Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin
2002; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.1093/emboj/21.11.2602
ISSN1460-2075
Autores Tópico(s)RNA Interference and Gene Delivery
ResumoArticle3 June 2002free access Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin Hsei-Wei Wang Hsei-Wei Wang The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Tyson V. Sharp Tyson V. Sharp The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Andrew Koumi Andrew Koumi The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Georgy Koentges Georgy Koentges The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Chris Boshoff Corresponding Author Chris Boshoff The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Hsei-Wei Wang Hsei-Wei Wang The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Tyson V. Sharp Tyson V. Sharp The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Andrew Koumi Andrew Koumi The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Georgy Koentges Georgy Koentges The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Chris Boshoff Corresponding Author Chris Boshoff The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK Search for more papers by this author Author Information Hsei-Wei Wang1, Tyson V. Sharp1, Andrew Koumi1, Georgy Koentges1 and Chris Boshoff 1 1The Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London, WC1E 6BT UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2602-2615https://doi.org/10.1093/emboj/21.11.2602 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-ΔEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin-ΔEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral anti-apoptotic protein and survivin-ΔEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-ΔEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor-of-apoptosis protein). Introduction Apoptotic cell death is a major antiviral cellular response to block the propagation of viruses (Tschopp et al., 1998b; Boya et al., 2001). Two apoptotic pathways in mammalian cells are employed to destroy virus-infected cells: the extrinsic death-receptor pathway and the intrinsic mitochondrial pathway. Both pathways converge to activate caspases, in particular the effector caspase-3 (Thornberry and Lazebnik, 1998). In the extrinsic pathway, death receptors belonging to the tumour necrosis factor (TNF) receptor gene superfamily, such as Fas/CD95 or TNFR1, utilize protein interaction modules to assemble receptor-signalling complexes that recruit and activate procaspases 8 and 10 (Ashkenazi and Dixit, 1998; Wallach et al., 1999). Activated caspase-8 and -10 then activate a cascade of caspases, which finally results in the activation of downstream effector caspases (such as caspase-3, -6 and -7), resulting in dismantling and removal of the cell (Thornberry and Lazebnik, 1998; Hengartner, 2000). This is one of the pathways activated by cytotoxic T-lymphocytes to kill virus-infected cells (Harty et al., 2000; Guidotti and Chisari, 2001). The intrinsic protection mechanism involves the participation of mitochondria, which release caspase-activating proteins. The transcriptional activation of Bcl-2 homology domain 3 (BH3) containing pro-apoptotic proteins of the Bcl-2 family (such as Bax) results in alternations in mitochondrial membrane potential, leading to the release of pro-apoptotic molecules such as cytochrome c (Adams and Cory, 1998; Green and Reed, 1998). Cytochrome c activates caspases by binding to and activating Apaf-1, inducing it to associate with procaspase-9, thereby triggering procaspase-9 activation and initiating effector caspases. When an infected cell senses the unscheduled activation of the cell cycle by viral proteins or DNA damage induced by viral replication, the cell initiates this intrinsic pathway (Evan and Littlewood, 1998; Vousden, 2000). The activation of effector caspases by either pathway is blocked by cytoplasmic proteins, the inhibitor-of-apoptosis proteins (IAPs). IAPs contain at least one BIR (baculovirus IAP repeat) domain that binds to procaspase-9 and activated effector caspase-3 and -7 to inhibit their activity (Deveraux and Reed, 1999; Miller, 1999). The BIR core includes a C2HC motif chelating a zinc ion that plays an essential role in inhibiting apoptosis (Deveraux et al., 1997; Roy et al., 1997; Vucic et al., 1998; Hinds et al., 1999). IAPs can also suppress apoptosis through caspase-independent mechanisms, which involve transcription factors such as NF-κB (Chu et al., 1997; You et al., 