PhD Andreas Villunger
email: andreas.villunger@i-med.ac.at

Francesca Grespi

Claudia Soratroi

Gerhard Krumschnabel

Benedicte Sohm

Christian Ploner

Stephan Geley

Ludger Hengst

Georg Haecker

Georg Häcker

BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis

Keywords: apoptosis, Bcl-2 family, BH3-only proteins, Bmf

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The Bcl-2 family comprises a number of cell death-and survival-promoting proteins that are characterized by certain structural motives, called Bcl-2 homology (BH) domains. The prosurvival family members such as Bcl-2 itself, Bcl-xL, Bcl-w or Mcl-1 contain up to four homology domains (BH1-4) whereas pro-apoptotic members of the same family either possess three out of four BH-domains (BH123 proteins), e.g. Bax and Bak or only the BH3domain, such as Bim, Bad or Bmf. According to this structural feature these proteins are referred to as BH3-only proteins and at least eight members have been described to trigger cell death in mammals [START_REF] Giam | BH3-only proteins and their roles in programmed cell death[END_REF]. While their exact mode of cell killing is still subject of intense investigations genetic evidence supports the notion that these molecules act upstream of Bax and Bak in cell death signalling, since mouse embryonic fibroblasts (MEFs) deficient for both of these molecules no longer die in response to BH3-only protein overexpression [START_REF] Zong | BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak[END_REF].

Biochemical evidence suggests that BH3-only proteins can interact with and antagonize partially overlapping sub-sets of Bcl-2 homologues by direct interaction, allowing for activation of Bax and/or Bak, leading to their oligomerization, subsequent destabilization of mitochondrial integrity, release of apoptogenic factors, caspase activation and cell death [START_REF] Willis | Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak[END_REF].

In an alternative model, members of different groups of BH3-only proteins compete for binding to Bcl-2-like anti-apoptotic molecules. Excess of so-called "de-repressors" (e.g. Bad, Bmf, Noxa) prevents sequestration of "direct activators" (Bim, Bid and Puma) by pro-survival homologues of Bcl-2 allowing them to directly interact with and activate Bax/Bak-like molecules [START_REF] Letai | Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics[END_REF][START_REF] Kuwana | BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly[END_REF]. Recent genetic evidence gained from BH3-domain exchange mutants in Bim suggest that BH3-only protein-mediated killing depends on aspects of both models in vivo [START_REF] Merino | The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins[END_REF].

The role of BH3-only proteins in normal physiology has been addressed primarily by analyzing mouse models lacking the individual genes [START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF]. Loss of Puma protects neurons, primary lymphocytes, myeloid cells and mouse embryonic fibroblasts (MEF) from DNAdamage induced apoptosis but also from certain p53-independent cell death stimuli such as glucocorticoids or serum-deprivation whereas Noxa appears to play a more restricted role in mediating DNA-damage responses [START_REF] Jeffers | Puma is an essential mediator of p53-dependent and -independent apoptotic pathways[END_REF][START_REF] Villunger | p53-and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa[END_REF]. Absence of Bid prevents liver failure in response to FAS-ligation, confirming the role of Bid as the connecting element between extrinsic and intrinsic cell death pathways [START_REF] Jost | XIAP discriminates between type I and type II FAS-induced apoptosis[END_REF][START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF]. Loss of Bim causes lymphadenopathy and subsequent autoimmunity mainly due to negative selection defects and acts as a suppressor of c-myc driven lymphomagenesis [START_REF] Pinon | Bim and Bmf in tissue homeostasis and malignant disease[END_REF].

Little is known about the biology of Bmf that seems to be regulated similarly to Bim. Both molecules share a highly conserved dynein light chain (DLC)-binding motif near their Ntermini that targets Bmf to the actin cytoskeleton and Bim to microtubules [START_REF] Pinon | Bim and Bmf in tissue homeostasis and malignant disease[END_REF]. Bmf can be released from the cytoskeleton in response to UV-radiation or during detachment-induced apoptosis (anoikis), which prevents epithelial cells from colonizing elsewhere [START_REF] Puthalakath | Rapid selection against truncation mutants in yeast reverse two-hybrid screens[END_REF]. Both forms of cell death, however, proceed normally in MEF and/or gastrointestinal epithelial cell from bmf -/-mice, suggesting redundancy with other BH3-only proteins [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF], most likely with Bim [START_REF] Hubner | Functional cooperation of the proapoptotic Bcl2 family proteins Bmf and Bim in vivo[END_REF]. In hemopoietic cells, Bmf is widely expressed and has been implicated in cytokine withdrawal-induced apoptosis of granulocytes where the protein was reported to accumulate during in vitro culture [START_REF] Villunger | Essential role for the BH3-only protein Bim but redundant roles for Bax, Bcl-2, and Bcl-w in the control of granulocyte survival[END_REF]. Multiple isoforms can be detected in lymphocytes and hence alternative splicing of bmf may also contribute to regulate its in vivo function [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF]. Two splice variants of bmf (termed bmf II and III) are expressed in normal and malignant human B cells, derived from patients with B cell lymphocytic leukemia. Surprisingly, these novel variants of Bmf lacked a functional BH3-domain and Bmf III also contains a different carboxy terminus.

