tula affects cell death through a functional … · tula, we purified proteins that interact with...
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TULA AFFECTS CELL DEATH THROUGH A FUNCTIONAL INTERACTION WITHAIF, A KEY FACTOR OF CASPASE-INDEPENDENT APOPTOSIS*
Therese S. Collingwood ,§, Evgeniya V. Smirnova , Marina Bogush , Nick Carpino#, RolandS. Annan ,§, and Alexander Y. Tsygankov ,||
Department of Microbiology and Immunology, and ||Fels Institute for CancerResearch and Molecular Biology, Temple University School of Medicine,
Philadelphia, PA; §Proteomics and Biological Mass Spectrometry Laboratory,GlaxoSmithKline, King of Prussia, PA; #Department of Molecular Genetics andMicrobiology, State University of New York at Stony Brook, Stony Brook, NY
Running title: TULA interacts with AIF and affects cell death
Address correspondence to Alexander Y. Tsygankov, 1Department of Microbiology and Immunology,Temple University School of Medicine, Kresge Bldg., Rm. 506, 3400 N. Broad St., Philadelphia, PA19140; Tel. 215-707-1745; FAX 215-707-5205; E-Mail: [email protected]
The lymphoid protein TULA/Sts-2 isassociated with c-Cbl and ubiquitylated proteinsand has been implicated in the regulation ofsignaling mediated by protein tyrosine kinases.The results presented in this report indicate thatTULA facilitates T-cell apoptosis independent ofeither T-cell receptor/CD3-mediated signaling orcaspase activity. Mass spectrometry-basedanalysis of protein-protein interactions of TULAdemonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously beenshown to function as a key factor of caspase-independent apoptosis. Using RNAi, wedemonstrate that AIF is essential for theapoptotic effect of TULA. Analysis of the sub-cellular localization of TULA and AIF togetherwith the functional analysis of TULA mutants isconsistent with the idea that TULA enhances theapoptotic effect of AIF by facilitating theinteractions of AIF with its apoptotic co-factors,which remain to be identified. Overall, ourresults shed new light on the biological functionsof TULA, a recently discovered protein,describing its role as one of very few knownfunctional interactors of AIF.
We recently identified TULA among multipleproteins that co-purified with c-Cbl from T-lymphoblastoid cells (1). TULA contains an N-terminal UBA domain, a centrally positioned SH3domain and a region of homology to
phosphoglyceromutases (PGM), which wasinitially termed HCD (Figure 1; (1,2)). TULAbinds to c-Cbl through its SH3 domain and toubiquitin and ubiquitylated proteins through itsUBA domain (1,3). Dimerization of TULAthrough its PGM domain has also been shown(3). Analysis of cell and tissue expression ofTULA demonstrates that this protein is expressedprimarily in T and B lymphocytes and islocalized both in the cytoplasm and in the nucleus(1,4).
A mouse orthologue of TULA (Sts-2) wasrecently identified (4), as was a second memberof the family, Sts-1 (5). Unlike TULA, Sts-1 isexpressed ubiquitously (4,5). (In this report wewill use the term TULA for consistency.)
TULA has been implicated in the regulationof cell signaling mediated by protein tyrosinekinases (PTKs). On the one hand, TULA wasreported to increase activity of receptor PTKs byinhibiting c-Cbl-driven downregulation of theiractivated forms. This appears to be mediated bypreventing interactions between ubiquitylatedforms of activated PTKs and proteins recruitingthem to the degradation pathway and, possibly,by decreasing the level of c-Cbl (1,3). On theother, the lack of both proteins of the TULA/Stsfamily resulted in hyper-reactivity of Tlymphocytes correlated with an increase in theactivity of Zap-70, the molecular basis of whichremained unclear (4). These results implied that
http://www.jbc.org/cgi/doi/10.1074/jbc.M706870200The latest version is at JBC Papers in Press. Published on August 20, 2007 as Manuscript M706870200
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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the effect of TULA on PTKs might not be the onlymechanism through which TULA exerts itsbiological effect. Indeed, the presence in TULA ofmultiple functional domains and extensive stretchesof amino acid sequences with unknown functionssuggested that TULA might exert effects unrelatedto either c-Cbl or PTKs.
In an effort to discover novel functions ofTULA, we purified proteins that interact withTULA and identified among them Apoptosis-Inducing Factor (AIF). AIF is a key factor ofcaspase-independent apoptosis (6-8). In the absenceof cellular stress signals, AIF is localized to theinternal mitochondrial membrane, where itfunctions as a FAD-dependent NADH oxidase,which is required for normal oxidativephosphorylation (9) and maintenance ofmitochondrial structure (10). Under conditionsinducing apoptosis, AIF is released frommitochondria (11-14) and translocated to thenucleus, where it induces caspase-independentapoptotic events through binding to DNA (15).These two functions of AIF are mediated bydistinct structural domains (15,16) and can bedissociated (6,10,17).
Overall, the molecular mechanism of theapoptotic effect of AIF remains poorly understood,and in particular, few functional interaction partnersof AIF have been identified (18-21). Our work,presented here, demonstrates that TULA and AIFare interaction partners and establishes a functionallink between them in inducing caspase-independentapoptosis. These results shed new light on themechanism of the apoptotic effect of AIF andreveal a novel biological function of TULA.