1997). The mammalian prototype of the IAP family is survivin, which is the smallest known IAP family protein (∼16 KDa) and contains a single BIR domain with which it binds caspases and prevents caspase-induced apoptosis (Tamm et al., 1998; Verhagen et al., 2001). Altered expression of survivin appears to be a common event associated with the pathogenesis of human cancer; survivin is overexpressed in many transformed cell lines and in common cancers, such as those of the lung, colon, liver, prostate and breast (Ambrosini et al., 1997; Ito et al., 2000). Reduced survivin expression causes apoptosis and sensitization to anticancer drugs, suggesting that survivin expression is important for cell survival or chemoresistance of certain carcinomas (Ambrosini et al., 1998; Olie et al., 2000; Satyamoorthy et al., 2001). An alternative anti-apoptotic mechanism is mediated by members of the Bcl-2 anti-apoptotic family, which possess two conserved motifs known as Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Adams and Cory, 1998; Chao and Korsmeyer, 1998). Coalescence of the α-helices in the BH1 and BH2 regions create an elongated hydrophobic cleft to form homo- or heterodimers with other proteins in this family (Muchmore et al., 1996; Sattler et al., 1997). The E1B-19K protein of adenovirus is the only known protein of this family that contains only a BH1, but no BH2 domain (Adams and Cory, 1998). However, no protein with only a BH2 domain has yet been described. Bcl-2 family members act by forming or controlling pores on the outer membrane of mitochondria, through which the release of cytochrome c and other intermembrane pro-apoptotic proteins is regulated. In addition, the anti-apoptotic Bcl-2 members might function directly to regulate caspase activities by binding to an adaptor molecule (Hengartner, 2000). These adaptors either bridge Bcl-2/Bcl-XL and Apaf-1, thereby inhibiting Apaf-1 activation, or bind procaspase-8 to Bcl-2/Bcl-XL, preventing its activation (Ng et al., 1997; Chau et al., 2000; Zhang et al., 2000). No effector caspase-binding adaptor has yet been identified. Viruses employ an arsenal of proteins to interfere with pro-apoptotic signalling pathways, affording a selective advantage for them to maintain a persistent infection or to prolong the survival of lytically infected cells, allowing maximum virus progeny production (O'Brien, 1998; Tschopp et al., 1998b). Many viruses express anti-apoptotic proteins, including IAP caspase inhibitors, Bcl-2 homologues and death-effector-domain-containing proteins termed vFLIPs [viral FLICE (Fas-associated death-domain-like IL-1 β-converting enzyme)-inhibitory proteins]. The degenerated caspase homologue vFLIP blocks the death-receptor pathway and the viral Bcl-2 homologues protect cells from death by maintaining mitochondrial integrity (Tschopp et al., 1998a; Boya et al., 2001). Studies of viral apoptosis inhibitors provide a valuable insight into the biochemical processes involved in the execution of cell death (Mosialos et al., 1995; O'Brien, 1998). Viral FLIPs led to the identification of cellular homologues (cFLIPs) (Irmler et al., 1997), while the BIR-containing caspase inhibitor (p35) of baculovirus guided the identification of the mammalian IAP family (Seshagiri et al., 1999). KSHV (Kaposi's sarcoma-associated herpesvirus; human herpesvirus-8) is an oncogenic virus closely associated with the pathogenesis of Kaposi's sarcoma and certain lymphoproliferations such as primary effusion lymphoma (PEL) and multicentric Castleman's disease (Boshoff and Weiss, 2001; Moore and Chang, 2001). Two KSHV anti-apoptotic proteins have been identified: the vBcl-2 encoded by open reading frame (ORF) 16 and the vFLIP encoded by ORF K13. However, no IAP homologue has yet been identified in KSHV or any other known herpesvirus. The K7 protein of KSHV is encoded by the putative ORF K7, which is unique to KSHV. K7 has no obvious homologue in other γ2 herpesviruses, including the close relative rhesus rhadinovirus (McGeoch and Davison, 1999; Alexander et al., 2000). This suggests that KSHV may have acquired this specific gene from its human host genome relatively recently. Here we report that the K7 protein is structurally and functionally related to a recently identified spliced variant of human survivin, survivin-ΔEx3 (Mahotka et al., 1999; Krieg et al., 2002). Both proteins protect cells from apoptosis and possess similar functional domains, therefore K7 is a new viral anti-apoptotic protein and survivin-ΔEx3 