Apoptosis Inducing Factor Binding Protein PGAM5 Triggers

Article pubs.acs.org/biochemistry

Apoptosis Inducing Factor Binding Protein PGAM5 Triggers Mitophagic Cell Death That Is Inhibited by the Ubiquitin Ligase Activity of X‑Linked Inhibitor of Apoptosis Audrey M. Lenhausen,†,§,# Amanda S. Wilkinson,‡,§ Eric M. Lewis,†,⊥ Kaitlin M. Dailey,‡ Andrew J. Scott,‡ Shahzeb Khan,‡ and John C. Wilkinson*,‡ †

Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, United States Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108, United States

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ABSTRACT: Apoptosis inducing factor (AIF) plays a well-defined role in controlling cell death but is also a critical factor for maintaining mitochondrial energy homeostasis; how these dueling activities are balanced has remained largely elusive. To identify new AIF binding partners that may define the continuum of AIF cellular regulation, a biochemical screen was performed that identified the mitochondrial phosphoglycerate mutase 5 (PGAM5) as an AIF associated factor. AIF binds both the short and long isoforms of PGAM5 and can reduce the ability of PGAM5 to control antioxidant responses. Transient overexpression of either PGAM5 isoform triggers caspase activation and cell death, and while AIF could reduce this caspase activation neither AIF expression nor caspase activity is required for PGAM5-mediated death. PGAM5 toxicity morphologically and biochemically resembles mitophagic cell death and is inhibited by the AIF binding protein X-linked inhibitor of apoptosis (XIAP) in a manner that depends on the ubiquitin ligase activity of XIAP. The phosphatase activity of PGAM5 was not required for cell death, and comparison of phosphatase activity between short and long PGAM5 isoforms suggested that only the long isoform is catalytically competent. This property correlated with an increased ability of PGAM5L to form dimers and/or higher order oligomers in intact cells compared to PGAM5S. Overall this study identifies an AIF/PGAM5/XIAP axis that can regulate PGAM5 activities related to the antioxidant response and mitophagy.

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Despite these functional implications related to its name, AIF has also emerged as a critical regulator of cell growth and survival through control of mitochondrial homeostasis, structure, and metabolic activity.11,12 AIF has been shown to possess NADH-oxidase activity in vitro,13 and multiple gene targeting experiments have demonstrated that loss of AIF in vivo results in a spectrum of abnormalities ranging from embryonic lethality to metabolic disorders consistent with deficiencies in cellular oxidative phosphorylation.11,14−16 More recently deleterious mutations in the AIF gene have been linked to patients with respiratory chain and redox imbalance disorders with diverse clinical manifestations that include mitochondrial encephalomyopathy,17 prenatal ventriculomegaly,18 and Cowchock syndrome.19 Contrasting these loss-ofAIF phenotypes, we and others have shown that in a variety of human cancers elevated AIF expression promotes a metabolic state that favors tumor cell growth and survival.20−22 Overall these studies suggest that the primary physiological activity of AIF is to promote cell survival through controlling redox homeostasis and energy metabolism, yet the complete

ell death is critical to the development and homeostatic maintenance of multicellular systems and is dysregulated during the pathogenesis of many diseases, yet the processes by which cells die are not defined by a singular set of rules. Indeed, while predominant cell death pathways such as apoptosis and necrosis have been well characterized at the biochemical and genetic levels, a variety of atypical cell death forms have been reported that present with novel morphological and enzymological phenotypes, many of which employ overlapping mechanisms in order to drive cellular demise.1 Thus, the identification and characterization of proteins with overlapping activity among multiple types of cell death are of great physiological significance. One such protein is apoptosis inducing factor (AIF), a mitochondrial flavoprotein first discovered for its ability to promote cell death through the triggering of chromatin condensation and DNA cleavage.2−6 AIF-mediated death is notably caspase independent, suggesting that the actual mechanisms through which AIF kills are not apoptotic and instead more closely resemble necrosis or necroptosis. The pro-death activities of AIF also appear restricted to specific cellular contexts: AIF-mediated death is only pivotal to certain cell types such as neurons and cardiomyocytes, and is limited to specific stimuli, including DNA-damaging agents, engagement of death receptors, oxidative stress, excitotoxins, and hypoxia−ischemia.7−10 © XXXX American Chemical Society

Received: April 3, 2016 Revised: May 19, 2016

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DOI: 10.1021/acs.biochem.6b00306 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

supplemented with 2 mM Glutamax at 37 °C in an atmosphere of 95% air and 5% CO2. Transfections were performed by the method of calcium phosphate precipitation as described previously.30 pEBB-AIF-TAP (full-length) and pEBB-N012AIF-TAP were generated by subcloning WT and N102 AIF into the pEBB tandem-affinity purification (TAP) plasmid.31 pCDNA3-ASK1-HA32 was obtained from Dr. Jonathan D. Ashwell (National Institutes of Health). pEBB-ANT2, pEBB PGAM5S, and pEBB-PGAM5L were generated by PCR using expressed sequence tag clones containing human ANT2 (Image clone #3867331, Life Technologies), human PGAM5S (Image clone #3048106, Open Biosystems), and human PGAM5L (Image clone #4525757, ATCC). pEBB-PGAM5S and pEBBPGAM5L were used to further subclone pEBB-HA-PGAM5S, pEBB-PGAM5 S -HA, pEBB-Flag-PGAM5 S , pEBB-HAPGAM5L, pEBB-PGAM5L-HA, and pEBB-Flag-PGAM5L. pEBB-PGAM5(S/L)-H105A mutants were generated by sitedirected mutagenesis (Stratagene) using pEBB-PGAM5S-HA and PGAM5L-HA as templates. pARE-Ti-Luciferase was obtained from Dr. William Fahl (University of Wisconsin). pEBB-PGAM5S-YN, pEBB-PGAM5S-YC, pEBB-PGAM5L-YN, and pEBB-PGAM5 L-YC were generated by subcloning PGAM5(S/L) into pEBB-BiFC plasmids.31 All remaining plasmids used in this study (pEBB-AIF-FLAG, AIF-GST, AIF-TB, Ub-Δ54-AIF-FLAG, Ub-Δ102-AIF-FLAG, pCW7 His-Myc-wildtype-ubiquitin, pCW7 His-Myc-K48R-Ubiquitin, pEBB-FLAG-XIAP, pEBB-FLAG-H467A-XIAP, pCDNA3-Bax) have been reported previously.31,33−35 Cell Lysis, Immunoblot Analysis, and Immunoprecipitations. Cell lysates were prepared in Laemmli buffer (625 nM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% βmercaptoethanol), urea lysis buffer (8 M urea, 300 mM NaCl, 0.5% NP40, 50 mM Na2HPO4, 50 mM Tris, pH 8.0, 1 ng/mL aprotinin, 10 ng/mL leupeptin) or 0.5% SDS lysis buffer (50 mM Tris-HCl, 0.5% SDS, 150 mM NaCl, 1% Nadeoxycholate, 1% Triton X-100, 10 mM EDTA, 1 mM PMSF) (all lysis buffers were supplemented with 1 mM PMSF, and 1 protease inhibitor cocktail tablet per 10 mL prior to use), normalized for protein content, and then separated by SDSpolyacrylamide gel electrophoresis (PAGE)26 using 4−12% gradient SDS-polyacrylamide gels (Invitrogen). For immunoblot analysis, SDS-PAGE was followed by transfer to nitrocellulose membranes (Invitrogen), which were then blocked with 5% milk in Tris-buffered saline containing 0.02 to 0.2% Tween, followed by incubation with the indicated antibodies for 1 h at room temperature. After washing, membranes were incubated with HRP-conjugated antimouse IgG or antirabbit IgG secondary antibodies for 45 min at room temperature and visualized by enhanced chemiluminescence. For immunoprecipitation experiments, cell lysates (0.5% SDS lysis buffer) were normalized for protein content and incubated with indicated antibodies for 2 h at 4 °C. Protein G-coupled agarose beads were then added and incubated for 1 h. For precipitation of GST-tagged proteins glutathione-agarose beads were added, and the samples were incubated at 4 °C for 2 h. Agarose beads were recovered by centrifugation and washed in 0.5% SDS buffer, and precipitated proteins were eluted by adding lithium dodecyl sulfate (LDS) sample buffer and heating the mixture to 95 °C for 5 min. Urea lysates used for Ni-NTA precipitation were subjected to sonication post lysis (Branson Sonifier 250, output control 2.5, 75% duty cycle, ∼25 pulses) normalized for protein content, then incubated with Ni-NTA agarose at 25 °C for 2 h. Beads were washed three times in urea wash buffer (8