EXPERIMENTAL PROCEDURES
DNA constructs and mutagenesis - cDNAencoding the full-length TULA or its N-terminalhalf (TULA-N1/2) was subcloned into the pFLAG5a vector (Sigma, St. Louis, MO) using theAdvantage-Hf2 polymerase (Clontech, MountainView, CA). The forward primer (5’ CAG GATATC ATG GCA GCG GGG GAG 3’) annealed tonucleotides at the N-terminal end of TULA andincluded a unique EcoRV restriction site. Thereverse primers (5’ TAG GGT ACC ATC CGTGTA GTT TTC C 3’ and 5’ TAG GGT ACC GTTGCC TGA GAT CCA GTT 3’) annealed tonucleotides 893 to 908 (TULA-N1/2) or 1863 to
1880 (full-length TULA) within the TULA short(1) protein sequence and included a unique KpnIrestriction site. These restriction sites wereincluded to create compatible ends for ligatingthe fragments into the pFLAG 5a vector. Theobtained constructs were confirmed bysequencing.
To introduce mutations, two syntheticoligonucleotides complementary to the oppositestrands of double-stranded DNA containing thesequence to be mutated were designed to contain15-18 nucleotides on either side of the mutationsite. The oligonucleotides were gel purified (IDTTechnologies, Coralville, IA). The mutagenesisreactions were performed using the QuikChangeSite-Directed Mutagenesis Kit according to themanufacturer’s recommendations (Stratagene, LaJolla, CA).
Cells - HEK293T and HeLa cells werecultured in DMEM supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/mlstreptomycin and 10% fetal bovine serum (FBS)(complete medium). HEK293T cells were plated24 hours before transfection to be 80% confluenton the day of transfection in antibiotic-freemedium. Purified plasmid DNA was transfectedinto HEK293T cells (10-20 mg per 2x106 cells)using Lipofectamine-2000 (Invitrogen/LifeTechnologies, Carlsbad, CA) according to themanufacturer’s recommendations. After a total of48 hours, transfected cells were harvested andwashed with phosphate-buffered saline (PBS).Cells were lysed in CelLytic buffer (Sigma) for15 minutes at room temperature, and cell debriswas removed by centrifugation. HeLa cells weretransfected in the same fashion, but usingLipofectin (Invitrogen/Life Technologies) orFugene6 (Roche, Indianapolis, IN).
Jurkat-Tag cells were cultured in RPMI1640supplemented with 20 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/mlstreptomycin and 10% FBS (complete medium).The cells were grown in antibiotic-free mediumfor 24 hours prior to electroporation. Cells werecentrifuged and resuspended at a final density of2x107 cells/ml in antibiotic-free medium. DNA(10 mg) was added to a 4-mm cuvette followed byaddition of 1x107 cells in 500 ml medium. Themix was pulsed at 310V, 10ms, in anelectroporator (ECM 830 from BTX, Holliston,
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MA). After electroporation, cells were cultured incomplete medium for 48 hours. The efficiency ofelectroporation was ~70%.
In several experiments Jurkat-Tag cells weretransfected using DMRIE-C (3 mg of DNA per5x106 cells) according to the manufacturer’srecommendations. Since the efficiency of DMRIE-C-mediated transfection did not exceed 10%, aGFP-encoding expression plasmid (pEGFP-C2,Clontech) was co-transfected in each sample at aratio of 1:15 to the total DNA, and only GFP+ cellswere analyzed using flow cytometry. Stable Jurkatcells with a reduced TULA expression level and thecorresponding control cells were generated usingthe shRNA-encoding or empty control lentiviralvector (1).
Z-VAD.fmk and Z-IETD.fmk (Biomol,Plymouth Meeting, PA), camptothecin andetoposide (Sigma) were added to finalconcentrations of 100, 4, 5 and 10 mM,respectively. Growth factor withdrawal of Jurkat-Tag cells was carried out in medium supplementedwith 0.5% FBS. For anti-CD3 stimulation, wells ofa 24-well plate were pre-coated with the mousemonoclonal antibody OKT3 at 10 mg/ml in PBSovernight at +4oC.
Isolation of TULA-associated proteins - 1-3mg of total protein from FLAG-TULA-expressingor vector-transfected HEK293T cells was incubatedwith 20 ml anti-FLAG M2 affinity gel (Sigma) andincubated at 4oC for 4 hours. The beads werewashed three times with lysis buffer, and anti-FLAG-bound proteins were eluted from the beadswith 0.1 M glycine (pH 3). Proteins eluted from theanti-FLAG beads were separated on a one-dimensional Bis-Tris minigel and stained in SimplyBlue Coomassie (Invitrogen). Each gel lane wasdivided and cut into 10 equal-sized gel slices.Proteins contained within each slice wereequilibrated in 100 mM ammonium bicarbonateand reduced, alkylated and digested with trypsin aspreviously described (22). One-tenth of eachunfractionated tryptic digest was analyzed by LC-ES MS/MS using a micro-column (Zorbax C18,75mm x 12 cm) reverse-phased HPLC interfacedwith an Agilent LC-MSD Ion Trap MS. ESMS/MS-based sequencing was performed on-line ina data-dependent manner, and two tandem massspectra were taken per survey scan as peptideseluted from the HPLC (23). Uninterpreted mass
spectra from each of the ten individual LC-MS/MS runs were collated and searched as asingle file against a human nonredundant proteindatabase using the Mascot search engine (MatrixScience) (24). Errors used were 2.0 Da on MSdata and 0.8 Da on MS/MS data.