appears to be its cellular homologue. Results K7 similarity to human survivin-ΔEx3 By searching the public databases, K7 was found to harbour a plant-form mitochondrial-targeting signal (MTS) and a transmembrane domain (Figure 1A), which suggested that K7 might be a mitochondrial protein. Several putative post-translational modification sites were found in K7, including an N-glycosylation site, a myristoylation site and several phosphorylation sites (Figure 1A). Hydropathy plots predicted K7 to be a single-transmembrane protein that is highly hydrophobic in the N-terminus (data not shown). Furthermore, by using different fragments of K7 protein as bait probe, we identified a BIR domain in K7 (Figure 1B). The BIR domain is essential and sufficient for IAPs to protect cells from apoptosis (Vucic et al., 1998). The IAP family member most similar to K7 is human survivin (E = 0.014; Figure 1B, upper panel). Sequence alignment and phylogenic analyses of BIR domains also revealed that K7 and survivin display the greatest similarity (Figure 1D and E). The K7 and survivin BIR domains are also comparable in three-dimensional (3D) structure predictions (Figure 1B) (Chantalat et al., 2000; Muchmore et al., 2000; Verdecia et al., 2000). However, K7 possesses the N-terminal half of a BIR motif and lacks the C-terminal-half C2HC Zn-binding fold of other BIR domains, and in this respect K7 is most similar to a naturally occurring splice variant of survivin, survivin-ΔEx3 (Figure 1C), which has been shown to be expressed in cancer cells and to be a functional anti-apoptotic protein (Mahotka et al., 1999; Krieg et al., 2002). Figure 1.K7 has sequence and predictive structural similarities to survivin-ΔEx3. (A) Schematic representation of the structure of K7. The MTS, the transmembrane (TM) domain, the BIR-like domain (N-BIR) and the putative BH2 domain of K7 are indicated. Several putative post-translational modification sites of K7, such as the N-myristoylation site (N-myr), SH3 motif, protein kinase C phosphorylation sites (PKC) and the N-glycosylation site (N-Gly), are also shown. (B). Comparison between K7 and survivin BIR domains. Sequence alignment of K7 and survivin BIR domains. (Upper panel) The BIR domain of survivin is underlined. (Bottom left) Ribbon representation of the K7 BIR domain. The α-helices, β-strands and turns are represented as red coils, yellow arrows and blue loops, respectively, and the corresponding amino acids are shown in the upper panel. (Bottom right) Superimposed image of K7 and survivin BIR domains. K7 is represented by red strands and survivin by blue strands. (C) Endogenous expression of survivin and survivin-ΔEx3, and the structures of these two isoforms. The kinetochore-binding domain of survivin, which is essential for chromosome segregation and cytokinesis, is indicated. R/K-rich, Arg/Lys-rich region. PCR analysis for cDNAs of survivin isoforms in a human fetal cDNA library is also shown (bottom panel). (D) Alignment of different BIR domains. The species designations used for the alignment, and the primary accession number or database entry number for each sequence are as follows, with the indicated accession numbers: K7, AAC57096. Human BIR domain proteins: survivin, 2315863; XIAP (X-linked IAP), P98170; cIAP1, NP_001157; cIAP2, NP_001156. Mouse: BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), CAA76720; TIAP, BAA28266; NAIP (neuronal apoptosis inhibitory protein), AAB69223. Drosophila: Deterin, XP_081836. Schizosaccharomyces pombe BIR protein SpIAP, NP_587866. Caenorhabditis elegans BIR protein CeIAP, NP_505949. Numbers to the left of the sequences indicate the positions of the amino acids in each protein. Residues conserved in the majority of the sequences are in black boxes, whereas similar sequences are in grey. (E) Phylogenic relationship of different BIR domains. The alignment data shown in (D) was used to construct a Phylogenic tree, which was derived by maximum likelihood searching by the TreeView program. The divergence scale of the across-page branches is indicated. (F) Alignment of the BH2 domain of Bcl-2 family members with K7 and survivin-ΔEx3. The following human and viral proteins are used in this alignment: Bax-β (AAA03620), Bax-α (AAA03619), Bcl-2 (AAA35591), Bcl-XL (CAA80661), Bcl-W (NP_004041), EBV (Epstein–Barr virus Bcl-2; CAA01638), HVP (herpesvirus papio Bcl-2; AAF99596), KSHV (KSHV Bcl-2; AAB62596), RHV (Rhesus herpesvirus Bcl-2; AAD21342), HVS (herpesvirus simiri Bcl-2; CAA45639), AHV3 (Ateline