mechanistic picture of AIF-mediated mitochondrial control remains elusive, and it is largely unknown how balance is achieved between the pro-death and pro-survival activities of AIF. In an effort to distinguish the pro-life functions of AIF from its pro-death activities, we performed a biochemical screen to isolate proteins that interact with AIF in healthy mitochondria under normal conditions. Among the proteins identified is the mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5),23,24 which has been implicated in a variety of signaling nodes including the antioxidant response, programmed necrosis, and mitochondrial quality control via mitophagy.25−28 Two isoforms of PGAM5 have been reported, PGAM5S and PGAM5L, which contain identical amino termini (239 residues) but differ in their carboxy terminal regions;29 functional distinctions between PGAM5S and PGAM5L remain unclear. We find that AIF binds both PGAM5 isoforms, with preference for PGAM5S. This binding allows AIF to attenuate PGAM5 control of the antioxidant response. Unexpectedly, we observed that transient expression of either PGAM5 isoform triggered cell death. While PGAM5 triggered caspase activation, neither caspase activity nor AIF expression was necessary for PGAM5-mediated cell killing, and both morphological and biochemical criteria suggested cell death was mitophagic in nature. While AIF did not regulate this death pathway, we found that the AIF binding protein X-linked inhibitor of apoptosis (XIAP) was selectively capable of blocking PGAM5L cell killing in a ubiquitination dependent manner. Assessment of phosphatase activity suggested that while PGAM5L was highly active, this activity was not necessary for cell death. In contrast PGAM5S failed to exhibit detectable phosphatase activity, which correlated with a reduced ability of PGAM5S to form dimers in intact cells. Overall these data demonstrate the existence of an AIF/PGAM5/XIAP axis that tunes cellular antioxidant responses and cell death progression, and further highlights significant functional differences between PGAM5 isoforms.

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EXPERIMENTAL PROCEDURES Materials. Reagents were obtained as follows: protein Gcoupled agarose, glutathione agarose, Ni-NTA agarose, Glutamax, tetramethylrhodamine methyl ester (TMRM), and phosphate-buffered saline from Invitrogen; calmodulin-Sepharose 4B, and immunoglobulin G (IgG)-Sepharose from Amersham; fetal bovine serum from HyClone; Dulbecco modified Eagle medium (DMEM) from Mediatech; Ser/Thr phosphatase assay system from Promega; DEVD-7-amino-4trifluoromethyl coumarin from BioMol; zVAD-fmk from Enzyme Systems Products; and site-directed mutagenesis kit from Stratagene. All other chemicals were from Sigma. Antibodies were obtained as follows: anti-AIF (#SC13116) and anti-ASK1 (#SC-7931) from Santa Cruz Biotechnology; antiphosphorylated JNK (#4668), anti PINK1 (#6946), and antiphosphorylated p38 (#9216) from Cell Signaling; antiPGAM5 (ab126534) from Abcam; anti-β-actin (A5316), horseradish peroxidase (HRP)-conjugated anti-FLAG (#A8592), HRP-conjugated antihemagglutinin (anti-HA) (#H6533) and anti-Flag (#F3165) from Sigma; anti-HA from Covance (#MMS-101P); and HRP-conjugated antimouse (#NA931V) and antirabbit (#NA934V) from Amersham. Cell Culture, Transfection, and Plasmids. HEK293T cells were grown in DMEM containing 10% fetal bovine serum B


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Biochemistry

manufacturer’s instructions. Briefly, cells were washed in PBS prior to lysis in reporter lysis buffer. Replicate samples were then loaded into 96-well assay plates, and both luciferase activity and GFP fluorescence were measured using a luminescence/fluorescence plate reader (BMG Labtech FLUOstar Omega). Luciferase activity was normalized against GFP fluorescence to control for interwell transfection efficiency. Viability Experiments. Cells were seeded at 300 000 cells per well in six-well plates and allowed to attach overnight. Cells were then transfected with the indicated plasmids and then incubated at 37 °C for 48 h. Cells were harvested, washed, and resuspended in PBS containing 2 μg/mL propidium iodide. Cell viability was then determined by flow cytometry using an Accuri C6 flow cytometer. Mitochondrial Membrane Potential. Cells were seeded in six-well plates, transfected 24 h later with the indicated plasmids, and then incubated at 37 °C for an additional 24 h. Floating and attached cells were then harvested, and mitochondrial membrane potential was assessed by resuspending cells in 200 nM tetramethylrhodamine methyl ester (TMRM) and measuring fluorescence with an Accuri C6 flow cytometer. Caspase Activity Assay and Inhibition. Cells were seeded at 300 00 cells per well in six-well plates and allowed to attached overnight. Cells were transfected with the indicated plasmids prior to incubation at 37 °C for an additional 48 h. Floating and attached cells were harvested, and caspase 3 assays were performed as described previously.39 For caspase inhibition experiments, samples were plated and transfected as described above. Ten hours post-transfection zVAD-fmk was added to each well (50 μM final). Forty-eight hours posttransfection cells were harvested, and caspase activity was determined as described.39 Phosphatase Assay. Cells were seeded in 10 cm plates, allowed to attach overnight, and then transfected with the indicated plasmids. Cell lysates prepared 24 h later (0.5% SDS lysis buffer) were normalized for protein content and incubated with indicated antibodies for 2 h at 4 °C. Protein G-coupled agarose beads were then added and incubated for 1 h. Agarose beads were recovered by centrifugation and phosphatase activity measured according to manufacturer’s instructions (Promega) using PPase-2C buffer (250 mM imidazole pH 7.2, 1 mM EGTA, 25 mM MgCl2, 0.1% β-mercaptoethanol, 0.5 mg/mL BSA). Transmission Electron Microscopy. Cells were seeded in 10 cm plates, allowed to attach overnight, and then transfected with control, PGAM5S, or PGAM5L plasmids. Cells were harvested 36 h post-transfection, fixed in 2.5% glutaraldehyde, and embedded in Spurr’s resin. Sections were cut with a Reichert Ultracut E ultramicrotome and counterstained with lead citrate and uranyl acetate. Digital images were taken using a FEI Tecnai Spirit 12 Bio Twin TEM at 80 kV adapted with an AMT 2 Vu digital camera and AMT proprietary software. Bimolecular Fluorescence Complementation. Cells were seeded in six-well plates, allowed to attach overnight, and then transfected with a total of 2 μg (1 μg each) of either control plasmids or plasmids encoding PGAM5S-YN, PGAM5SYC, PGAM5L-YN, and PGAM5L-YC in various combinations. Twenty-four hours following transfection cells were harvested by trypsinization and fluorescent populations within each sample were identified by flow cytometry using an Accuri C6 flow cytometer.

M urea, 300 mM NaCl, 0.5% NP40, 50 mM Na2HPO4, 50 mM Tris, pH 8.0) then once in Triton buffer (25 mM Hepes pH 7.9, 100 mM NaCl, 1% Triton-X100, 1 mM EDTA, 10% glycerol, 1 mM NaF, 1 mM NaVO4, 1 mM DTT), and recovered proteins were eluted as described above. For all precipitation experiments, recovered proteins were then separated by electrophoresis, and immunoblot analysis was carried out as described above. Chemical Cross-Linking, Coomassie Staining. Beads were cross-linked as described.36 IgG-sepharose beads (2 mL of 50% slurry) were washed twice in 0.2 M sodium borate (pH 9.0) and then resuspended in 0.2 M sodium borate containing 20 mM dimethylpimelimidate prior to incubation at RT for 30 min. Beads were then washed twice in 0.2 M ethanolamine (pH 8.0) and incubated at RT for an additional 2 h. Beads were then washed twice with 1 M NaCl, 50 mM Tris (pH 8.0), twice with PBS, and resuspended in PBS containing 0.01% sodium azide for storage until use. To test cross-linking efficiency, beads were washed three times with RIPA lysis buffer (phosphate-buffered saline containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) prior to elution in LDS buffer as described above. Eluates were separated by SDS-PAGE, and protein content of each eluate was visualized by Coomassie staining. Tandem Affinity Purification. Cells were seeded into two groups of five 15 cm plates each and then transfected with 12 μg of pEBB-AIF-TAP (full-length) or pEBB-AIF-N102-TAP per plate. The AIF-N102 construct produces a protein containing 102 amino acid residues corresponding to the amino-terminus of AIF in fusion with the full TAP tag, and was used as a background control for proteomic analysis. Two days after transfection cells were fractionated by dounce homogenization, protein was extracted from the crude mitochondrial fraction, and extracts were subjected to tandem affinity purification as described.37 Final purified samples were submitted to the Proteomics Centre at University of Victoria for further processing including trypsin digestion, HPLC separation, and MS/MS to determine peptide sequences. Lentiviral Knockdown. Lentiviral-mediated shRNA knockdown of AIF in HEK293T cells was performed as previously described.38 Briefly, FG12 derived plasmids were combined with equal amounts of lentiviral packaging plasmids and transfected into HEK293T cells by calcium phosphate precipitation as described.37 Forty-eight hours after transfection, supernatants were harvested, filtered using 0.45-μmpore size Millex HV PVDF filter units (Millipore), and concentrated by centrifugation at 60000g for 90 min. The supernatants were aspirated, and virus-containing pellets were resuspended in PBS overnight at 4 °C. One day before infection, target cells were seeded at 60 000 cells/well in 12well plates. At the time of infection, Polybrene was added to a final concentration of 25 mm, resuspended virus was added, and cells were incubated for 4 h at 37 °C in an atmosphere of 93% air, 7% CO2. Virus containing supernatants were then removed, fresh medium was added to cells, and cells were incubated for an additional 2−3 days in an atmosphere of 95% air, 5% CO2. Stable incorporation of FG12-based lentiviral DNA was determined by immunoblot analysis. Reporter Gene Activity Assays. HEK293T cells were plated in six-well plates prior to transfection with the indicated plasmids by calcium phosphate precipitation. Twenty-four hours post-transfection luciferase activity was determined using a luciferase assay kit (Promega) according to the C