Overexpression of Bmf II or Bmf III in HeLa cells increased their colony forming potential, whereas Bmf, as previously reported, showed pro-apoptotic activity [START_REF] Morales | Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells[END_REF]. B-CLL cells undergo rapid apoptosis in vitro and this cell death correlated with enhanced mRNA expression of the full-length form of Bmf, containing the BH3-domain necessary for apoptosis induction, but not the other two isoforms [START_REF] Morales | Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells[END_REF]. Consistently, genetic ablation of all Bmf isoforms expressed in mice revealed a role in the regulation of normal B cell homeostasis and irradiation [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF] as well as oncogene-driven tumorigenesis [START_REF] Frenzel | Suppression of Bcell lymphomagenesis by the BH3-only proteins Bmf and Bad[END_REF]. Finally, a role for Bmf in necroptotic cell death, triggered by TNF in the absence of functional caspases in L929 fibroblasts was reported, but its exact role in this process remains to be fully understood [START_REF] Hitomi | Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway[END_REF].

Here we characterize the bmf gene locus in detail, report the molecular basis of the generation of the two major isoforms of Bmf, designated Bmf S and Bmf CUG , and provide evidence that Bmf can act as a sensor for stress that associates with the repression of the conventional CAP-dependent translation machinery.

Results

:

An additional conserved translation start site in the bmf mRNA sequence allows expression of a longer Bmf isoform, Bmf CUG .

Blasting the mbmf cDNA obtained in the initial library screen [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF] against the NCBI-HTGS database identified RP23-266K3 as one BAC clone that contains the mouse bmf locus, annotated to chromosome 2. Further analysis of intron-exon boundaries revealed a possible alternative start codon only 4 base pairs upstream of the 5´end of the longest cDNA clone that was originally identified as putative transcription start site (TSS) (Fig. 1a). Inclusion of this additional 5´sequence indicated that the mouse bmf mRNA sequence (4.8kb in size) now contains a significantly longer ORF (816bp) as the one initially published (558bp) and used for the initial characterization of Bmf protein [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF]. Translation of this alternative open reading frame in silicio predicted a longer version of the mouse Bmf protein, here designated Bmflong (Bmf L ), with an alternative extended N-terminus, approximately 30kDa in size, but otherwise identical to the originally published protein sequence, referred to as Bmf-short (Bmf S ).

Screening of the NCBI mouse EST-database confirmed that transcripts containing this upstream ATG can be found in cDNA libraries, e.g. derived from mouse E13 embryonic heart muscle tissue or E12 mouse ovary, suggesting that this longer ORF may be used in vivo to generate Bmf L . Consistently, two major isoform of Bmf can be found by Western analysis in lymphocyte extracts from adult mice [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF] or human leukemia cells [START_REF] Mackus | Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation[END_REF] and cell lines [START_REF] Ploner | The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia[END_REF].

Immunoprecipitation using Bmf-specific antibodies and subsequent immunoblotting revealed that two protein products were precipitated from mouse splenocytes (Fig. 1b). The smaller product, migrating at approximately 22kDa, presumably represented the originally described mBmf S, isoform, as indicated by comparison with mBmf S transiently expressed in 293T cells (Fig. 1b). The slower migrating band was approximately 26kDa in size and we initially considered it to represent Bmf L . Interestingly, two additional bands were detected in lysates and IP-complexes from Bcl-2 overexpressing lymphocytes (Fig. 1b, sFig.1a), 24kDa and 20kDa in size, that are absent or only poorly expressed in most wild type lymphocyte subsets analyzed [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF]. These proteins may represent either low abundance splice variants of bmf, post-translationally modified versions of the protein(s) or degradation products of the two major isoforms (see below). Phosphatase treatment of cell lysates excluded phosphorylation as a possible modification (sFig. 1a). Together, these observations suggest that (co)transcriptional modifications such as differential splicing, alternative promoter and/or start site usage, or alternative post-translational modifications must account for the generation of the multiple Bmf isoforms.

To verify that both putative start-site ATGs contained in the mbmf mRNA can be used, we generated and subcloned a cDNA encoding the ORF for Bmf L into pSK-bluescript and performed a T7-driven coupled in vitro transcription/translation reaction (IVTT) in rabbit reticulocyte lysates in the presence or absence of 35 S-methionine. The reaction products were separated by SDS-PAGE, exposed to x-ray film or subjected to immunoblotting using an anti-Bmf specific mAb. Three major protein products of comparable size were detected by both methods (Fig. 1c,d, not shown). Notably, two of those products migrated with the same behaviours as those recognized in lysates from mouse thymocytes. However, the longest product generated by IVTT did not appear to have an endogenous counterpart (Fig. 1c).

Mutation of the second, internal ATG2 led to the disappearance of the shortest Bmf product, indicating that only Bmf S is translated from this internal ATG in exon 3 and excluding the possibility that the other two products may arise form post-translational modification of Bmf S (Fig. 1d). In contrast, deletion of the first ATG1, as expected, led to the disappearance of the largest protein product but the two other products, resembling in size the endogenous proteins were still produced. Since there was no other ATG in-frame with the ORF of Bmf S , we searched for the presence of alternative start sites and discovered a CTG which was flanked by a strong Kozak sequence in frame with the downstream ATG2 that gives rise to Bmf S . Site-directed mutagenesis confirmed that this CTG is recognized efficiently by the translation machinery and can be used to generate a longer isoform of Bmf, designated herein as Bmf CUG , since mutation of this CTG caused loss of the 26kDa product which was still produced when both ATGs in this cDNA were mutated (Fig. 1d). Overexpression of a single transcript containing this putative translation initiating CTG in the context of the surrounding Kozak sequence in 293T cells allowed efficient co-expression of Bmf CUG and Bmf S (lane 5), while in its absence only Bmf S was translated into protein (lane 2) (Fig. 1e).