herpesvirus 3 Bcl-2; NP_047987), BHV4 (bovine herpesvirus 4 Bcl-2; AAD34361), Bak (Q16611), and ASFV (African swine fever virus Bcl-2; NP_042735). The R/K-rich region in survivin-ΔEx3 is underlined. (G) Phylogenic tree for BH2 domain-containing apoptotic regulators. The same BH2 domains shown in Figure 1F were used to construct this tree. Download figure Download PowerPoint survivin-ΔEx3 mRNA is a product of alternative splicing and lacks the exon 3 of survivin (Mahotka et al., 1999). The skipping of exon 3 in survivin-ΔEx3 mRNA results in an interrupted BIR domain followed by an additional frame shift in exon 4. Consequently, the survivin-ΔEx3 ORF ends in the 3′ untranslated region of survivin and generates a novel 63-amino-acid-long C-terminal tail. survivin-ΔEx3 has been shown to be present in cancer tissues (Mahotka et al., 1999; Krieg et al., 2002), and when we attempted to clone survivin from a human fetal tissue library, we amplified an additional smaller product (Figure 1C). Direct sequencing of this amplicon revealed that the sequences of survivin-ΔEx3 and survivin in the fetal tissue library are identical to those described in cancer cells (data not shown). To gain further insight into survivin-ΔEx3, we performed protein domain analysis and secondary structure predictions. We found that in the new C-terminus of survivin-ΔEx3, a transmembrane domain and a novel potential plant-form MTS are created, similar to the N-terminal part of K7 (Figure 1A and C). In addition, there is a putative BH2 domain on both K7 and survivin-ΔEx3 (Figure 1A and C). Comparison of the BH2 domain sequences of K7 and survivin-ΔEx3 with other known Bcl-2 family members indicated that they share limited similarity to the BH2 domains of known apoptotic regulators (Figure 1F), and that both proteins clustered together in phylogenetic analysis (Figure 1G). Neither K7 nor survivin-ΔEx3 has a BH1 domain, which normally cooperates with the BH2 domain for anti-apoptotic function (Adams and Cory, 1998; Chao and Korsmeyer, 1998). The highly structural similarities between K7 and human survivin-ΔEx3, together with the finding of putative BH2 and BIR domains on K7 (Figure 1A and C), suggest that K7 is an anti-apoptotic protein. Expression of K7 gene in PEL cells Microarray analyses have shown previously that K7 is either expressed in latency or is highly inducible upon n-butyrate induction of KSHV latently infected lymphoma cells (Jenner et al., 2001; Paulose-Murphy et al., 2001). However, the probes used in these array studies could have cross-hybridized with the PAN (polyadenylated nuclear) RNA gene, which is highly expressed in the virus lytic cycle and overlaps with the K7 ORF (Figure 2A). We performed northern blot analysis to detect K7-specific RNA in KSHV-positive PEL cell lines by using a K7-specific probe (5′-UTR probe; Figure 2A). Three K7-specific RNAs were identified in KSHV-positive BCP-1 PEL cells (Figure 2B, left) and two K7-specific transcripts in another PEL cell line BC-3 (1.8- and 5-kb species; data not shown). These RNA species could not be detected in KSHV-negative B-cell lines such as Ramos (Figure 2B, right) and DG75 cells (not shown). The 1.8-kb RNA transcript was detectable within 2 h after 12-O-tetradecaylphorbol 13-acetate (TPA) induction, followed by the 3 and 5 kb RNA species (Figure 2B). These RNAs increased with time and reached a peak 24 h post-treatment (Figure 2B). To control for loading and integrity of the mRNA, the same blots were re-probed with GAPDH (Figure 2B). Hybridizing the same blots with a probe to a KSHV lytic gene product, PAN (Sun et al., 1996, 1999), confirmed successful induction of the viral lytic cycle (Figure 2B). Figure 2.K7 is a lytic viral gene. (A) Schematic representation of the structure of the K7 gene. The cDNA probe used in northern blot analysis and the K7-specific primers used in RT–PCRs are shown. (B) Northern blot analyses of K7 RNAs. The KSHV latently infected PEL cell line BCP-1 and the KSHV-negative Ramos cell line were lytically induced by TPA, and total RNA was extracted at the indicated time points. Blots were first probed against K7 mRNA, then reprobed with a GAPDH probe or with another KSHV gene, PAN, to indicate the induction of viral lytic cycle. (C) RT–PCR analysis for K7 transcripts. RNA from TPA-treated, KSHV-infected PEL cells (BC3) was collected at the indicated time points. M, 100-bp DNA fragment markers. (+), RNA from TPA-treated BCP-1 cells (lane 2) and cDNA of K7 (lane 3) were used as positive