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Biochemistry

Figure 1. Proteomic TAP screen identifies novel AIF binding proteins. (A) Schematic showing domain organization and TAP tag position for fulllength and N102-AIF proteins. Arrows indicate sites of proteolytic cleavage that occurs during maturation (thin line at position 54/55) or cell death (thick line at position 102/103), numbering corresponds to the human AIF protein sequence. MLS, mitochondrial localization sequence; FAD, split FAD binding domain; NAD(H), NAD(H) binding domain; Ct, carboxy-terminal domain. (B) IgG sepharose beads were either left untreated (non cross-linked) or cross-linked as described in materials and methods. Beads were then washed and eluted. Wash and eluate fractions were separated by SDS-PAGE and protein content of each sample was determined by Coomassie staining. Note the abundance of protein contained within non crosslinked eluate compared to cross-linked eluate. (C) HEK293T cells were transiently transfected with plasmids encoding either control (N102-AIFTAP, top panel) or full-length AIF-TAP (middle, bottom panels) fusion proteins. Forty-eight hours after transfection cells were fractionated, and mitochondrial extracts were prepared. Tagged proteins were then captured using cross-linked IgG beads, eluted by TEV protease digestion, and captured a second time using calmodulin beads. Final eluates from calmodulin binding were then precipitated with trichloroacetic acid prior to proteomic analysis. Shown is immunoblot analysis of samples following each step of the TAP procedure. Immunoblotting was performed using antiAIF followed by HRP secondary (top, middle panels), or HRP secondary alone (bottom panel). Note the HRP secondary will detect the protein A domain of the TAP tag and is the source of signal observed in the top panel, not AIF immunoreactivity.

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RESULTS Identification of Novel AIF Binding Proteins through Tandem Affinity Purification. The mechanisms through which AIF regulates cellular life-death balance remain elusive more than a decade since initial discovery of the AIF protein.3 In order to identify novel factors that may further define the spectrum of cellular activities controlled by AIF, we employed a biochemical screen to identify potential AIF binding proteins using the tandem affinity purification (TAP) method.40,41 In previous experiences employing this approach we frequently observed the presence of nonspecific IgG-derived proteins in our captured fractions. In order to avoid such contamination in our mitochondrial extracts, IgG-conjugated beads were chemically cross-linked prior to use for TAP. As shown in Figure 1B, cross-linking substantially reduced the presence of IgG material that eluted during subsequent wash steps. As bait this screen employed either full-length AIF in fusion with a carboxyterminal TAP tag (AIF-TAP) or a truncated AIF protein containing only the first 102 amino terminal residues (N102TAP) (Figure 1A). N102 serves as a negative control for nonspecific binding since this molecule is localized to mitochondria in a similar manner to both wildtype AIF and our full-length AIF-TAP fusion proteins, but lacks the residues

contained within the mature AIF protein. To focus on those factors most likely to participate in cellular regulation under healthy conditions, we prepared extracts only from mitochondrial fractions following physical disruption of AIF-TAP expressing cells. Following transfection and expression in HEK293T cells, mitochondrial extracts were prepared and then tagged proteins were captured using cross-linked IgG beads, eluted by TEV protease digestion, and captured a second time using calmodulin beads. Figure 1C follows the purification of AIF containing complexes through the TAP process. Purified material was precipitated from calmodulin binding eluates by trichloroacetic acid, digested with trypsin, and potential AIFinteracting proteins were identified by liquid chromatographytandem mass spectrometry analysis of generated peptides. This approach identified a number of novel AIF-associated proteins (Table 1), including proteins involved in such varied processes as control of oxidative stress, necrosis, facilitation of mitochondrial solute transport, and the control of transcription and translation of mitochondrial genes. Validation of AIF Binding Candidates ANT2 and PGAM5. To confirm the protein−protein interactions indicated by our TAP screen we employed a coimmunoprecipitation approach and focused our validation efforts on two factors: adenine nucleotide transporter 2 (ANT2) and D


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Biochemistry

reported to encode two distinct protein isoforms as a result of alternative splicing,25 both isoforms were tested for binding to AIF. When either full-length AIF or the Δ54 (mature) form of AIF were coexpressed along with ANT2 containing either a carboxy- or amino-terminal HA tag, coprecipitation was observed between all combinations of AIF and ANT2 in correlation with protein expression levels (Figure 2A). Similarly, when full-length or Δ54 AIF was coexpressed with either PGAM5S or PGAM5L coprecipitation was also observed, albeit the association between full-length AIF and PGAM5L was substantially weaker (based on the faint band intensity) than the other combinations tested (Figure 2B). Next the ability of ANT2 and PGAM5 to compete for binding to AIF was tested. An AIF-GST fusion protein was expressed in cells along with ANT2 alone, PGAM5 alone, or both ANT2 and PGAM5 prior to precipitation with glutathione-conjugated beads. As shown in Figure 2C, when either ANT2 or PGAM5 were expressed alone, a robust association with AIF was detected. Interestingly, whereas PGAM5 binding was not affected by the coexpression of ANT2, the ability of ANT2 to bind AIF was diminished when PGAM5 was present, suggesting the possibility that the regions within AIF to which ANT2 and PGAM5 bind at least partially overlap, and that PGAM5 is capable of displacing ANT2. As the

Table 1. Novel AIF Binding Proteins Identified by TandemAffinity Purification name

function

AIFM1, apoptosis inducing factor (AIF) HSPA9, mitochondrial heat shock protein 70 PGAM5, phosphoglycerate mutase 5 SLC25A5, ADP/ATP carrier protein 2 SFXN1, sideroflexin 1 SLC25A11, 2-oxoglutarate/malate carrier SLC25A3, phosphate carrier MRPL18, mitochondrial ribosomal protein L18 TUFM, mitochondrial Elongation factor Tu

bait heat shock, mitochondrial protein import antioxidant response mitochondrial nucleotide transport mitochondrial solute transport mitochondrial solute transport mitochondrial solute transport mitochondrial transcription/ translation mitochondrial transcription/ translation

phosphoglycerate mutase 5 (PGAM5). We chose to concentrate on ANT2 and PGAM5 based on the known functions of each molecule: ANT2 assists in the transport of adenine nucleotides (ATP, ADP) across the inner membrane of the mitochondria and thereby supports oxidative phosphorylation,42 and PGAM5 has been implicated in controlling antioxidant responses, cell death, and mitophagic processing of mitochondria.24−29,43 Since the PGAM5 gene has been