Epitope tagging allowed for the efficient expression of Bmf S or Bmf CUG individually (lanes 3 & 4). We concluded that these two protein products most likely represent the endogenous proteins recognized by our anti-Bmf antibodies in hemopoietic cells or after IVTT (Fig. 1b,d).

Sequence alignment revealed that this CTG site was conserved across all mammalian species where genomic DNA of this region was contained in the Ensemble database (Fig. 2), allowing for translation of Bmf CUG , containing 24 additional amino acids at its N-terminus (Fig. 1f). In contrast, the first ATG found in exon I was either not present as in horse (equus), or, in other species such as humans and chimpanzee, this ATG1 was not in frame with the downstream ATG2, used to translate Bmf S (Fig. 2).

Taken together this indicates that at least two Bmf isoforms can be translated from a single mRNA [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF], due to alternative start site usage from a common open reading frame.

Importantly, all these isoforms appear to contain a functional BH3-domain, since they could be co-immunoprecipitated with Bcl-2 from splenocytes overexpressing a bcl-2 transgene (sFig. 1b)

Bmf CUG and Bmf S bind the same Bcl-2 family proteins and show comparable apoptotic potential as well as subcellular localization

Having identified the nature of the two major isoforms expressed in lymphatic tissue we investigated if there were substantial differences in their potential to interact with known partners of the Bcl-2 family as well as in their ability to kill cells. Therefore, constructs that allow expression of either isoform alone (not shown) or both isoforms at the same time (Fig. 1e) were transiently transfected together with FLAG-tagged versions of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, or the viral Bcl-2 homologues BHRF1 and KS-Bcl-2 (Fig. 3a). Co-immunoprecipitation analysis revealed that both isoforms effectively interacted with all mammalian prosurvival proteins tested, but not with their viral counterparts (Fig. 3b). As expected, introduction of a leucine to alanine mutation in the BH3-domain (L/A) abrogated interaction with Bcl-2 but we repeatedly observed residual binding of both Bmf isoforms to Mcl-1 suggesting that amino acids outside the BH3-domaine are involved in this interaction (Fig. 3b).

To assess possible differences in their proapoptotic potential we generated a series of IL3dependent BaF3 cells that stably expressed above-mentioned pro-survival Bcl-2 family proteins alone, or together with one or the other isoform of Bmf. Upon IL3-deprivation, BaF3 cells undergo rapid cell death that can be blocked by overexpression of anti-apoptotic Bcl-2 proteins (sFig. 2a), unless these are themselves blocked by BH3-only proteins [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF]. In line with their ability to bind to Bcl-2, Bcl-xL or Mcl-1 in our IP-analysis, Bmf CUG or Bmf S antagonized the pro-survival effect of these Bcl-2 family members upon IL3-deprivation, triggering massive apoptosis in BaF3 cells (sFig. 2). Bmf overexpression failed to antagonize the anti-apoptotic effect of the two viral Bcl-2-like proteins KS-Bcl2 and BHRF1, consistent with a lack of direct interaction in our IP-analysis (Fig. 3b). Notably, BaF3 cells co-expressing Bcl-w together with Bmf S or Bmf CUG resisted apoptosis upon IL3-deprivation (sFig. 2), which may have been due to extremely levels of transgenic Bcl-w that, presumably, was not neutralized effectively by overexpressed Bmf (not shown).

The collective data suggested that both Bmf isoforms are equally potent in killing but since we were unable to generate BaF3 cells that overexpressed either isoform alone (not shown) it remained possible that differences may be masked by simultaneous high-level expression of a pro-survival Bcl-2 homologue. To extend our observations to a different model system, we performed lentiviral transduction of apoptosis-sensitive U2OS osteosarcoma cells with constructs encoding for Bmf S or Bmf CUG, as well as the Bmf S L138A or Bmf CUG L162A , carrying a point mutation in the BH3-domain. Parallel transduction using a GFP-encoding virus served as a control to quantify infection efficiency (usually >90%) and treatment related cell death (usually ~ 20%). While both wild type isoforms of Bmf induced comparable apoptotic cell death, assessed 24h after transduction by Annexin-V staining or sub-G1 analysis, BH3domain point-mutant versions of Bmf failed to induce cell death of U2OS significantly, when compared to GFP-transduced control cells (Fig. 4). Seeding identical numbers of transduced U2OS cells confirmed that both isoforms of Bmf, but not its L/A-mutant versions, strongly reduced the colony formation potential of these cells, as monitored by crystal violet staining after 5 days of culture. Again, both proteins did so with comparable efficiency (Fig. 5a).

