. The proapoptotic BH3 domain (helix 3) is indicated in yellow. (b) Amino acid sequence of full length human Bid. Amino acids of tbidn are numbered. Helices (H) 18 are annotated based on Chou et al. [1999]. The Caspase8 cleavage site is indicated with the large arrowhead. (c) Amino acid sequence of tbidn in various species. Cysteine residues are indicated in red, the conserved T17 is underlined.

60 Chapter 3 62 49 38 28 18 14 6 TAP tbidn TAP 23 tbidn TAP TAP + HA Ubiquitin tbidn TAP 23 tbidn TAP tbidn TAP 23 tbidn TAP Blot: antiha 49 38 28 17 14 IgG Cal antiha blot antibid blot antibid 17 tbidn TEV TAP Supplementary Figure 2. The Bid sequence is target for ubiquitination. HA ubiquitin was coexpressed in HeLa cells with the TAP empty vector, tbidn TAP and tbidn TAP lacking the first 23 amino acids of Bid (Δ23). Proteins were purified on the TAP tag, separated by SDSPAGE and probed by immunoblotting for ubiquitin and Bid with antiha and antibid antibodies, respectively. Data show that ubiquitination is specific for Bid, since the TAP tag alone does not carry ubiquitin and deletion mutation of Bid abrogates ubiquitination. MG132: + CHX: 0 15 30 45 45 min HA tbidn TEV30 CHX: 0 10 20 30 40 min HA tbidn TEV30 wildtype (7.5 kd) wildtype C15;C28 C3;C15;C28 wildtype C3; C15; T17; S27;C28;S29 T17; S27;C28;S29 TCL, antiha blot C15;T17; S27;C28;S29 wildtype S9; S10 S9; S10; C15;T17; S27;C28;S29 TCL, antiha blot Supplementary Figure 3. Effects of point mutations on tbidn stability. Alanine substitutions mutations were generated in HA tbidn TEV30. Wildtype and mutant proteins were expressed in HeLa cells, which were incubated for the indicated time periods with CHX with or without MG132. Total cell lysates (TCL) were examined by antiha immunoblotting for levels of HA tbidn TEV30. Lines indicate samples tested together in one experiment.

Unconventional ubiquitination allows Bid activity 61 188 98 62 49 38 28 17 14 6 C isolate C ME ph7.4 ph12 C isolate C ME ph7.4 ph12 Blot: antiubiquitin (P4D1) antiring1b Ring1b Supplementary Figure 4. Control for hydrolysis and reduction of ubiquitination. A complex of Ring1b and Bmi1 was subjected to in vitro ubiquitination. Ubiquitin is attached to lysine on Ring1b, as shown by mass spectrometry (Buchwald et al. 2006). The in vitro ubiquitinated sample without further treatment is the control (C) shown in the first lane. The other lanes show material that was recovered by precipitation with Ni agarose beads (isolate) after it had been exposed to the indicated conditions: buffer (C), reduction with βmercaptoethanol (βme), mild hydrolysis at ph12 and incubation at ph7.4 as control. The precise procedures are outlined in the Materials and Methods section. Recovered materials were resolved by Nu PAGE and immunoblotted as indicated to detect Ring1b and its ubiquitinated species. The arrow denotes the nonubiquitinated Ring 1b protein. Lower MW species (bracket) are Ring1b breakdown products. Myc (Nterminus) GFP (Cterminus) Overlay + MitoTracker control 15 m TNF 2 h 20 m 4 h (40% of cells) 15 m 8 h (80% of cells) 15 m Supplementary Figure 5. Time course of tbidn disappearance. MCF7 Bid RNAi cells expressing Myc Bid GFP were stimulated with TNFα for 2, 4 or 8 h or not (control), stained to detect mitochondria (MitoTracker) and the Nterminal Myc tag and examined by CLSM. The 4 h time point represents cells in which the GFP signal localized at mitochondria (about 40% of the population). At 8 h, the frequency of cells with such localisation was about 80%. In the other cells, localisation was as depicted for the 2 h time point.