Figure 2. PGAM5 competes with ANT2 for binding to AIF; AIF attenuates PGAM5 antioxidant responses. (A) Plasmids encoding empty vector (control), FLAG-tagged full-length AIF (FL-AIF) or FLAG-tagged mature AIF (Δ54 AIF) along with plasmids encoding either amino- or carboxyterminal HA-tagged ANT2 were cotransfected into HEK293T cells. AIF was immunoprecipitated using anti-FLAG, lysates and immunoprecipitates were separated by SDS-PAGE prior to immunoblot analysis for the presence of ANT2 (HA) and AIF (FLAG). (B) Plasmids encoding an empty vector (control), HA-tagged PGAM5S or HA-tagged PGAM5L were cotransfected with either an empty vector (control), FLAG-tagged full-length AIF (FL AIF), or Flag-tagged mature AIF (Δ54 AIF) in HEK293T cells. AIF was immunoprecipitated using anti-Flag 24 h post-transfection. Input lysates and immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblot for the presence of PGAM5 using anti-HA. (C) HEK293T cells were transfected with a plasmid encoding AIF-GST along with either control, PGAM5-HA alone, ANT2-FLAG alone, or PGAM5HA and ANT2-FLAG expressing plasmids. AIF was precipitated using glutathione beads, and the presence of PGAM5 and ANT2 in precipitated complexes was determined by immunoblot analysis. (D) HEK29T3 cells were transfected with the indicated plasmids along with a luciferase-based ARE reporter. Cells were left untreated or treated with tBHQ, and antioxidant responses were determined following measurement of luciferase activity; data were derived from three independent experiments and are presented as the means ± SD. E


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Biochemistry

Figure 3. PGAM5 triggers caspase-independent cell death. (A) HEK293T cells were transfected with an empty vector (control), PGAM5S, PGAM5L or the pro-apoptotic Bcl-2 family member, Bax. Forty-eight hours following transfection, cell viability was assessed using propidium iodide staining and flow cytometry. Data were derived from three independent experiments and are presented as the means ± SD. (B) Mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine (TMRM) and flow cytometry following overnight transfection. Data were derived from triplicate samples and represented as percentage of cells maintaining high MMP (ΔΨm). (C) HEK293T cells were transfected with empty vector (control), PGAM5S, or PGAM5L. Cell lysates were subjected to a caspase 3 DEVD-AFC assay 48 h post transfection, data presented as the means ± SD obtained from three individual experiments. (D) Empty vector (control), PGAM5S, or PGAM5L was transfected into HEK293T cells. Cells were treated with 50 μM ZVAD at 10 h post-transfection. Cell lysates were subjected to a caspase 3 DEVD-AFC assay 48 h post transfection, data presented as the means ± SD obtained from three individual experiments. (E) Empty vector (control), PGAM5S, or PGAM5L was transfected into HEK293T cells. Cells were treated with 50 μM ZVAD at 10 h post-transfection. Forty-eight hours post-transfection cells were harvested, and viability was measured using propidium iodide and flow cytometry. (F) HEK293T cells were transiently transfected with an empty vector (control), ASK1, PGAM5S, or PGAM5L. Whole cell lysates were prepared at 48 h post-transfection, and Western blot analysis was performed using phosphorylated p38 and JNK antibodies.

were then left untreated or treated with tert-butylhydroquinone (tBHQ), a robust stimulator of antioxidant response genes. As expected, neither control transfected cells nor cells expressing AIF alone exhibited a substantial increase in antioxidant responses following treatment (Figure 2D). In contrast, expression of either PGAM5 isoform significantly potentiated tBHQ stimulation in the absence of AIF. Importantly the coexpression of AIF and PGAM5S resulted in a substantially reduced antioxidant response following treatment whereas PGAM5L appeared unaffected by AIF coexpression. These data are in agreement with the coprecipitation studies shown above (Figure 2B) indicating that AIF binds more robustly to

stronger binding partner, we focused subsequent functional efforts on evaluation of the AIF/PGAM5 association. Initial functional characterizations of the PGAM5 protein25,29 suggested an ability of PGAM5 to regulate the antioxidant response, a protective gene expression program that assists cells under conditions of oxidative stress. To determine if AIF was capable of influencing PGAM5 activity following oxidative stress we employed a luciferase-based approach to assess antioxidant responses. HEK293T cells were transiently transfected with a control plasmid or plasmids encoding AIF, PGAM5S, PGAM5L, AIF + PGAM5S, or AIF + PGAM5L along with a plasmid encoding firefly luciferase under control of the antioxidant response promotor. Following transfection cells F


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Biochemistry

Figure 4. AIF reduces PGAM5 caspase activation, fails to block PGAM5-mediated death. (A) Protein expression of AIF in the H293T parental, shLacZ, and shAIF cell lines was detected by immunoblotting with AIF antibody. β-Actin was detected as a loading control. (B) H293T parental, shLacZ and shAIF cells were transfected with empty vector (control), PGAM5S, or PGAM5L. Forty-eight hours post transfection cells were harvested and viability was measured using propidium iodide and flow cytometry, data presented as the means ± SD obtained from three individual experiments. (C and E) Empty vector (control), full-length AIF (FL-AIF), mature AIF (Δ54 AIF), or apoptotic AIF (Δ102 AIF) were transfected with or without PGAM5S in HEK293T cells. Cell lysates were subjected to a caspase 3 DEVD-AFC (C) and cell viability (E) assay 48 h post transfection. Insets in each panel indicate immunoblot analysis confirming PGAM5 protein expression. Black line through immunoblot image in panel C inset indicates the position at which irrelevant lanes were removed from the image for purposes of clarity; remaining portions of image received identical treatment during imaging process. Data presented as the means ± SD obtained from three individual experiments. (D and F) Empty vector (control), full-length AIF (FL-AIF), mature AIF (Δ54 AIF), or apoptotic AIF (Δ102 AIF) were transfected with or without PGAM5L in HEK293T cells. Cell lysates were subjected to a caspase 3 DEVD-AFC (D) and cell viability (F) assay 48 h post transfection; data presented as the means ± SD obtained from three individual experiments.

antioxidant responses, we observed via phase-contrast microscopy that at 48 h post-transfection (a time frame beyond the window required for luciferase measurements of antioxidant activity) cells expressing either PGAM5S or PGAM5L began to exhibit morphological changes indicative of apoptosis. To

PGAM5S and suggest that AIF can regulate the antioxidant response activities of PGAM5S. Elevated Levels of PGAM5S and PGAM5L Trigger Caspase-Independent Cell Death. While investigating the functional interactions between AIF and PGAM5-mediated G


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Biochemistry

on either pathway (Figure 3F). These data conflict with what has been previously reported for PGAM5L24 and suggests the activation of ASK1 is not involved in PGAM5-mediated cell death. The reasons for this discrepancy are unclear, but may reflect cell type dependent differences in the ability of PGAM5L to promote activation of p38 and JNK. PGAM5-Induced Death Is AIF Independent. Although apoptosis is generally considered a caspase-dependent process1 there are alternative models, notably AIF-induced cell death, which can occur in the absence of caspase activation.48 Since both PGAM5 isoforms associate with AIF, a well-known caspase-independent pro-apoptotic protein, PGAM5S and PGAM5L could be promoting cell death through interaction with AIF. To determine if AIF plays a role in PGAM5-induced cell death, cell viability following PGAM5 expression was assessed in AIF deficient HEK293T cells. Lentivirus was used to stably infect HEK293T cells with shRNA sequences targeting either AIF or LacZ as a negative control.37 Cells stably incorporating hairpin DNA were detected by GFP fluorescence and AIF ablation was confirmed by immunoblot analysis (Figure 4A). Once knockdown of AIF was confirmed, the parental (uninfected, AIF proficient), shLacZ (infected negative control, AIF proficient) and shAIF (AIF deficient) cell lines were transfected with PGAM5S or PGAM5L expression plasmids and cell viability was assessed at 48 h posttransfection. Both PGAM5 isoforms were able to promote a significant loss in cell viability in the absence and presence of AIF (Figure 4B), suggesting PGAM5 does not induce caspaseindependent cell death through its association with AIF. It was notable, however, that AIF does have the ability to partially inhibit caspase-3 activation resulting from expression of either PGAM5 isoform: the unprocessed form (F.L), mature form (Δ54), or apoptotic form (Δ102) of AIF were coexpressed with PGAM5S and caspase activity was assessed 48 h post-transfection. All three AIF variants reduced caspase activity following elevated expression of either PGAM5 isoform (Figure 4C,D), yet this reduction in caspase activation failed to increase cell viability (Figure 4E,F) and is consistent with the caspase-independent nature of PGAM5-mediated cell killing observed above (Figure 3E). Overall, these data reveal an unexpected role for AIF in attenuating caspase-activation, yet this activity does not prevent PGAM5-mediated cell death, indicating that the presence of AIF is not essential for either PGAM5 isoform to kill and that the physical association between AIF and PGAM5 may be more relevant to the maintenance of cellular redox balance and homeostasis. PGAM5S and PGAM5L Promote Mitochondrial Abnormalities and Mitophagy. The data above demonstrate that elevated expression of either PGAM5 isoform is sufficient to trigger cell death, and while features of apoptotic cell death are apparent (MOMP, caspase activation), caspase activation is not required for PGAM5-mediated toxicity. It has been reported previously that transgenic expression of PGAM5 in the flight muscles of Drosophila promotes mitochondrial fragmentation, indicating PGAM5 plays a role in the process of mitochondrial fission.49 The ectopic expression of human PGAM5S and PGAM5L in COS-1 and HeLa cells also results in altered mitochondrial morphology, further supporting a role for PGAM5 in regulating mitochondrial dynamics.29 In an effort to establish if elevated PGAM5 expression is disrupting mitochondrial dynamics to the detriment of the cell, transmission electron microscopy (TEM) was performed on HEK293T cells 36 h post-transfection with an empty vector