Finally, we wondered whether Bmf CUG and Bmf S would have different preferences for Bax or Bak, in order to kill cells. Therefore, SV40-MEF expressing or lacking bak, bax or both genes were transduced with the two Bmf isoforms alone or a construct that allows simultaneous expression of both proteins. The colony formation potential of these MEF was monitored upon selection with puromycin for one week. As shown in Fig. 5b, both isoforms reduced the colony formation of wt, Bax and Bak-deficient cells suggesting that both BH123 proteins can be engaged to mediate Bmf-induced apoptosis. Bmf S appeared to kill SV40 MEF somewhat more effectively and preferentially in a Bax-dependent manner. Notably, bax -/-bak -/-MEF, despite being highly resistant to the effects of etoposide treatment, also showed reduced colony formation upon transduction with Bmf when both isoforms were expressed simultaneously from a single plasmid (Fig. 5b).

To investigate if the additional 24 aa found in Bmf CUG , predicted to contain an additional αhelix, may define differences in subcellular localization we transiently transfected HA-tagged versions of the individual isoforms into 293T HEK cells or NIH3T3 fibroblasts, followed by immunofluorescence analysis using an epitope tag-specific antibody. Both isoforms colocalized predominantly to mitochondria, in both cell types analyzed (Fig. 6a and data not shown). Since the anti-Bmf antibodies currently available are not suitable to distinguish between isoforms by immunofluorescence, we decided to study Bmf localization performing biochemical subcellular fractionation experiments. Analysis of cytoplasmic and mitochondrial fractions from NIH3T3 cells that were left untreated or incubated with cytochalasin D for 16h, a stimulus reported to induce translocation of Bmf from the actin-cytoskeleton to mitochondria [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF], confirmed that endogenous Bmf can already be found in the mitochondrial fraction prior treatment (Fig. 6b). Alkaline extraction also indicated that Bmf is not simply loosely attached to the outer mitochondrial membrane but, similar to Bim (23), must have been actively inserted into it (Fig. 6b). Notably, the C-term end of Bmf, fused to GFP, allows for active import of this fusion protein into isolated yeast mitochondria (GH, unpublished observations).

Finally, we also investigated whether both proteins may display differences in protein stability but again, at least under stady-state conditions, both isoforms were equally sensitive to CHXtreatment showing a half-life of approximately 3-4h, as assessed in WEHI231 pre-B cells (sFig. 1c), primary lymphocytes or NIH3T3 fibroblasts (not shown).

In summary these data suggest that both isoforms show comparable half-life, identical subcellular localization, killing potential and do not differ in their ability to counteract Bcl-2 prosurvival proteins tested. Furthermore, Bmf can engage both, Bax and Bak to exert its proapoptotic function.

Bmf CUG and Bmf S can be translated in a CAP-independent manner

Since non-ATG dependent initiation often associates with translation of proteins from intraribosomal entry sites (IRES) [START_REF] Touriol | Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons[END_REF], we wondered whether the RNA sequence between both translation start sites possesses the capacity to enable CAP-independent translation. To test this hypothesis, we first subcloned different cDNA fragments encoding different portions of the 5´end of the bmf mRNA into a bicistronic luciferase reporter system where a minimal SV40

promoter drives expression of one mRNA endoding the Renilla (RN) luciferase, followed by a spacer and the ORF encoding for Firefly (FF) luciferase. Inserting the 258bp TSS-ATG2 fragment or the 72bp CTG-ATG2 fragment into this spacer region revealed that the longer fragment was able to promote significant expression of the FF-reporter while insertion of a non-related sequence from the GAPDH gene did not (Fig. 7a). Finally, when the same experiment was performed using modified constructs that carry an additional stem-loop structure between the SV40 promoter and the open reading frame for Renilla, hindering CAPdependent translation, a clear shift in the ratio between RN/FF was observed in the presence of the TSS-ATG2 fragment contained in the bmf mRNA (Fig. 7a). The well-described IRES element from the cyclin dependent kinase inhibitor p27 was used as an internal control allowing comparable expression of FF-luciferase [START_REF] Kullmann | ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR[END_REF]. To exclude residual promoter activity in the tested cDNA fragments to be responsible for the observed FF activity in our dual reporter assay, the same DNA elements were also inserted into the promoter-less pGL2luc luciferase expression vector and its activity was compared to that of a bona-fide bmf promoter fragment encoding nucleotides -600 to +70 relation to the TSS. While the latter fragment induced significant luciferase activity upon overexpression in 293T cells, all other DNA fragments did not, arguing against this possibility (Fig. 7b). To confirm this observation in a different 3c) that should not become visible, if both proteins were synthesized in a strictly CAPdependent manner. Finally, we transcribed bmf cDNA including a polyA tail in vitro without adding a 5´CAP and used this mRNA in a separate translation reaction. cDNA encoding GFP served as a control. The translation reactions were then separated by SDS-PAGE and immunoblotting was performed using anti-Bmf-specific and anti-GFP-specific antibodies.

While the IVT of GFP mRNA did not generate any detectable protein expression, due to the lack of the 5´CAP-structure, we observed that Bmf S was effectively translated from the uncapped mRNA, but, surprisingly, we also detected a weaker signal resembling Bmf CUG (Fig. 7c).

Together, these findings strongly suggest that the bmf mRNA contains features of an IRES element in its 5´region allowing for alternative start-site usage and, presumably, expression of both Bmf-isoforms.