Immunoprecipitation and immunoblotting -1-3 mg of total protein from whole cell lysate wasimmunoprecipitated with 1-3mg of anti-TULA-N(GETQLYAKVSNKLKSRSSPS) (ProteintechGroup Inc., Chicago, IL) in a total volume of 1ml as described previously (1). Then proteinswere separated using SDS-PAGE, transferred tonitrocellulose and probed with 1:1000 anti-FLAGM2 (Sigma), 1:1000 anti-TULA-N, or 1:500 anti-AIF (Santa Cruz Biotechnology, Santa Cruz,CA). After blots were washed, the appropriateperoxidase-conjugated secondary antibody wasadded, and proteins were visualized using theECL Plus Kit and the Typhoon FluorescentImager (GE Healthcare Life Sciences,Piscataway, NJ).
Annexin-V staining - Electroporated Jurkat-Tag cells were washed and resuspended in 100 mlannexin-V binding buffer (10 mM HEPES, 140mM NaCl, 2.5 mM CaCl2, pH 7.4). Then 5 ml of0.1 mg/ml propidium iodide and 5 ml annexin-Vallophycocyanin conjugate (Molecular Probes,Eugene, OR) were added to the cells. After cellswere incubated for 15 minutes at roomtemperature, 400 ml annexin binding buffer wasadded, and cells were analyzed using flowcytometry. DMRIE-C-transfected Jurkat-Tagcells and TULA-knockdown Jurkat cells wereanalyzed using an annexin V-Cy5 apoptosis kitfrom Biovision (Mountain View, CA).
Transfection of small interfering RNAs(siRNAs) – To deplete endogenous AIF andsimultaneously overexpress TULA, a 21-merannealed AIF-targeting siRNA and scrambledcontrol (Ambion, Austin, TX) were resuspendedin water at a final concentration of 100 mM. Thesense sequence of the AIF-specific siRNAcorresponded to nucleotides 1540-1558 in theAIF sequence. (Several AIF-specific siRNAswere tested in pilot experiments, and this siRNAwas selected as the most efficient one.) siRNAwas electroporated into Jurkat-Tag cells (100 nMsiRNA and 1x105 cells in 75m l Opti-MEM(Gibco/Life Technologies) using 1-mm cuvettes
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in the BTX Electroporator at 150V for 100 ms. Tosimultaneously electroporate siRNA and DNA,Flag-TULA expression or control plasmid (2 mg)was added to siRNA. After recovery in completemedium for 48 hours, transfected cells were eithercultured in complete RPMI1640 medium orsubjected to serum deprivation in RPMI1640supplemented with 0.5% FBS for an additional 24hours. At that time overall cell death was measuredusing trypan blue exclusion. To deplete endogenousTULA, the same electroporation procedure wasdone using TULA-specific siRNA SMARTpool L-008616-00 from Dharmacon (Lafayette, CO).
Sub-cellular distribution - To obtainimmunofluorescence images, HeLa cells wereseeded onto fibronectin-coated coverslips (BDBiocoat) at a confluency of 50% in DMEMmedium containing 10% FBS without antibiotics.On the following day, the cells were transfected toexpress Flag-TULA and/or Myc-AIF (3 mg of eachconstruct per coverslip) using Fugene6 as permanufacturer’s recommendations. Forty-eighthours post-transfection the cells were washed, fixedwith 4% paraformaldehyde in PBS, washed again,and permeabilized with 0.2% Triton X-100 in PBSfor 5 minutes at room temperature. Cells wereblocked with 1% BSA and washed twice with PBS.FITC-conjugated anti-Flag (5-10 mg/ml) and Cy3-conjugated anti-Myc (1 mg/ml) (Sigma) were addedas appropriate. The antibodies were incubated withthe cells overnight at +4oC in the dark. The cellswere washed three times with PBS before mountingthe coverslips onto a slide with anti-fade mountingsolution including DAPI stain (Molecular Probes).Cell images were obtained using the Leica DMIRE2 confocal microscope with a 100x objective.
For sub-cellular fractionation, 293T cells weretransfected with either empty or TULA expressionvector (10 m g per 75-cm2 flask) usingLipofectamine-2000. Sub-cellular fractions wereobtained from transfected cells at 48 hours post-transfection using a Qproteome Cell Compartmentkit (Qiagen, Valencia, CA).
RESULTS
AIF is a novel TULA interacting protein- Tosearch for novel functions of TULA we sought toidentify TULA interaction partners via a
proteomics approach. For this purpose, FLAG-tagged full-length TULA and TULA-N1/2 (1-299), a truncation mutant lacking the C-terminalhalf, but containing both binding domains ofTULA (UBA and SH3) (see Figure 1), weretransiently overexpressed in HEK293T cells andimmunoprecipitated with anti-FLAG antibody.The eluted immune complexes were separated bySDS-PAGE and proteins associated with theseforms of TULA were identified using liquidchromatography-tandem mass spectrometry (LC-MS/MS). Several proteins were identified in theTULA and TULA-N1/2 immunoprecipitates andnot in the vector control, and one of these was c-Cbl (8 unique peptides), a previouslycharacterized TULA-interacting protein (1,3). Asecond protein identified with 15 unique peptideswas Apoptosis-Inducing Factor (AIF). Originally,we identified AIF only in the TULA-N1/2immunoprecipitates. However, the molecularmass of AIF suggested that it co-migrates withfull-length TULA, which is a very large band onthe SDS-PAGE gel. Since co-migration withTULA was likely to hinder identification of AIFin this system, we targeted four unique AIFpeptides for mass spectrometry-based sequencingin the gel band corresponding to full-lengthTULA and identified AIF from all four peptidesequencing events. We performed theseexperiments using TULA and TULA-N1/2 intriplicate, and AIF was identified each time withmore than 10 peptides in each trial(Supplementary Table 1). Interestingly, c-Cblwas only identified in the immune complexeswith full-length TULA.