quantify cell death following elevation of PGAM5S and PGAM5L expression, a propidium iodide fluorescence exclusion assay was performed. HEK293T cells were transfected with an empty vector (control), PGAM5S, PGAM5L, or the Bcl-2 associated protein, Bax. Bax is a pro-apoptotic member of the Bcl-2 family and was used as a positive control.44 Following transfection, cells were harvested, resuspended in propidium iodide, and cell viability was determined by flow cytometry. Expression of both PGAM5S and PGAM5L resulted in a more than 50% loss of cell viability compared to control (Figure 3A), similar to Bax expression, confirming the morphological changes following PGAM5 overexpression are indicative of a cell death process. Previous studies have implicated PGAM5S and PGAM5L as regulatory factors in the control of cell death via apoptotic and necrotic pathways,26,45 yet to our knowledge no studies have been reported indicating that PGAM5 overexpression alone is sufficient for triggering cell death, and a definitive molecular characterization of the role PGAM5 plays in cell death has yet to be reported. To further define the mechanism of PGAM5 toxicity we assessed integrity of the outer mitochondrial membrane. Permeabilization of the outer mitochondrial membrane (MOMP) is a common mechanism for triggering apoptosis, and MOMP is sometimes a nonspecific consequence of necrotic forms of cell death. To assess the extent to which MOMP occurs following PGAM5 expression, HEK293T cells were transfected with control, PGAM5S, PGAM5L, or Bax encoding plasmids. Cells were then incubated with tetramethylrhodamine methyl ester (TMRM), a fluorescent indicator of mitochondrial membrane potential (ΔΨm) that can be quantified by flow cytometry.46 A decrease or loss of membrane potential serves as an indirect assessment of MOMP. As shown in Figure 3B, the expression of both PGAM5 isoforms led to a substantial decrease in ΔΨm compared to control cells, suggesting integrity of the outer mitochondrial membrane has been lost. During apoptosis a common consequence of MOMP is caspase activation resulting from cytosolic release of cytochrome c, an obligate step in the biochemical completion of the apoptotic pathway. To determine the extent of caspase activation following PGAM5-mediated MOMP, a caspase assay was performed. The activity of caspase-3 was assessed 48 h post-transfection using an AFC-derived caspase-3 substrate.39 Both PGAM5 isoforms were able to induce a significant amount of caspase-3 activity compared to control (Figure 3C). Interestingly, when cells were incubated with the pan-caspase inhibitor zVAD-fmk following transfection with PGAM5, no change in cell killing was observed following expression of either PGAM5 isoform despite complete inhibition of caspase activity (Figure 3D,E). These data suggest that while elevated PGAM5 expression triggers cell death with features of apoptosis, the apoptotic machinery (specifically caspases) is not required in order for cell death to proceed. The phosphatase activity of PGAM5L has been shown to trigger the ser/thr kinase ASK1, which plays a significant role in regulating the p38 and JNK pathways.32,45 Activation of these pathways can trigger a variety of cellular responses depending on stimulus and cell type, and often results in cell death.47 To determine if the cell death being triggered by PGAM5S and PGAM5L is a result of increased activation of these pathways, we assessed activation of p38 and JNK by immunoblot against their phosphorylated forms. The overexpression of ASK1 resulted in increased phosphorylation of both p38 and JNK, while PGAM5S and PGAM5L expression did not have an effect H


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spherical mitochondria (Figure 5C) with abnormal or absent cristae formation (Figure 5D); compared to another cell population which contained a limited number of large, spherical mitochondria (Figure 5E) also exhibiting highly aberrant or absent cristae (Figure 5F). A small population of cells expressing PGAM5S demonstrated a perinuclear aggregation of irregular mitochondria with the outer mitochondrial membrane either indiscernible or visibly fragmented (data not shown). This aggregation was also seen following the expression of PGAM5L (Figure 5G,H) along with another population of cells containing an abundance of large, spherical mitochondria distributed throughout the intracellular space containing abnormal or deficient cristae (data not shown), that resemble those observed in Figure 5E,F (following PGAM5s expression). Taken together these data demonstrate dramatic alterations to mitochondrial number, distribution, morphology, and structure following upregulation of PGAM5S and PGAM5L. A likely explanation is that elevated PGAM5 expression leads to alterations in the mitochondrial quality control pathway known as mitophagy, which is an autophagic mechanism specific to mitochondria that allows for the clearance and recycling of this organelle following damage or dysfunction, and that can lead to cell death when dysregulated. While the mechanisms that promote mitophagy are complex, a significant node to controlling mitophagic signaling is regulated by the cellular kinase PTEN-induced putative protein kinase 1 (PINK1).50 Under conditions of mitochondrial stress PINK1 protein levels rise, leading to increased phosphorylation of the PINK1 substrate Parkin51 and subsequently the mitochondrial recruitment of proteins such as p62 and ULK1 that carry out the mitophagic process.52,53 Thus, elevated levels of PINK1 serve as a marker for cells undergoing mitophagy. In order to support our electron microscopy observations and test the hypothesis that PGAM5-mediated cell death is mitophagic in nature, we assessed PINK1 expression levels following PGAM5 transfection. Notably all cells transfected with either PGAM5 isoform exhibited significant elevation in PINK1 expression (Figure 5I, note PINK1 is undetectable by immunoblot in control cells), indicating that PGAM5 drives mitophagic cell death in this model system. It is also notable that phosphatase deficient mutants (H105A) of either PGAM5 isoform retained the ability to promote PINK1 stabilization (Figure 5I) and trigger cell death, as described below. X-Linked Inhibitor of Apoptosis Prevents PGAM5Mediated Cell Killing via Noncanonical Ubiquitination. X-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of cell death that promotes cell survival through the direct inhibition of caspase activity but also controls cellular signaling through an intrinsic E3 ubiquitin ligase activity.30,54 Interestingly AIF is an XIAP substrate,31 and we have previously demonstrated that XIAP can attenuate AIF-mediated cell death via noncanonical ubiquitination.55 A recent screen for additional XIAP substrates identified PGAM5 as a target for both XIAP and the XIAP homologue cIAP-1,56 yet a detailed analysis of XIAP-mediated PGAM5 ubiquitination was not undertaken, and the physiological significance of this modification remains unclear. In light of the AIF-PGAM5 interaction and the ability of PGAM5 to promote cell death, we hypothesized that the modification of PGAM5 by XIAP may promote cell survival. We began by testing the extent to which PGAM5 isoforms are ubiquitinated by XIAP in intact cells. HEK293T cells were transfected with a plasmid encoding his-tagged ubiquitin along with control or PGAM5 expression plasmids in the absence or

(control), PGAM5S, or PGAM5L. HEK293T cells transfected with an empty vector displayed an abundance of mitochondria distributed throughout each cell (Figure 5A) that exhibited healthy dynamics and normal cristae formation (Figure 5B). Upon exogenous expression of PGAM5S, cells displayed a range of phenotypes; all containing highly abnormal mitochondria. One population of cells possessed an abundance of small,

Figure 5. PGAM5 induces mitophagic cell death. Panels A−H: HEK293T cells were transiently transfected with control (panel A, B), PGAM5S (panels C−F) or PGAM5L (panel G, H). Thirty-six hours after transfection whole cell transmission electron microscopy was then performed to determine cellular architecture. Right column images are magnification left column images as indicated by boxes. Asterisks and lower case (m) designated mitochondrial; circle/oval highlights mitochondrial aggregation. Arrows highlight cristae formation, with triangles highlighting a lack of cristae. Scale bars are as shown; each image is representative of at least 10 images per sample. (I) HEK293T cells were transiently transfected with the indicated plasmids. Forty-eight hours after transfection lysates were prepared and immunoblotted for the presence of PINK1 and PGAM5. Immunoblotting for β-actin was used as a loading control. I