Accumulation of Bmf protein and Bmf-dependent apoptosis upon inhibition of CAPdependent translation

Keeping the previous findings in mind, we investigated if Bmf protein can be induced under conditions that favor CAP-independent and, hence, IRES-mediated protein translation. To test this, we deprived NIH3T3 cells and HC11 mammary epithelial cells of serum or exposed them to hypoxia, both conditions known to favor CAP-independent translation [START_REF] Spriggs | Re-programming of translation following cell stress allows IRES-mediated translation to predominate[END_REF]. Furthermore, CAP-dependent translation was compromised by inhibition of the PI3K/AKT pathway or mTOR using LY294002 or rapamycin, respectively [START_REF] Wang | The mTOR pathway in the control of protein synthesis[END_REF]. Finally, we directly interfered with conventional translation using 4EGI-1, an inhibitor of the eukaryotic initiation factor (eIF) 4E -eIF4G interaction and hence, CAP-dependent translation [START_REF] Moerke | Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G[END_REF]. Notably, all these stimuli caused accumulation of both Bmf isoforms, while treatment of cells with the ER-stressor tunicamycin, the DNA-damaging drug etoposide or the pan-kinase inhibitor staurosporine had no such effect (Fig. 8a; not shown). Notably, treatment of both cell types with the actindepolymerizing agent cytochalasin D also lead to an increase in Bmf protein levels (Fig. 8a; sFig. 1d).

In order to evaluate whether Bmf is also important for cell death induction under these conditions, apoptosis was quantified in NIH3T3 cells stably expressing an shRNA targeting bmf mRNA for degradation by RNAi (Fig. 8b). Consistent with a rate-limiting pro-apoptotic role of Bmf under these conditions, its knockdown rendered NIH3T3 cells refractory to cell death induced by stimuli that interfere with CAP-dependent translation including serum deprivation or inhibition of PI3K/AKT/mTOR network. Knock-down of Bmf also delayed cell death induced by cytochalasin D treatment, which does not associate with CAP-independent translation, but apoptosis induced by UV-radiation, anoikis, the proteasome inhibitor Velcade or treatment with the histone deacetylase inhibitor Trichostatin A occurred normal (Fig. 8b and sFig. 4).

Notably, cell death induced by rapamycin, inhibiting mTOR complex 1 activity, or the PI3Kinhibitor LY294002 was both only partially blocked. This may have been due to incomplete knockdown of Bmf expression or involvement of other pro-apoptotic factors. Therefore, we wanted to confirm our observations in a clean genetic back-ground and investigated whether Bmf could be induced in SV40 immortalized MEF, which usually do not express significant levels Bmf protein (AV, unpublished), and whether absence of the protein in bmf -/-MEF could confer resistance to apoptosis induction by these stimuli. Serum-deprived wt MEF, as well as cells treated with inhibitors of the PI3K/AKT/mTOR network all induced Bmf protein. When wt MEF were first serum-deprived for 24h, triggering Bmf accumulation, subsequent exposure to cycloheximide prevented further protein accumulation more potently than inhibition of transcription with actinomycin D (Fig. 9a). Similar observations were made in MEF treated with rapamycin or L294002 (Fig. 9b). Together these observations again supported the idea that inhibition of CAP-dependent translation favors Bmf protein accumulation under these conditions, at least in part by IRES-mediated translation. Consistently, inhibition of the PI3K/AKT/mTOR network by LY294002 or rapamycin triggered cell death in bmf -/-MEF less effectively. Notably, these cells still succumbed to apoptosis equally fast, or even faster upon treatment with staurosporine (Fig. 9c). Serum deprivation yielded inconsistent results in the different SV40 MEF clones tested and the observed differences did not yield statistically significant differences (Fig. 9c) suggesting that some of the observed effects are cell type specific or depend of the type of immortalization.

Since viral infection often associates with the inhibition of CAP-dependent translation, we wondered if loss of Bmf prevents attrition of T cells observed to occur early in such anti-viral immune responses [START_REF] Bahl | IFN-induced attrition of CD8 T cells in the presence or absence of cognate antigen during the early stages of viral infections[END_REF]. This situation can be mimicked to a certain degree by injection of polyIC that triggers the typ I IFN-dependent apoptotic loss of CD8 + T cells early in an immune response [START_REF] Bahl | IFN-induced attrition of CD8 T cells in the presence or absence of cognate antigen during the early stages of viral infections[END_REF]. Therefore, wt and bmf -/-mice were injected with a single dose of polyIC and the percentage of CD8 + T cells was quantified 20 h later. CD8 + T cell numbers declined but naïve CD8 + CD44 -T cells were most affected by polyIC treatment in wt animals. In contrast, in mice lacking Bmf that presented with an overall reduced percentage of naïve T cells, polyIC treatment had no such effect (Fig. 9d). Together our results support the hypothesis that Bmf can act as a sentinel for cell stress that associates with the inhibition of CAP-dependent translation in a cell type and stimulus dependent manner.