To verify association of TULA and AIF andto identify the region of TULA involved in AIFbinding, we transiently overexpressed TULA andTULA mutants in HEK293T cells,immunoprecipitated them, and analyzed theobtained immune complexes using Westernblotting. Consistent with the mass spectrometryresults, co-immunoprecipitation of AIF wasclearly detectable (Figure 2A). Immunoblottingalso showed that TULA-N1/2 binds to AIF betterthan full-length TULA does. To assure that thedifference in the amount of co-immunoprecipitated AIF was not due todifferences in the cellular levels of AIF in cellsoverexpressing full-length TULA and TULA-N1/2 (as well as other TULA mutant forms - see
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below), we immunoblotted AIF in whole celllysates and demonstrated that its level did not varysignificantly between samples (Figure 2B). Sincethe TULA-N1/2 mutant contains an SH3 domainand since AIF has several putative SH3-bindingmotifs (PXXP) including 545PSTPAVPQAP554, wehypothesized that TULA binds to AIF through theSH3 domain. However, the mutant form of TULAlacking a functional SH3 domain as a result of theW279L point mutation bound AIF with the sameefficiency as wild-type TULA did (Figure 2A).Furthermore, the SH3-deleted forms of bothTULA-N1/2 and full-length TULA bound AIF tothe extent characteristic of the binding of AIF byTULA-N1/2 (data not shown). Likewise, deletionof the UBA domain had no effect on AIF binding(Figure 2A). Finally, the TULA-C1/2 truncatedform (amino acids 300-623) did not bind to AIF(data not shown). Taken together these findingsindicate that the N-terminal half of TULA isnecessary for AIF binding, but that neither the SH3nor the UBA domain is critical.
Since c-Cbl is well characterized as a TULAbinding partner in T cells, we examined its bindingto TULA relative to AIF. Interestingly, highbinding of c-Cbl to various forms of TULA wasinvariably linked to the low AIF binding to themand vice versa (Figure 3), thus being in agreementwith the findings of our mass spectrometry-basedexperiments (see above). This mutual exclusion isunlikely to be due to a direct competition of c-Cbland AIF for the same binding site, since c-Cblbinds to the SH3 domain of TULA (1), which isdispensable for AIF binding (see Figure 2). It ismore likely that c-Cbl and AIF bind to alternativeconformation states of TULA or induce such statesupon binding.
We also evaluated the possibility that AIFbinds to the TULA homologue Sts-1/TULA-2. Bothproteins were co-expressed in 293T cells, Sts-1 wasimmunoprecipitated and the obtained immunecomplexes were analyzed using Western blotting.Co-immunoprecipitation of AIF and TULA-2 wasnot detected (data not shown), in spite of their highlevels of expression, indicating that the interactionof TULA with AIF is specific for this particularfamily member.
TULA facilitates T-cell apoptosis- Ourmultiple attempts to generate stable TULA-overexpressing T- or B-cell lines using lentiviraltransduction have failed, in spite of the generally
high success rate for the lentiviral system used(25,26) and the fact that vector control stabletransductants were consistently generated (datanot shown), suggesting that constitutive highexpression of TULA is detrimental for cellviability. Since AIF has been shown to be a keyfactor of caspase-independent apoptosis, wedecided to explore the effect of TULA expressionon T-cell apoptosis and to analyze the functionallink between TULA and AIF in this event.
First, we analyzed the effect of reducingendogenous levels of TULA on T-cell apoptosis.A stable variant of Jurkat cells with a reducedlevel of TULA (“TULA-knockdown”) wasgenerated using a shRNA-encoding lentiviralvector (1). In these cells, the level of TULAprotein was reduced ~4-fold as compared to theparental cells and cells expressing a controlshRNA (Figure 4A). (A decrease in TULAmRNA in these cells has been shown in ourprevious report (1).) Apoptosis in the TULAknockdown and control cells was induced usinganti-CD3 (a T cell-specific apoptotic stimulusmimicking biological TCR/CD3-mediatedsignaling), etoposide (an apoptosis-inducingdrug), and growth factor withdrawal (serumdeprivation was used, since Jurkat cells are IL-2-independent). Early apoptosis of Jurkat cells wasassessed using annexin-V staining indicative ofphosphatidyl serine exposure on the outer leaf ofthe plasma membrane. Results from theseexperiments show that TULA expression iscritical for apoptosis induced by serumdeprivation, but not by anti-CD3 or etoposidetreatment (Figure 4B).
To ascertain that the effect of TULA shRNAwas not due simply to clonal variability, wedetermined whether depletion of TULA achievedusing transient transfection of TULA-targetingsiRNA (Figure 4C) would produce a similareffect. These experiments indicated that TULA-specific siRNA substantially reduces serumdeprivation-induced cell death (Figure 4D). Thefinding that the effect of transient transfection issomewhat lower than that of stable TULA-specific shRNA expression is likely to beexplained by a higher residual level of TULA insiRNA-treated cells as compared to shRNA-expressing cells (Figure 4A vs. 4C).
To further establish a role for TULA in T-cell apoptosis and to compare the pro-apoptotic
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potential of mutant forms of TULA, we employedan independent experimental approach, transientlyoverexpressing wild-type TULA and its mutantslacking known functional domains in Jurkat cellsand comparing sensitivity of these cells to apoptosisinduced with anti-CD3 or camptothecin. Thepercentage of cells undergoing apoptosis in thecontrol and the camptothecin-treated cells increasedsubstantially when wild-type TULA wasoverexpressed, although no synergism betweenTULA overexpression and camptothecin treatmentwas observed. In contrast, overexpression of TULAdid not significantly modify sensitivity of Jurkatcells to anti-CD3-induced apoptosis; they werehighly sensitive regardless of TULAoverexpression (Figure 5A). These results takentogether with those shown in Figure 4 indicate thatthe pro-apoptotic effect of TULA is not linked toTCR/CD3-mediated signaling.