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Figure 6. PGAM5L, but not PGAM5S, is inhibited by noncanonical XIAP-mediated ubiquitination. (A) HEK293T cells were transfected with a plasmid encoding His6-Myc-Ubiquitin (His-wt-UB) along with empty plasmid (control) or plasmids encoding PGAM5L-HA or PGAM5S-HA in the absence or presence of XIAP. Cell lysates were incubated with Ni-NTA agarose, and precipitated material was immunoblotted for the HA epitope (for PGAM5) and XIAP. (B) HEK293T cells were transfected with His-UB and PGAM5L-HA plasmids in the absence and presence of XIAP as described in panel A. Twenty-four hours later cells were treated with MG132 for 4 h prior to lysis and precipitation as described above. Immonoblotting was then performed using anti-HA (PGAM5), anti-Flag (XIAP) and antiubiquitin. (C) HEK293T cells were transfected with a plasmids encoding PGAM5L-HA along with either wildtype or K48R ubiquitin in the absence and presence of XIAP. Lysates were precipitated and analyzed as described in panel A. (D) HEK293T cells were transfected with the indicated plasmids. Forty-eight hours following transfection, cell viability was assessed using propidium iodide staining and flow cytometry. Data were derived from three independent experiments and are presented as the means ± SD. (E) HEK293T cells were transfected with plasmids encoding His-wt-UB and PGAM5L-HA in the absence and presence of either wildtype or H467A XIAP. Lysates were precipitated and analyzed as described in panel A.

the abundance of ubiquitinated PGAM5 species. Interestingly, while both isoforms of PGAM5 displayed significant ubiquitination laddering under basal conditions (Figure 6A) only PGAM5L exhibited increased ubiquitination following expres-

presence of a plasmid encoding wildtype XIAP. Cell lysates were prepared, and ubiquitinated proteins were precipitated using Ni-NTA beads. Immunoblot analysis for PGAM5 levels was then used to determine the extent to which XIAP increased J


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Figure 7. Phosphatase activity is restricted to PGAM5L and is dispensable for cell death. (A) Empty plasmid (control), PGAM5L wild-type, and PGAM5L H105A were transiently expressed in HEK293T cells. PGAM5L proteins were precipitated at 24 h post-transfection and incubated with a phosphorylated ser/thr peptide substrate. Phosphatase activity was measured as the amount of free phosphate available following 15 min of incubation. Inset, immunoblot analysis of PGAM5L wildtype and H105A expression in phosphatase assay. (B) Empty plasmid (control), Flag-tagged PGAM5S wild-type, and Flag-tagged PGAM5L wild-type were transiently expressed in HEK293T cells. PGAM5 proteins were precipitated at 24 h post-transfection and incubated with a phosphorylated peptide substrate. Phosphatase activity was measured as the amount of free phosphate available following 15 min of incubation. Inset, immunoblot confirming expression and precipitation of PGAM5S and PGAML prior to incubation with phosphorylated substrate. (C) HEK293T cells were transfected with empty vector (control), PGAM5L wildtype, or PGAM5L H105A. Cell lysates were subjected to a caspase 3 DEVD-AFC assay 48 h post transfection, data presented as the means ± SEM obtained from three individual experiments. (D) PGAM5L variants or plasmid control were transiently expressed in HEK293T cells. Forty-eight hours after transfection cells were harvested and viability measured using propidium iodide and flow cytometry. (E) HEK293T cells were transfected with empty vector (control), PGAM5S wildtype, or PGAM5S H105A. Cell lysates were subjected to a caspase 3 DEVD-AFC assay 48 h post transfection, data presented as the means ± SD obtained from three individual experiments. (F) PGAM5S variants or plasmid control were transiently expressed in HEK293T cells. 48h after transfection cells were harvested and viability measured using propidium iodide and flow cytometry. Data presented in each panel represents the means ± SD obtained from three individual replicates of each experiment.

sion of XIAP. This observation is notable since the screen in which PGAM5 was identified as an XIAP substrate did not assess the short isoform of PGAM5. The most common form of ubiquitination involves the covalent attachment of ubiquitin to lysyl residues within the target protein followed by polyubiquitin branching that extends from lysine 48 of ubiquitin itself. This “K48” polyubiquitination allows the proteasome to recognize and degrade the targeted protein. While this mechanism accounts for the majority of cellular ubiquitination events, other forms of ubiquitin branches that employ alternative lysyl residues within the ubiquitin

polypeptide have been reported, and these noncanonical modifications generally do not result in protein degradation. We previously reported that AIF is a substrate of noncanonical ubiquitination mediated by XIAP,55 and therefore tested whether PGAM5 is modified in a similar manner. To determine if PGAM5L is degraded following ubiquitination, as would be the case if K48 polyubiquitin occurs, we repeated the Hisubiquitin precipitation in the presence of the proteasomal inhibitor MG132, which will prevent degradation of K48 ubiquitinated proteins if this linkage is employed. As shown in Figure 6B, we did not observe increased PGAM5L levels K


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Biochemistry

Figure 8. PGAM5L forms dimers in live cells and cell extracts. (A) HEK293T cells were transiently transfected with the indicated plasmids prior to lysis and immunoprecipitation with anti-FLAG. Immunoblot analysis was performed using anti-HA and anti-FLAG antibodies in order to detect the presence of PGAM5L homodimers, PGAM5S homodimers, and PGAM5L/PGAM5S heterodimers. (B−E) HEK293T cells were transfected with plasmids encoding the indicated Bi-FC tagged PGAM5 proteins. 24 h post-transfection cells were harvested by trypsinization and fluorescent complexes were analyzed and quantified by flow cytometry. Representative raw data histograms are shown for each BiFC combination tested. Marker lines indicate the fraction of each cell population that is positive for fluorescence, indicating protein−protein interaction. (F) Multiple replicates of each BiFC combination shown in panels B−E were quantified to determine extent of BiFC detection among PGAM5 isoforms. Data presented represents mean ± SD obtained from three individual replicates of each group.

inhibited by XIAP (Figure 6D). Moreover, the ability of XIAP to prevent PGAM5L from triggering death was dependent on ubiquitin ligase activity, as the XIAP mutant H467A, which lacks the ability to ubiquitinate substrates but retains the ability to inhibit caspases31,35,57 (Figure 6E) failed to prevent PGAM5L-mediated death. Overall these data demonstrate that XIAP serves as an isoform specific E3 ubiquitin ligase of PGAM5L, which prevents the long isoform from triggering cell death through a ubiquitin dependent mechanism. PGAM5S Lacks Detectable Phosphatase Activity in Vitro, PGAM5L Phosphatase Activity Is Dispensable for Inducing Cell Death. To date several studies have demonstrated intrinsic phosphatase activity for the long isoform of PGAM5, as substrates including ASK1, Drp1, and FUNDC1 have been identified.24,27,28 Interestingly the ability of PGAM5S to serve in a similar catalytic capacity has been largely inferred

following MG132 treatment, suggesting that XIAP employs noncanonical ubiquitination. To further confirm this observation we employed an alternative form of ubiquitin in our precipitation assay in which the lysine 48 has been mutated to arginine. This mutation will block ubiquitination if a canonical linkage is employed. Figure 6C demonstrates that XIAP is equally capable of using K48R ubiquitin as a substrate when modifying PGAM5L, confirming the noncanonical nature by which XIAP ubiquitinates PGAM5L. To determine the functional significance of PGAM5 ubiquitination we assessed the ability of XIAP to block PGAM5-mediated cell death. Following elevated expression of either PGAM5 isoform we observed significant cell death, consistent with our viability assessments shown in Figure 3. Interestingly, whereas XIAP had no effect on the ability of PGAM5S to kill, PGAM5L mediated cell death was significantly L