Discussion:

BH3-only proteins are essential regulators of cell death that sense countless endogenous and exogenous stress signals translating them into the activation of the apoptotic cell death machinery. In order to provide a cell with the biggest possible flexibility to respond to highly diverse forms of stress, multiple modes of regulation are usually required to control the activation and function of individual BH3-only proteins. For example, DNA-damage can trigger the transcription of Puma and Noxa [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF][START_REF] Yu | PUMA induces the rapid apoptosis of colorectal cancer cells[END_REF][START_REF] Oda | Noxa, a BH3only member of the bcl-2 family and candidate mediator of p53-induced apoptosis[END_REF], while phosphorylation/dephosphorylation events upon growth factor receptor-engagement can modulate the activity of Bad by cytoplasmic sequestration [START_REF] Datta | Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery[END_REF][START_REF] Del Peso | Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt[END_REF] or abundance of Bim via the proteasome [START_REF] Ley | Regulatory phosphorylation of Bim: sorting out the ERK from the JNK[END_REF]. Furthermore, Nmyristylation of Bid secures its proper mitochondrial targeting and caspase-cleavage its proapoptotic activity [START_REF] Zha | Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis[END_REF]. Protein-protein interaction, e.g. with motor complex associated proteins such as dynein-light chain molecules can lead to distinct subcellular localization of Bim or Bmf, at least in some cell types that depend on integrin signals and/or contact to the ECM for survival [START_REF] Pinon | Bim and Bmf in tissue homeostasis and malignant disease[END_REF]. Alternative splicing as a means of regulation of BH3-only protein activity has been described, e.g. for Puma [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF], Bim (37) but also Bmf [START_REF] Morales | Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells[END_REF]. However, none of the major isoforms of Bmf described here is generated by differential splicing but by different start site usage and, therefore, do not represent equivalents of human BMF II or BMF III, found in CLL cells [START_REF] Morales | Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells[END_REF]. BMF II and BMF III as well as the γand δ-isoforms of Puma, do lack a functional BH3-domain [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF] and their physiological functions remains unclear. In contrast, all Bmf isoforms detected in our study could be co-immunoprecipitated together with Bcl-2 from lymphocytes, indicating that they all must contain a functional BH3-domain (sFig. 1b).

Whether the additional 24 aa encoded in the N-terminus of Bmf CUG can serve as an interface for specific protein-protein interactions outside the Bcl-2 family is subject of our current investigations.

Functional analysis failed to reveal any differences in the binding pattern of Bmf isoforms to pro-survival Bcl-2 proteins, similar to observations made using alternatively spliced isoforms of Bim [START_REF] O'connor | Bim: a novel member of the Bcl-2 family that promotes apoptosis[END_REF]. Bmf CUG bound to Bcl-2, Bcl-x, Bcl-w and Mcl-1, as initially also reported for Bmf S [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF], but neither isoform bound effectively to the viral Bcl-2-like proteins BHRF1 or KS-Bcl2 in overexpression studies (Fig. 3). Although Biacore analysis defined a low affinity of the Bmf BH3-domain for Mcl-1 [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF], this interaction can be readily detected under conditions of overexpression (Fig. 3 and ( 20)). Recently, we also observed interaction of endogenous Bmf with endogenous Mcl-1 in mouse B lymphoma cells (AV, unpublished), suggesting that the use of BH3-domain peptides cannot recapitulate entirely the interaction behaviour of the fulllength protein.

Similar to the major Bim isoforms, shorter Bmf S appeared somewhat more potent in reducing the colony forming potential of MEF, however this effect was not observed in U2OS cells.

Both isoforms can engage Bax as well as Bak to promote cell killing but Bmf S seems to have a preference for Bax over Bak (Fig. 5). To our surprise, we observed that MEF lacking Bax and Bak also showed reduced colony formation when both isoforms were overexpressed from a single plasmid and at the moment, we can only speculate that these cells may either activate Bok under these conditions or die by necroptosis [START_REF] Hitomi | Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway[END_REF]. However, a possible impact of Bmf isoform overexpression on the proliferation of these MEF has not been formally excluded. Hence, the physiological functions of both isoforms remain largely elusive and will require the generation of knock-in mutant mouse strains expressing only one or the other isoform of Bmf.

Our observation that the generation of Bmf CUG involves translation initiation from a CUG, encoded in exon 2 suggested that IRES-mediated translation might serve as a mechanism to initiate translation. This possible feature was overlooked initially when the short isoform of Bmf that starts from an ATG encoded at the beginning of exon 3 was first described [START_REF] Puthalakath | Bmf: a pro-apoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[END_REF]. Our follow up analysis suggests that actually both major isoforms appear to be translated in an IRES-dependent manner from alternative start-sites contained in the bmf mRNA (Figs. 2,7).

Alternative start site usage has also been reported for Bad, leading to the expression of two main isoforms, but in this case ATG serves for initiation of translation in both cases [START_REF] Ranger | Bad-deficient mice develop diffuse large B cell lymphoma[END_REF].