These experiments also demonstrated thatTULA mutants lacking the UBA domain or afunctional SH3 domain were unable to facilitate T-cell apoptosis in spite of being expressed at a levelcomparable to that of TULA (Figure 5A, bottompanel). Considering that proteins of a single familymay have similar functions, we evaluated the effectof Sts-1 (TULA-2) on apoptosis of Jurkat cells.Unlike TULA, its ubiquitous homologue did notfacilitate apoptosis either in untreated cells or incells treated with camptothecin (Figure 5B). Thisresult indicates that the pro-apoptotic effect ofTULA is specific for this family member.
TULA exerts its apoptotic effect through AIF-The results shown in Figures 4 and 5 indicate thatsensitivity of Jurkat cells to TCR/CD3-mediatedapoptosis remains unchanged by either knockdownor overexpression of TULA, thus ruling out that thepossibility that TULA affects apoptosis byregulating TCR/CD3 signaling. Given that TULAand AIF bind in vivo and that AIF inducesapoptosis, we considered the possibility that TULAexerts its pro-apoptotic effect through AIF.
First, we addressed this issue by evaluatingcaspase dependence of the observed effect ofTULA, because TCR/CD3-induced apoptosisrequires caspase activation (29-32), while AIF hasan established role in caspase-independentapoptosis (6-8). To determine whether or notcaspases play a role in TULA’s pro-apoptoticeffects, we determined the effects of Z-VAD, apan-caspase inhibitor, on cell death in our
experimental system. These experimentsindicated that inhibition of caspases reducesdeath of TULA-overexpressing Jurkat cellsneither in complete medium (Figure 6A) norunder serum deprivation (Figure 6B), whileinhibiting camptothecin-induced death of Jurkatcells under both conditions. Likewise, the effectof TULA was insensitive to Z-IETD, a caspase-8inhibitor (data not shown). Therefore, the pro-apoptotic effect of TULA is largely caspase-independent in agreement with the idea thatTULA exerts this effect through AIF.
We then directly evaluated the possibilitythat the pro-apoptotic effect of TULA requiresAIF. We transiently transfected Jurkat cells withAIF-specific siRNA and TULA-encoding vectorto simultaneously reduce the endogenous level ofAIF and to overexpress TULA and subjectedthem to serum deprivation to stimulate apoptosis.As expected, a decrease in the level of AIFreduced cell death, whereas overexpression ofTULA caused an increase. Consistent with a co-operative role of these proteins in apoptosis, adecrease in AIF expression significantly inhibitedthe effect of TULA overexpression on serumdeprivation-induced cell death (Figure 7). (Itshould be noted that in this system TULAoverexpression exerted only a minor pro-apoptotic effect in complete medium (cell deathwas 15.8+/-3.1% and 21.0+/-1.1% for vectorcontrol and TULA-overexpressing cells,respectively), and therefore, serum deprivationwas essential for revealing the observed effects.This variation between the results shown inFigures 5 and 7 is probably due to the differencesbetween the basal levels of cell death in thesystems utilized; it is higher for electroporatedcells (Figure 7) than for transfected cells (Figure5).)
Taken together, our results indicate thatTULA physically interacts with AIF and exertsits pro-apoptotic effect in an AIF-dependentfashion. However, it remains unclear how theinteraction of TULA and AIF facilitatesapoptosis, since these proteins appear to reside indifferent compartments inside the cell; AIF islocalized to mitochondria under normalphysiological conditions (6-8,15,33,34), but afterapoptotic stimulation it is released to the cytosoland subsequently translocates to the nucleus (11-14), whereas no mitochondrial localization has
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been shown for TULA. The latter is localizedprimarily to the cytoplasm with some amount of itbeing present in the nucleus (1). To determinewhether or not AIF and TULA can co-localize, wetransiently co-expressed epitope-tagged constructsof both proteins in HeLa cells and examined theirlocalization under normal and stressed cultureconditions using confocal microscopy. Undernormal conditions, no significant co-localization ofTULA and AIF was detectable (Figure 8).However, their co-localization became apparent inthe cytoplasm when cells were subjected to astaurosporine treatment, which induces apoptosismediated by the release of pro-apoptotic factorsfrom mitochondria (Figure 8). The apoptosisinduction under these conditions is visualized byconsiderable morphological disturbances of thenuclei in staurosporine-treated cells. The lack of co-localization of TULA and AIF prior to the stresstreatment argues that TULA is unlikely to facilitateapoptosis by promoting the release of AIF frommitochondria.
To address this issue directly, we determinedthe effect of TULA overexpression on sub-cellularlocalization of AIF. HEK293T cells weretransfected with wild-type and mutant forms ofTULA, and distribution of AIF was examined usingsub-cellular fractionation followed by Westernblotting. AIF is largely localized to the membranein a manner consistent with its mitochondrialtargeting, while a very small fraction of it is presentin the cytosol and in the nucleus (Figure 9A),probably due to the transfection-induced cell stress,and not to cross-contamination of fractions, whichwas minimal (Figure 9B). This pattern of sub-cellular distribution of AIF was not affected byoverexpression of any form of TULA (Figure 9C).Therefore, the results shown in Figures 8 and 9argue that (a) the functional interaction of TULAwith AIF occurs when AIF has already beenreleased from mitochondria and (b) translocation ofAIF to the nucleus is not the major target ofTULA’s pro-apoptotic effect.