controls. Download figure Download PowerPoint We also used RT–PCR to detect K7 RNA and to confirm that K7 is expressed only during the lytic viral cycle. The sense primer used in PCR is upstream of the PAN gene but within the K7 ORF, thus avoiding amplification of PAN (Figure 2A). In TPA-untreated latent PEL cells, a small amount of K7 RNA could be detected (Figure 2C), and this possibly arises from the 2–3% of lytic cells present in untreated cells (Dupin et al., 1999). We conclude that the K7 gene is a lytic gene expressed in PEL cells. Expression and characterization of K7 protein in transfected cells We cloned K7 ORF cDNA from BC3 cDNA library and tagged it at both the N- and C-termini with the influenza virus haemagglutinin (HA) epitope. HeLa and 293 cells were transiently transfected with this construct and western blotting revealed two K7-specific signals (Figure 3A). The predicted molecular mass of tagged K7 is 15.8 kDa, correlating to the 16 kDa band detected (Figure 3A). In addition, a 19 kDa band was also detected (Figure 3A). To determine whether this band was due to post-translational modification, we treated K7-transfected HeLa cells with tunicamycin, a potent inhibitor of N-linked glycosylation, and this led to a dose-dependent loss in the appearance of processed K7 (gp19; Figure 3B, left). Mutation in the N-glycosylation site of K7 (mutant N108Q) totally removed the higher modified form (Figure 3B, right), confirming that the 19 kDa band was due to the N-glycosylation of K7. The N-glycosylation of K7 was enhanced after TNF-α treatment (Figure 3C), further implying an important physiological role of this modification. Figure 3.K7 expressed as a predicted 16 kDa protein and as a 19 kDa glycoprotein. Cells were transfected with the indicated plasmids, and protein extracts were collected 48 h after transfection and probed by western blots with an anti-HA mAb. (A) K7 expression pattern. Two different forms of K7 were detected in transfected cells. (B) The 19 kDa form of K7 is a glycoprotein. (Left panel) HeLa cells transfected with K7 were treated with different doses of tunicamycin to inhibit N-glycosylation for 24 h before extraction. (Right panel) A point mutation (N108Q) on the N-glycosylation site of K7 could prevent K7 from modification. (C) The N-glycosylation of K7 is increased after TNF-α treatment. HeLa cells transfected with a K7-expressing plasmid or with vector alone were exposed 24 h post-transfection to 10 ng/ml TNF-α plus 1 μg/ml CHX. (D) K7 has a short half-life. K7-transfected HeLa cells were treated with 10 μg/ml CHX at 48 h post-transfection to block protein synthesis. Cell extracts were collected at indicated time points. (Lower panel) Same blot stained with Coomassie Blue to show equal loading of protein in each lane. (E) K7 forms homodimers. HeLa cell extracts, which contain transfected K7, were incubated with GST, GST-K7-CT or GST-K7-BIR. Specifically bound proteins were eluted from beads with SDS–PAGE sample buffer before analysis. Download figure Download PowerPoint To determine the half-life of K7, transfected cells were treated with 10 μg/ml of the protein synthesis inhibitor cycloheximide (CHX). At this concentration, new cellular translation stops and only the existing proteins can be detected (Akgul et al., 2000). Forty-eight hours after transfection, the transfected HeLa cells were treated with CHX for the indicated periods before cell extracts were collected and analysed. K7 was undetectable within 2 h of treatment, indicating that K7 is a labile, short half-life protein, similar to some other apoptotic proteins (Figure 3D) (Somia et al., 1999; Akgul et al., 2000; Duriez et al., 2000). We next tested whether K7, like Bcl-2 family members and similar to human survivin (Adams and Cory, 1998; Chantalat et al., 2000; Muchmore et al., 2000), can associate with itself to form homodimers. The C-terminal (CT) 56 amino acids of K7, which contains the putative BIR and BH2 domains, and the BIR domain of K7 were fused to a glutathione S-transferase (GST) protein domain, expressed in Escherichia coli to give GST-K7-CT/GST-K7-BIR recombinant proteins, and then tested for interaction with full-length K7 by in vitro GST pull-down assays. Figure 3E shows that both GST-K7-CT and GST-K7-BIR recombinant proteins precipitate full-length K7. K7 localizes to mitochondria, endoplasmic reticulum and the nuclear membrane We examined the subcellular localization of K7 in cells by immunofluorescence assay. K7 is a cytosolic protein with a