During apoptosis, eukaryotic initiation factors are modified early on, e.g. by dephosphorylation of 4E-BP1 and subsequent caspase-cleavage of eukaryotic eIF4G, both enforcing CAPindependent translation [START_REF] Spriggs | Re-programming of translation following cell stress allows IRES-mediated translation to predominate[END_REF]. Consistently, a number of apoptosis regulators including p53, Bcl-2, Apaf-1 and XIAP were reported to be generated from IRES-elements under conditions of stress, thought to maintain a functional apoptotic machinery when CAP-dependent translation is compromised [START_REF] Graber | Cap-independent regulation of gene expression in apoptosis[END_REF]. However, some of these proposed IRES-elements were subsequently proven to be false and the activity observed in dual reporter systems due to cryptic splicing events generating a 3´cistron of the second reporter [START_REF] Kozak | A second look at cellular mRNA sequences said to function as internal ribosome entry sites[END_REF][START_REF] Baranick | Splicing mediates the activity of four putative cellular internal ribosome entry sites[END_REF][START_REF] Saffran | The XIAP IRES activates 3' cistron expression by inducing production of monocistronic mRNA in the {beta}gal/CAT bicistronic reporter system[END_REF]. To strengthen our claim that Bmf can be translated in an IRES-mediated manner, we have (i) excluded residual promoter activity in the DNA sequences tested, (ii) demonstrated that insertion of a hair-pin structure favours expression of the second cistron in dual reporter assays and, most importantly, performed (iii) sequential transcription translation reaction using uncapped mRNA that confirmed translation of Bmf protein in the absence of a 5´CAP structure (Fig. 7).

Consistently, endogenous Bmf expression was clearly induced by different stimuli that associate with the repression of CAP-dependent translation (Figs. 8,9). The latter observation proposes a role for Bmf isoforms as sentinels that become activated in response to stress that associates with reprogramming of the translation machinery from CAP-dependent to CAPindependent translation such as growth factor deprivation, hypoxia or inhibition of the PI3K/AKT/mTOR signaling axis (Fig. 8,9; sFig.1). Consistent with a role for Bmf as a sensor of this type of stress, knock-down or absence of Bmf interfered but ultimately failed to prevent apoptosis in response to some of the tested stimuli suggesting that it must collaborate with other cell death regulators, presumably other BH3-only proteins, such as Bim (15) in a cell type and stimulus dependent manner. Whether Bmf plays also a rate-limiting role for apoptosis induced upon physiological triggers of CAP-independent translation in vivo, e.g.

during viral infection or hypoxia-induced tumor cell death will be subject of our future investigations.

Material & Methods

Cell culture, primary cells and reagents.

All cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 . Mouse embryonic fibroblasts were isolated from E14.5 embryos after removal of internal organs, brain and fetal liver by trypsin digestion of the remaining carcass. All experiments were performed using MEFs that were immortalized with the SV40 large T antigen. U2OS cells, MEFs and NIH3T3 murine fibroblasts were cultured in the DME medium supplemented with 10% FCS (PAA), 250µM L-glutamine (Invitrogen) and penicillin/streptomycin (1U/ml, Sigma-Aldrich). IL3-dependent BaF3 pro-B lymphoid cells cells were cultured in RPMI 1640 medium, supplemented with 250µM L-glutamine, 50µM 2-mercaptoethanol, penicillin/streptomycin (1U/ml) and 10% FCS, supplemented with 15% of WEHI-3B cell supernatant. HC11 mouse mammary epithelial cells were cultured in RPMI 1640 medium, supplemented with 5µg/ml bovine insulin (Sigma), 10ng/ml murine EGF (Peprotec), 250µM L-glutamine, 50µM

2-mercaptoethanol, penicillin/streptomycin (1U/ml) and 10% FCS. The generation of vav-bcl-2 transgenic (44) and bmf -/-mice has been described [START_REF] Labi | Loss of the BH3only protein Bmf impairs B cell homeostasis and accelerates gamma irradiationinduced thymic lymphoma development[END_REF].

Apoptosis-induction, sub-G1 and AnnexinV/propidium iodide staining

For the induction of cell death the following reagents were used: 2µM Cytochalasin D (Sigma), 10µM LY-294002 (InvivoGen), 500nM rapamycin (Alexis), 50µM 4EGI-1 (Alexis), 50nM Staurosporine (Sigma), 1µg/ml Tunicamycine (Sigma). Cells cultured under hypoxic conditions were cultured in a humidified atmosphere containing 1% O 2 .

For sub-G1 analysis, cells were collected, along with their supernatant, centrifuged for 5 minutes at 1500rpm, 4°C. Then the pellet was washed once with PBS and centrifuged again.

After supernatant removal, pellet was resuspended in 500µl 70% Methanol (in PBS) and fixed at -20°C. Cells were then washed with PBS and centrifuged for 5 minutes at 3000rpm, 4°C.

The pellet was resuspended in 475µl PBS with RNAseA (Sigma; final concentration 10µg/ml) and incubated at 37°C for 20 minutes. After that, 25µl of propidium iodide (final concentration 2µg/ml) were added to the cell suspension, which was incubated for 10 minutes at 4°C before analysis in flow cytometer, FACScan (Becton Dickinson). Alternatively, the percentage of viable cells in culture was determined by staining them with 2µg/mL propidium iodide plus APC-or FITC-coupled Annexin-V in Annexin binding buffer (Becton Dickinson) and subsequent flow cytometric analysis.

Individual in vitro transcription and in vitro translation

cDNA encoding mbmf L or GFP cDNA was subcloned in the pTEX plasmid containing a polyA sequence downstream of the multiple cloning site. DNA was linearized with NotI, run on a 1.5% agarose TAE gel and then purified using a gel extraction kit (Qiagen). In vitro transcription using 25% volume of eluted DNA was performed using the SP6 MEGAscript Kit (Ambion). RNA was recovered by LiCl precipitation (final concentration 4.5M). RNA concentration was quantified in a spectrophotometer and 1µg of in vitro transcribed RNA was subsequently translated using the Retic Lysate IVT kit (Ambion) according to the manufacturers recommendation, in 25mM or 50mM potassium-acetate containing reaction buffer. Protein was precipitated using 12% TCA and resuspended in 2x Laemmli buffer and separated by SDS-PAGE prior immunoblotting.