DISCUSSION
Taken together, our results obtained usingseveral independent approaches demonstrate thatTULA is involved in T-cell apoptosis (Figures 4,5). Furthermore, our results indicate that TULA
affects apoptosis through a mechanismindependent of either TCR/CD3-mediatedsignaling or caspase activation (Figures 4-6).Finally, we have shown that TULA binds to AIF(Figure 2, Supplementary Table 1), a key factorof caspase-independent apoptotic events. Bindingof AIF to TULA together with the caspaseindependence of the pro-apoptotic effect ofTULA have provided an initial argument in favorof the idea that TULA affects apoptosis throughits interaction with AIF.
Indeed, further experiments indicated thatAIF is essential for the pro-apoptotic effect ofTULA (Figure 7). Notably, the effect of AIF-specific siRNA on the pro-apoptotic activity ofTULA was higher than its effect on the proteinlevel of AIF. This apparent discrepancy mayreflect the fact that the pro-apoptotic effect ofTULA is highly sensitive to the level of AIF orthat siRNA differentially affects the level of AIFin various cellular compartments – the fraction ofAIF that mediates the effect of TULA may bedepleted more than others. It should also be notedthat AIF depletion reduced not only TULA-facilitated apoptosis, but also apoptosis in theabsence of overexpressed TULA. This may bedue to the role endogenous TULA plays in thebasal apoptosis in Jurkat cells.
The essential role of AIF in TULA-facilitated apoptosis and the physical interactionof TULA with AIF, taken together, stronglysuggest that binding of TULA to AIF is crucialfor the pro-apoptotic effect of TULA. Indeed,overexpression of the TULA mutant lacking theN-terminal domain (TULA-C1/2) and incapableof binding to AIF does not promote apoptosis inJurkat cells and even decreases it (data notshown). This finding is consistent with the notionthat TULA-AIF binding is essential for the pro-apoptotic effect of TULA, but may also beexplained by the lack of TULA UBA and SH3,which are essential for this effect, while notrequired for TULA-AIF binding (Figure 2).However, since our mutational studies implicatedmultiple sites within the N-terminal half ofTULA in binding to AIF, thus not permitting usto obtain a specific mutant of TULA defective inAIF binding, but fully functional otherwise (datanot shown), evidence of the essential role ofTULA-AIF interactions in the effect of TULA
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based on the mutational disruption of theseinteractions remains to be provided.
Several findings presented in this report allowus to outline the molecular basis of the pro-apoptotic effect of TULA. First of all, the caspase-independent nature of TULA’s effect argues thatTULA does not act by permeabilizingmitochondrial membrane, since this would result inthe release of multiple apoptotic factors actingthrough caspases. Therefore, considering that theeffect of TULA is mediated by AIF, TULA mayfacilitate (a) release of AIF from mitochondria, (b)transfer of AIF to the nucleus and (c) interactions ofAIF with its co-factors. Since TULA and AIF arenot substantially co-localized in unstressed cells(Figure 8) and since overexpression of TULA doesnot alter sub-cellular distribution of AIF inunstressed cells (Figure 9), it is unlikely that TULAacts by inducing the release of AIF frommitochondria. Likewise, the lack of a significanteffect of TULA overexpression on the nuclearlocalization of AIF (Figure 9) provides no supportfor the idea that TULA facilitates apoptosis byincreasing the nuclear translocation of AIF.Therefore, it is more likely that TULA promotesAIF-dependent apoptosis by facilitating interactionsbetween AIF and its apoptotic co-factors (6). Thispossibility is supported by the following findings:UBA- and SH3-deficient forms of TULA bind toAIF, but lack a pro-apoptotic effect (Figures 2 and5), suggesting that interactions of the UBA and/orSH3 domains of TULA with proteins other thanAIF are required for this effect. Although theidentity of co-factors whose binding to AIF isfacilitated by TULA remains to be elucidated, ourresults argue that c-Cbl is unlikely to be essentialfor the AIF-TULA cooperation, since the ability ofseveral forms of TULA to bind to AIF and c-Cblshowed a clear inverse correlation (Figure 3).
Analysis of the mechanism by which TULAcooperates with AIF is hindered by the lack ofclarity regarding the molecular mechanism of theapoptotic effect of AIF. It has been shown that thenuclear transfer of AIF is essential for this effect,that AIF exerts its effect through binding to DNA,and that the initial step of AIF-induced apoptosis islikely to be chromatin condensation (6-8,15,33,34).However, little is known about proteins cooperatingwith AIF in apoptosis. Although it was suggestedthat endonuclease G and cyclophilin A mightcooperate with AIF in apoptosis (19,21), the
involvement of these two proteins in the effect ofAIF remains to be established.