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Biochemistry

approach. Epitope-tagged versions of both PGAM5L and PGAM5S containing either the FLAG or HA tag were cotransfected into HEK293T cells in the following combinations: PGAM5L-FLAG + PGAM5L-HA (long homodimer), PGAM5 S -FLAG + PGAM5 S -HA (short homodimer), PGAM5S-FLAG + PGAM5L-HA (long/short heterodimer), PGAM5L-FLAG + PGAM5S-HA (long/short heterodimer). Cell lysates were then prepared and precipitated with antibodies to the FLAG epitope prior to immunoblot analysis for HA. As shown in Figure 8A, coprecipitation identified a robust interaction between tagged PGAM5L proteins, suggesting the formation of at least a dimeric complex by the long isoform. A comparatively modest complex indicative of a possible heterodimer between PGAM5L and PGAM5S was also identified by this approach. Complexes suggesting a possible homodimer between tagged PGAM5S proteins were also detected, with signal intensities that were reduced relative to the long/long and long/short combinations. This latter result may indicate a decreased ability for PGAM5S to form dimeric complexes, or may alternatively be the result of decreased extraction efficiency of the short PGAM5 isoform.26 While the above coprecipitation approach is valuable for the preliminary identification of PGAM5 containing complexes, the nature of coprecipitation makes it possible for protein−protein interactions to occur postlysis. Subcellular localization of PGAM5 has been reported by several groups, and while consensus suggests that PGAM5 is mitochondrial,29,43 agreement about the specific location within the mitochondria (outer membrane, inner membrane space, inner membrane, or matrix) is lacking. Thus, is it likely that different pools of PGAM5 exist, and that postlysis conditions allows interactions between PGAM5 isomers to occur that would not normally be possible. This is especially true for the short/long heterodimer combination. To circumvent the postlysis issue, as well as to observe PGAM5 complexes within living cells, we employed the bimolecular fluorescence complementation approach (BiFC)31,59 as an alternative to coprecipitation. The basis of this method is the separation of yellow fluorescent protein (YFP) into amino (YN) and carboxy (YC) terminal domains, neither of which is fluorescent when coexpressed in cells. When separately fused to two interacting molecules, these YFP domains may recombine, resulting in cellular fluorescence. Thus, this approach detects protein−protein interactions within a living cell, maintaining barriers between organelles and avoiding potential lysis-induced artifacts. BiFC combinations of PGAM5 L -YC, PGAM5 L -YN, PGAM5S-YC, and PGAM5S-YN were generated and transfected into HEK293T cells prior to analysis by flow cytometry. As indicated by raw data histograms shown in Figure 8B−E, neither negative control samples (Figure 8B) nor the combination of PGAM5S-YC and PGAM5S-YN (Figure 8C) yielded an increase in fluorescence above background. A small population of cells expression the combination of PGAM5L-YC and PGAM5S-YN exhibited an increase in fluorescence intensity (∼5%, Figure 8D), whereas the combination of PGAM5L-YC/PGAM5L-YN yielded a significant population (∼20%, Figure 8E) of cells with increased fluorescence when compared to controls. Quantification of replicate samples from each group is shown in Figure 8F. It should be noted that neither our coprecipitation nor BiFC approaches can distinguish between dimers and higher order oligomeric structures. However, when taken together these experiments suggest that (1) PGAM5L has a strong tendency to form

based on sequence similarity to PGAM5L, yet no definitive studies have demonstrated intrinsic phosphatase activity for PGAM5S. PGAM5L-dependent dephosphorylation of the ser/ thr kinase ASK1 has been reported to trigger the p38 and JNK pathways leading to cell death,32,45 yet as described above (Figure 3) we did not observe p38 or JNK activation following elevated expression of either PGAM5 isoform. Distinct from regulating ASK1, the phosphatase activity of PGAM5L has been implicated in the activation of Drp1 during necroptosis, offering potential insight into the mechanism of PGAM5-mediated cell killing.26 To determine the role of PGAM5 phosphatase activity in controlling cell death in our model system we generated mutants of both PGAM5 isoforms in which the histidine residue at position 105 was mutated to alanine, which has been shown to ablate the phosphatase activity of PGAM5L.45 Since this histidine residue falls within the first 238 amino acids of each isoform it is therefore present in both PGAM5S and PGAM5L. To assess phosphatase activity, a control plasmid or plasmids encoding either wildtype or H105A PGAM5L were transfected into HEK293T cells. Twenty-four hours posttransfection PGAM5 was precipitated from cell lysates, and phosphatase activity was determined on beads using a phosphorylated peptide substrate in vitro. Under these experimental conditions, PGAM5L exhibited significant in vitro phosphatase activity while the H105A mutant was comparable to controls (Figure 7A). We carried out an identical series of experiments testing the phosphatase activity of PGAM5S. However, when compared to the robust activity exhibited by PGAM5L, the short PGAM5 isoform appears catalytically impaired (Figure 7B). We speculate that the lack of activity is likely related to differential dimer formation between the short and long isoforms (as discussed below). To rule out the possibility that PGAM5S is catalytically active in ways that were not detectable within our model system, and that this undetected activity may play a role in controlling cell death responses, we tested the ability of both H105A-PGAM5L and H105A-PGAM5S to trigger cell death responses. As shown in Figure 7C,E, both wildtype and H105A variants for both isoforms were equally capable of triggering caspase activation, and the viability of cells following elevated expression of either variant (wildtype, H105A) for both PGAM5 isoforms was similarly reduced (Figure 7D,F). Overall these data indicate that while the PGAM5L isoform exhibits significant phosphatase activity, this activity is not observed in the short isoform, and that mutation of H105 to alanine in either PGAM5L or PGAM5S (which ensures both are catalytically inactive) fails to prevent PGAM5-mediated cell death. Lack of Dimerization by PGAM5S. A recent report indicated that PGAM5L forms dimers and higher order oligomers in vitro dependent on the presence of a conserved amino acid motif within the amino terminus, and that formation of higher order oligomers is critical for maximal phosphatase activity.58 However, the presence of PGAM5 dimers, (long/long homodimers, short/short homodimers, long/short heterodimers, or higher order oligomers) has not been demonstrated in intact cells, and differences between the abilities of both isoforms to form dimers have not been established. We therefore questioned (1) whether dimerization between PGAM5 isoforms could be detected in intact cells and (2) whether an altered capacity to dimerize may explain our lack of observed phosphatase activity for PGAM5S. The ability of PGAM5 to dimerize was first tested by a coprecipitation M


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is not important, though we note that caspase activation following PGAM5 expression is inhibited by AIF. To our knowledge this is the first reported instance in which AIF expression inhibits caspase activity. While AIF does not influence PGAM5-mediated death, we found that the AIF binding protein XIAP significantly reduces the ability of PGAM5L to kill. As a high affinity caspase inhibitor XIAP exhibits a well-established role in controlling the process of apoptosis.30 Distinct from the ability to inhibit caspases, however, XIAP also possesses intrinsic E3 ubiquitin ligase activity that has been implicated in regulating a variety of substrates, including AIF.31 Indeed, as reported by Wells and co-workers,56 PGAM5 is a substrate of XIAP, though the functional significance of this modification remained unclear until now. We report that XIAP not only employs but in fact depends on its ubiquitin ligase activity to block PGAM5L: H467A-XIAP, which lacks E3 ligase activity but retains the ability to inhibit caspases, fails to inhibit PGAM5L. Moreover, the nature of XIAP-mediated PGAM5 ubiquitination is noncanonical: while most ubiquitination substrates undergo modification by the addition of polyubiquitin chains linked through lysine 48 of the ubiquitin polypeptide,68 thereby signaling proteasomal degradation, XIAP does not employ a K48 linkage for PGAM5. In addition, proteasomal inhibition does not lead to an increase in PGAM5 abundance as would be expected if the fate of ubiquitination was degradation. These observations are consistent with our previous studies exploring the ability of XIAP to modify AIF55 and suggest that PGAM5 joins AIF in the subclass of XIAP substrates that do not receive K48-ubiquitin linkages. We tested the role of PGAM5 phosphatase activity in our cell death model and observed that the H105A variants of both PGAM5S and PGAM5L retained the ability to trigger cell death. The phosphatase activity of PGAM5 L has been well documented in a variety of studies examining activity either in vitro using purified protein or from cell extracts.24,58 Similar activity for PGAM5S has been largely inferred based on the presence of the PGAM domain within each isoform;26 however it has yet to be demonstrated that such activity exists, largely due to difficulties in solubilizing the PGAM5S isoform.26,58 Several reports have indicated that when expressed in bacteria PGAM5S fails to solubilize under conditions that retain native structure,26,58 and reports have indicated that when expressed in eukaryotic cells PGAM5S exhibits significantly more hydrophobic character than the long isoform.26 We have also observed this phenomenon: detergent conditions used for solubilizing PGAM5S are much more stringent than those required for extraction of PGAM5L (data not shown), suggesting that when compared to PGAM5L the c-terminus of PGAM5S introduces significantly higher hydrophobic character and/or increases the affinity of PGAM5S for membrane imbedded proteins making extraction difficult. Under extractions conditions that both solubilized PGAM5S and retained phosphatase activity for PGAM5L we were unable to detect phosphate release by PGAM5S when offered a phosphorylated peptide substrate. While we cannot rule out the possibility that an alternative substrate may be dephosphorylated by PGAM5S, our data suggest this isoform possesses much lower catalytic activity than PGAM5L. A structure for PGAM5L has been deposited in the Molecular Modeling Database (MMDB,69 code 3MXO) and shows a dimeric complex that forms due to amino acids residues within the carboxy-terminal domain of PGAM5L that

complexes containing at least a dimer, (2) heteromeric complexes between PGAM5L and PGAM5S form with a much lower frequency, and (3) dimeric complexes composed exclusively of PGAM5S are essentially undetectable in intact cells. In light of our inability to detect phosphatase activity in PGAM5S and recent data suggesting that PGAM5L must oligomerize for full catalytic activity, these data suggest that a lack of dimer formation is a significant factor to reduced catalysis by PGAM5S.