nuclear membrane staining pattern (Figure 4A). Expression of K7 in cells revealed a punctate cytoplasmic and lacy reticular staining pattern (Figure 4A), which is similar to that of mitochondria, but distinct from Golgi, and lysosome staining (data not shown). Staining of K7-expressing cells with the mitochondria-specific dye MitoTracker Red verified that K7 colocalized with mitochondria (Figure 4B). However, not all K7 proteins in cells are distributed to mitochondria; some green K7 signals did not overlap with the red mitochondrial staining (Figure 4B). Cellular fractionation experiments confirmed that K7 was located in mitochondria and nuclear membrane fractions, and also showed that K7 resided in the endoplasmic reticulum (ER) (Figure 4C). In the ER, only the unglycosylated 16 kDa form of K7 was present (Figure 4C). The distribution pattern of K7 was similar to that of Bcl-2, but was distinct from that of Bax (Figure 4C), which is known to reside in the cytoplasm and only translocates to mitochondria in response to death signals (Iwahashi et al., 1997; Wolter et al., 1997; Gross et al., 1998). Figure 4.K7 localizes to the mitochondria and ER. (A) Confocal microscopy of HeLa cells transfected with HA-tagged K7. Transfected cells were stained with an anti-HA mAb at 48 h post-transfection. (B) Colocalization of K7 and mitochondria. K7-transfected HeLa cells were stained with antibody to HA (green), and mitochondria were stained with CMXRos (red). The cell nucleus was stained with Hoechst 33258 dye (blue). Partial colocalization is seen upon overlay of these images. (C) Subcellular fractionation of HeLa cells transfected with K7. Transfected cells were separated into nucleoplasmic (NP), cytoplasmic (C), mitochondria (M) and ER fractions. The fractions were probed by western blots with antibodies to HA (for tagged K7), human Bcl-2 and Bax. (D) The N-terminal 70 amino acids of K7 is sufficient and essential for mitochondrial targeting. HeLa cells transfected with the indicated plasmids were subjected to subcellular fractionation analysis. The fractions were probed by western blots with a polyclonal antibody to GST to detect each fusion protein. Download figure Download PowerPoint Computational analyses suggested a putative mitochondrial-targeting domain in the K7 N-terminus (Figure 1A). We next investigated whether this domain is functional. Full-length K7, as well as the N-terminus (amino acids 1–70) and C-terminus (amino acids 71–126), were fused with a GST protein domain in a eukaryotic expression vector, transfected into cells and then examined for intracellular localization. Immunoblot analysis of subcellular fractions demonstrated that the ectopically expressed GST–K7 fusion protein was located in the mitochondria and ER fractions, whereas the GST protein itself was cytosolic (Figure 4D). The N-terminus of K7 localized GST to the mitochondria and ER (Figure 4D, GST-N70), but the C-terminus did not (Figure 4D, GST-C56). K7 can protect cells from apoptosis induced by various stimuli The putative functions of K7 were first explored by transient transfection assays. We tested the cytoprotective function of K7 on apoptosis induced by different stimuli (Figure 5). In Figure 5A, apoptosis was induced by TNF-α or anti-Fas monoclonal antibody (mAb) in the presence of 1 μg/ml of CHX, and apoptotic cells were stained by Annexin V–fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining and counted by flow cytometry. In the presence of K7, apoptosis was reduced to 30–50% (Figure 5A), whereas GFP or LacZ protein did not protect transfected cells from apoptosis (not shown). Figure 5.K7 inhibits apoptosis. (A) K7 protects cells from death receptor mediated apoptosis. HeLa cells transfected with a K7-expressing plasmid or with vector alone were exposed 24 h post-transfection to 10 ng/ml TNF-α or 100 ng/ml anti-Fas mAb plus 1 μg/ml CHX. Apoptotic cells were double stained with Annexin V–FITC/PI and counted by flow cytometry. Results are expressed as the mean ± SD of three independent experiments. (B) K7 rescues Bax-induced apoptosis. HeLa cells were transfected with 100 ng of Bax-expression plasmid plus 400 ng (4:1), 900 ng (9:1) or 1800 ng (18:1) of the indicated constructs. Forty-eight hours post-transfection, all cells, both adherent and in suspension, were pooled, washed and stained with the mitochondrial membrane-specific dye CMXRosamine to assess the mitochondrial membrane potential (ΔΨm), and were th
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