As noted above, the caspase-independentnature of the pro-apoptotic effect of TULA(Figure 6), which is consistent with the body ofdata related to AIF, is one of argumentssupporting the functional cooperation betweenTULA and AIF in apoptosis induction. It remainsunclear what needs of the cell are served by theexistence of the AIF-dependent apoptoticmechanism alongside the well-characterizedcaspase-based mechanisms. Possibly, AIF plays acritical role in cell death in response to specificstimuli, including CD2- and CD44-mediatedsignaling (34,35) and certain drugs (7,36-43). Forinstance, comparison of the sensitivity of wild-type and AIF-null ES cells to various apoptogenicstimuli indicated that AIF is essential for some(growth factor withdrawal), but not the other(etoposide, azide, UV), pathways of deathinduction (44). Similarly, TULA does notinterfere with T-cell apoptosis induced byTCR/CD3 ligation, but plays a critical role ingrowth factor withdrawal-induced apoptosis(Figure 4), thus suggesting that the pro-apoptoticeffect of TULA is specific for certain types ofcell death. It is also possible that AIF functionallycooperates with caspases in the cell deathcascades, being responsible for the early stages ofapoptosis (34,39). In particular, a recent reportindicates that large-scale DNA degradation,which was thought to be a hallmark of theapoptotic effect of AIF, may be caspase-dependent, but agrees with the previous studiesthat AIF-dependent chromatin condensation isindependent of caspases and represents an earlystep of the cell death, which precedes its caspase-dependent steps (45).
To summarize, we have demonstrated, forthe first time, that TULA (Sts-2) exerts a pro-apoptotic effect and that this effect is mediated byAIF. The effect of TULA on cell death is specificfor particular death stimuli; it is significant forserum deprivation-induced apoptosis, but isnegligible for apoptosis induced by TCR/CD3ligation or several DNA-damaging drugs. Theeffect of TULA is largely caspase-independent,thus being entirely consistent with the idea that itis mediated by AIF, a key factor of caspase-independent apoptosis. It appears that the role ofTULA is primarily to amplify the apoptotic
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events dependent on AIF rather than act as anindependent inducer of apoptosis. Our resultssuggest that TULA promotes AIF-dependentapoptosis primarily by facilitating the interactions
between AIF and its co-factors. Further studiesshould reveal the molecular basis of the apoptoticeffect of TULA in detail.
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FOOTNOTES
*The authors thank Greg Harvey for excellent editorial assistance. This work was in part supported byNIH grant CA78499 (A.Y.T.)1The abbreviations used are: AIF, apoptosis inducing factor; FBS, fetal bovine serum; LC-MS, liquidchromatography-mass spectrometry; PBS, phosphate-buffered saline; PGM, phosphocruceromutase;PTK, protein tyrosine kinase; SH3, Src homology 3 domain; Sts, suppressor of T-cell receptor signaling;TCR, T-cell receptor; TULA, T-cell ubiquitin ligand; UBA, ubiquitin-associated domain
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FIGURE LEGENDS
Figure 1. Structures of TULA and AIF. Major structural elements of TULA and AIF are shown. UBA is aubiquitin-associated domain; SH3 is a Src-homology 3 domain, binding to proline-rich motifs;HCD/PGM is a region of homology to phosphoglyceromutases, MLS and NLS are mitochondrial andnuclear localization signals, respectively, FAD- and NAD-BD are FAD- and NAD-binding domains,respectively. Finally, mutated forms of TULA utilized in the experiments described below are shown.
Figure 2. Co-immunoprecipitation of TULA and AIF. 293T cells were transfected with wild-type (WT)and various mutant forms of TULA (10 mg of each construct per 75-cm2 flask, except for TULA-N1/2,which was transfected at a dose of 20 mg per 75-cm2 flask) using Lipofectamine-2000. Control cells weretransfected with the empty vector at a dose equal that of the expression constructs. Empty vector wasadded to transfections to make total amounts of DNA equal. Cells were lysed 48 hours after transfection.Cell lysates were subjected to immunoprecipitation (IP) with an indicated antibody (NRS=normal rabbitserum) (A) or analyzed as whole cell lysates (WCL) (B). Antibodies used for Western blotting (WB) areindicated. The proteins detected are indicated by arrowheads at the right. The molecular weight markersare indicated at the left.
Figure 3. Binding of TULA to AIF and c-Cbl. 293T cells were transfected with various mutants of TULAas described in the legend to Figure 2. Cells were lysed 48 hours after transfection. Cell lysates weresubjected to immunoprecipitation with anti-TULA. Immunoprecipitates were analyzed using Westernblotting (WB) with the antibodies indicated. The proteins detected are indicated by arrowheads at theright. The molecular weight markers are indicated at the left.
Figure 4. Effect of TULA on apoptosis of Jurkat cells: RNAi. (A) The level of TULA protein in parentalJurkat cells not transduced with an shRNA-encoding vector (“none”) and Jurkat cells transduced toexpress TULA-specific shRNA or a mutated form of this shRNA (“control”) was determined usingWestern blotting and normalized to the level of GAPDH. (B) Jurkat cells transduced to stably expressTULA-specific or control shRNA were subjected to serum deprivation or etoposide treatment for 4 hoursor to anti-CD3 stimulation for 8 hours, as indicated, and analyzed for Annexin-V staining using flowcytometry. (C) Jurkat cells were transiently transfected with TULA-targeting or scrambled (control)siRNA as indicated, and the level of TULA was determined as described in (A). (D) Jurkat cellstransiently transfected with TULA-targeting or scrambled siRNA were subjected to serum deprivation for24 hours and analyzed for viability. Results are shown as mean+/-sem of duplicate measurements in anindividual representative experiment.
Figure 5. Effect of TULA on apoptosis of Jurkat T cells: Overexpression. (A) Jurkat-Tag cells weretransfected with Flag-tagged TULA, wild-type (WT) and mutant, using DMRIE-C. A vector for GFPexpression was co-transfected in each case. Control cells were transfected with the empty vector at a doseequal that of the expression constructs. At 24 hours after transfection, cells were treated with the stimuliindicated at the bottom of the panel for an additional 8 hours and then analyzed for annexin-V stainingusing flow cytometry. The analysis was carried out for GFP-positive cells only. The bottom panels showWestern blotting of Jurkat-Tag whole cell lysates obtained 24 hours after transfection. The antibodiesused for Western blotting are indicated at the bottom of the corresponding panels. GAPDH was used as aloading control. The proteins detected are indicated by arrowheads at the right. The molecular weightmarkers are indicated at the left. (B) Jurkat-Tag cells were transfected with the TULA-2/Sts-1 expressionconstruct or empty vector and analyzed as described in (A). Results are shown as mean+/-sem ofduplicate measurements. Each panel shows an individual representative experiment.