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DISCUSSION AIF was originally discovered through its ability to trigger caspase-independent cell death in a variety of model systems.11 Although recognized for this apoptotic function, accumulating evidence suggests AIF plays a more significant role in cell survival. To date a handful of AIF binding proteins have been identified, including Hsp70, H2AX, XIAP, and CHCHD4,6,31,60,61 and the diversity of signaling capable through each of these factors is consistent with the multiple roles played by AIF in coordinating life/death balance decisions. Yet the ability of AIF to bind each of these factors does not completely explain the mechanisms by which AIF activities are regulated in either the pro-death or pro-survival settings. To further explore this issue, we employed a biochemical screen to uncover potential AIF associated proteins from healthy mitochondria. In addition to our identification of the AIF/PGAM5 association described here, it is worth noting that several of the AIF associated factors we identified include components of mitochondrial membrane transport as well as machinery necessary for expression of the mitochondrial genome.62 To find such factors in our screen is consistent with the established role for AIF in controlling mitochondrial energy metabolism and suggest additional avenues (nucleotide transport, mitochondrial gene expression) through which AIF may regulate this process. Further study will be needed to evaluate these possibilities. PGAM5 has been implicated in a diverse range of cellular activities related to control of signal transduction pathways. Through interaction with Keap1, PGAM5 can regulate levels of the Nrf2 transcription factor and thereby control cellular defenses against oxidative stress.25,29 As a mitochondrial proximal protein PGAM5 is ideally placed to sense and respond to changing cellular redox levels, and it is conceptually satisfying that AIF plays a role in this process. Our observation that PGAM5S is more sensitive to AIF is notable and further suggests a greater diversity between PGAM5 isoforms than is currently clear from published studies. As an oxidoreductase AIF has long been appreciated as a potential regulator of cellular oxidative stress,63 and the ability to control PGAM5mediated antioxidant responses represents an additional means through which AIF influences cellular redox balance. As a regulator of cell death PGAM5 has been separately implicated as a promotor and inhibitor of apoptosis, and as both a necessary component of necrotic cell death and an inhibitor of this process.24,26,64−67 As such no consensus paradigm for PGAM5 control of cell death exists, and there are likely context and stimulus dependent factors that determine whether PGAM5 acts as a killer or protector of cells. We now add to this body of evidence our observations that transient expression of either PGAM5 isoform triggers cell death with features of apoptosis, including MOMP and caspase activation, that morphologically and biochemically progresses through a mitophagic pathway. In this arena the PGAM5/AIF interaction N


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Biochemistry are absent in the short isoform.70 Hannink and co-workers recently reported that in order for PGAM5L to catalyze phosphate removal from substrates in vitro the protein must form higher order oligomers, which depends on a multimerization motif found within the amino-terminal region of the protein.58 Our coprecipitation and fluorescence complementation approaches confirm the presence of oligomeric structures containing at least a dimer both in cell extracts as well as live cells and are consistent with our observed catalytic activity for PGAM5L. Notably the formation of dimers for PGAM5S in cell extracts is greatly reduced compared to PGAM5L, and we do not detect the presence of PGAM5S dimers in living cells by fluorescence complementation. These data are consistent with a model in which some level of oligomerization is necessary for catalytic activity and demonstrate for the first time the existence of PGAM5 dimers/multimers in living cells. Due in part to a general focus on PGAM5L in the majority of published studies, isoform-specific PGAM5 functions have been only sparingly reported. Wang and co-workers26 reported sequential activity of PGAM5L and PGAM5S in the promotion of programmed necrosis in human cells, yet the generality of these observations remains unclear in the face of subsequent studies in mouse cells and tissues demonstrating that PGAM5 isoforms are not required in the necrotic pathway.66,67,71 While studies of PGAM5 deficient mice indicate that the PGAM5 gene is indispensable for mitophagy71 and regulates inflammatory responses,72 the presence of the PMGA5S isoform in mouse tissues remains unconfirmed. Indeed, a complete assessment of isoform expression across a variety of cell types in different organisms has yet to be reported. While experiments presented here do not address this question, we can now begin to directly compare PGAM5 isoform functions as expressed in cultured human cells. Properties common to both isoforms include AIF binding and the phosphatase independent promotion of mitophagic cell death. This later characteristic may involve phosphatase-independent regulation of PINK1, which is in agreement with a previous report demonstrating that the PGAM5L double mutant H99A/H105A retains the ability to promote PINK1 signaling.71 PGAM5S is selectively sensitive to AIF during oxidative stress and appears devoid of phosphatase activity, likely due to altered dimerization capabilities. In contrast to PGAM5S, PGAM5L is not affected by AIF during oxidative stress yet displays exquisite sensitivity to XIAP during mitophagic cell death. Moreover PGAM5L exhibits strong dimerization properties in intact cells and phosphatase activity in vitro. These observations lead us to propose that while there is functional overlap between PGAM5 isoforms in controlling the antioxidant response and mitophagic cell death, each isoform is regulated in mechanistically distinct ways, thus broadening the means in which the PGAM5 axis can be controlled in the context of intracellular signaling. In summary we report here the identification of novel AIF binding proteins representing various aspects of mitochondrial metabolic control, including the mitochondrial phosphatase PGAM5. Using transient expression approaches we show that while AIF can inhibit the ability of PGAM5S to regulate antioxidant responses, AIF does not influence PGAM5mediated cell death. This death exhibits characteristics of apoptosis but is mitophagic in both morphology and biochemical signature. Through its E3 ubiquitin ligase activity XIAP can block the ability of PGAM5L to trigger cell death. While not required for cell killing, the long isoform exhibited

robust phosphatase activity in vitro that was not detectable for PGAM5S likely due to observed differences in the ability of each PGAM5 isoform to form dimers and/or multimers in living cells. While the ectopic nature of our approaches prevents extension of our finding to all cell/tissue types, overall these data highlight the existence of an AIF/PGAM5/XIAP axis that regulates multiple aspects of cellular function.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Chemistry and Biochemistry, North Dakota State University, Dept. 2710, P.O. Box 6050, Fargo, ND 58108-6050. Tel: 701-231-6354. Fax: 701-231-8670. E-mail: [email protected]. Present Addresses #

Biologics, Inc., 120 Weston Oaks Ct, Cary, NC 27513. Department of Chemistry, Mathematics & Physics, 381 Grunenwald STC, Clarion University of Pennsylvania, Clarion, PA 16214.

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Author Contributions §

A.M.L. and A.S.W. contributed equally to this work.

Funding

This work was supported by Grant W81XWH-08-1-0045 (to J.C.W.) from the Department of Defense Prostate Cancer Research Program, grant RSG-09-166-01-CCG (to J.C.W.) from the American Cancer Society, and the State of North Dakota Experimental Program to Stimulate Competitive Research (ND-EPSCoR, project #FAR0022246, to J.C.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Drs. Jonathan Ashwell and William Fahl for kindly providing plasmids. ABBREVIATIONS AIF, apoptosis inducing factor; ANT2, adenine nucleotide transporter 2; BiFC, bimolecular fluorescence complementation; DMEM, Dulbecco modified Eagle medium; HA, hemagglutinin; HRP, horseradish peroxidase; JNK, c-Jun Nterminal kinase; LDS, lithium dodecyl sulfate; MOMP, outer mitochondrial membrane; PAGE, SDS-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PGAM5, phosphoglycerate mutase 5; PINK1, PTEN-induced putative protein kinase 1; SDS, sodium dodecyl sulfate; TAP, tandemaffinity purification; tBHQ, tert-butylhydroquinone; TEM, transmission electron microscopy; TMRM, tetramethylrhodamine methyl ester; XIAP, X-linked inhibitor of apoptosis; YFP, yellow fluorescent protein

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REFERENCES

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