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Figure 6. Contribution of caspases to the effect of TULA on T-cell apoptosis. Jurkat-Tag cells wereelectroporated to express TULA or treated with camptothecin for 24 hours as indicated in completemedium (A) or under conditions of serum deprivation (B). Z-VAD.fmk was added 48 hours post-electroporation for 24 hours; serum deprivation was initiated at the same time in (B). Results are shown asmean+/-sem of duplicate measurements. Each panel shows an individual representative experiment. Thelevels of cell death as defined by trypan blue exclusion were corrected for the cell death in vector controlcells cultured in complete medium and normalized to the effect of camptothecin in each panel (in order tocompare individual experiments).
Figure 7. Contribution of AIF to the effect of TULA on T-cell apoptosis. (A) Jurkat-Tag cells wereelectroporated with AIF-specific or scrambled (control) siRNA and lysed 48 hours afterwards. AIF in celllysates was analyzed using Western blotting. The amount of AIF normalized to the amount of GAPDH isshown at the bottom. (B) Jurkat-Tag cells were electroporated simultaneously with siRNA, scrambled (-)or AIF-specific (+), and DNA, TULA expression plasmid (+) or empty vector (-), as indicated and letrecover for 48 hours. Cell death was induced by serum deprivation for 24 hours and assessed using trypanblue exclusion. The difference in the numbers of dead cells between treated and untreated samples foreach electroporated culture is presented as mean+/-sem of three independent experiments. Statisticalanalysis was done using t-test and the p values for the relevant pairs are shown.
Figure 8. Co-localization of TULA and AIF. HeLa cells were grown on fibronectin-treated coverslips andtransfected to express Flag-TULA and/or Myc-AIF. After 48 hours, cells were either left untreated ortreated with 400 nM staurosporine (Sigma) for 1.5 hours. Cells were then fixed, permeabilized, stained toreveal Flag-TULA-N1/2 (FITC) and Myc-AIF (Cy3), and counterstained with DAPI, a nuclear stain, asdescribed in detail in Experimental Procedures. TULA, AIF and nuclear staining, as well as their overlay,is shown for representative fields for both untreated and staurosporine-treated cells.
Figure 9. Sub-cellular localization of AIF. (A) HEK293T cells were transfected to express wild-type ormutant TULA, as described in the legend to Fig. 2, and subjected to fractionation. The obtained fractionswere analyzed using Western blotting as shown, and the proteins detected are indicated by arrowheads atthe right. Left and right panels represent two different gels; to adjust the quantitative results cross-reference samples were added to each gel. (B) Sub-cellular fractions of cells transfected to express wild-type TULA were used to characterize the fractionation procedure using GAPDH, VLA-2a and lamin asproteins located primarily in the cytosol, membrane and nucleus, respectively. (C) Percentage of AIF inthe fractions analyzed in (A) was calculated based on the intensity of AIF bands and sample volumes.The sample volumes were equal with the exception of those for immunoblotting of membrane-localizedAIF, which were 10-fold smaller due to a dramatic difference between the amounts of AIF in fractions.For comparison, panel (B) shows anti-AIF immunoblotting of equal-size samples.
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SUPPLEMENTARY DATA
Supplementary Table 1AIF-specific peptides identified in TULA immune complexes
Immunoprecipitation with subsequent identification of associated proteins was carried out for full-lengthTULA and TULA-N1/2, a C-terminal truncation mutant of TULA. Three individual experiments wereperformed in each case. The number of experiments in which a particular peptide was detected in theTULA–N1/2 immunoprecipitates is shown in the third column. Peptides denoted with an asterisk werealso detected in the experiments with full-length TULA by targeted analysis.
Peptide sequence Amino acids # ExpISGLGLTPEQK 99-109 3/3
AALSASEGEEVPQDK 113-127 3/3 *VLIVSEDPELPYMRPPLSK 159-177 2/3
ELWFSDDPNVTK 178-189 3/3VVQLDVR 233-239 3/3
LNDGSQITYEK 245-255 3/3CLIATGGTPR 256-265 3/3
ALGTEVIQLFPEK 325-337 2/3ILPEYLSNWTMEK 343-355 2/3
VMPNAIVQSVGVSSGK 363-378 3/3 *KVETDHIVAAVGLEPNVELAK 388-408 1/3VETDHIVAAVGLEPNVELAK 389-408 1/3
TGGLEIDSDFGGFR 409-422 2/3VNAELQAR 423-430 3/3 *
RVEHHDHAVVSGR 451-463 3/3SATEQSGTGIR 519-529 3/3 *
GVIFYLR 563-569 2/3IIKDGEQHEDLNEVAK 591-606 1/3
DGEQHEDLNEVAK 594-606 3/3LFNIHED 607-613 2/3 by guest on February 18, 2020
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Annan and Alexander Y. TsygankovTherese S. Collingwood, Evgeniya V. Smirnova, Marina Bogush, Nick Carpino, Roland S.
caspase-independent apoptosisTula affects cell death through a functional interaction with AIF, a key factor of
published online August 20, 2007J. Biol. Chem.
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