Coupled in vitro transcription/translation reaction

Usually, 1µl plasmid DNA (100ng) was mixed with 10µl translation mix containing reticulocyte extract and, optional, 1µl 35 S methionine (Amersham). Samples were incubated for 90 minutes at 30°C and then frozen until SDS-PAGE was performed.

SDS-PAGE: 3µl of each sample were diluted in ONYX lysis buffer, mixed 1:3 with laemmli loading buffer and applied on a 14-20% precast gradient polyacrylamide gel. On one lane a protein size marker was loaded. Electrophoresis was performed at 70 volt in Tris-Glycine buffer. After electrophoresis the gel was vacuum-dried and exposed to an X-ray film for 2 days. Results were obtained after developing the film using AGFA Curix 60 photo imager.

Alternatively, non-radioactive reactions were electroblotted onto nitrocellulose membranes and subjected to immunoblotting using anti-Bmf mAb 17A9.

Luciferase assays

Lipofectamine was used to transiently transfect 293T cells with different dual-luciferase constructs for 6h. After additional 24h, cells were harvested and Renilla and Firefly-luciferase activity was quantified using the Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturers recommendation.

Immunofluorescence stainings

Cells were cultured on glass cover slips and 24h or 48h after lipofectamine transfection cultures were briefly washed with PBS and incubated for 30min at 37°C with 100nM

MitoTracker Red CMXRos (Molecular Probes, Invitrogen, Austria) in culture medium to stain the mitochondria. Then cultures were briefly washed with PBS, medium was removed and cells were fixed for 15min at room temperature with 4% PFA (in PBS). Cells were then washed 3 times with PBS and further incubated for 1h with PBS containing 0.1% Triton X-100, 1% bovine serum albumin and 10% fetal calf serum for permeabilization and blocking.

Incubation with primary antibody (mouse anti-HA clone: 12CA5, Sigma, 1:100 in blocking solution) was performed overnight at 4°C. After 3 further washes with PBS, cells were incubated with secondary antibody (Alexa-fluor-488-labelled anti-mouse, Molecular Probes, Invitrogen, Austria; 1:100 in blocking solution) for 1h at room temperature, then washed again 3 times and incubated with DAPI (1µg/ml, in PBS) for nuclear staining for 10min at room temperature. Finally, following another washing step, cells were fixed with Vectashield antifade mounting medium (Vector Laboratories Burlingarne, CA) and the glass cover slips were mounted on microscope slides. Cell images were collected with a confocal laser scanning microscope (Zeiss LSM 510 Axiovert 100M, 63x/1.4 oil immersion lens; Carl Zeiss MicroImaging, Inc.), with the resolution set to 1024 x1024 pixels, and pinholes set to acquire images of below 1 µm thickness. Further image processing, i.e. background correction, adjustment of brightness and contrast and export to tif-format, were made using Image J software.

For the fluorescence detection of IRES-mediated expression of YFP 293T HEK cells cultured on glass cover slips were transfected with plasmid constructs containing cDNA encoding a ECFP-Venus fusion protein, or a sequence where both open reading frames were separated by the putative Bmf-IRES, and were then exposed to 50 µM 4EGI-1, 0.5 µM rapamycin for 24h or were left untreated. Next, cells were fixed with 4% PFA in PBS at room temperature, washed with PBS for 3 times and mounted on microscope slides with Vectashield antifade mounting medium. Images were then acquired using a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) with a 63xglycerol immersion objective, applying the LAS AF acquisition software Version 2.1.0. Cells were alternately excited either with the 458nm or the 514 nm laser line of an Argon laser and emission filter sliders set to 460-495nm or to 520-590nm to detect CFP and YFP, respectively. Image resolution was set of 512 x 512 pixels and pinholes to 1 Airy unit. Relative emission intensities of acquired images were finally quantified with Image J software.

Mitochondrial subfractionation

After 16h of incubation with 2µM Cytochalasin D or solvent control, NIH3T3 cells were harvested in 300µl of isotonic mitochondrial buffer (MB) (70mM sucrose, 210mM mannitol, 1mM EDTA, 10mM Hepes pH7.5) supplemented with proteases inhibitor cocktail (Complete, Roche). Cells were left for 15min on ice and then they were broken by 7 passages repeated for 3 times through a 25G 0.5-by 25mm needle fitted on a 1ml syringe. Cells suspension were then centrifuged at 2000g at 4°C for 5 min to remove unbroken cells. Lysates were spun further for 10min at 13000g at 4°C to pellet mitochondria, ER and nuclei. The cytosolic supernatant fraction was subsequently centrifuged for 1h at 4°C at 39000rpm in an ultracentrifuge. Supernatant was then precipitated with 12%TCA and resuspended in 45µl of 2x Laemmli buffer prior boiling (5min, 95°C). The 13K pellet = heavy membrane fraction was resuspended in 65µl MB-EGTA buffer (containing 0,5mM EGTA instead of EDTA) and further centrifuged at 500g for 3min at 4°C to separate the nuclear fraction. The supernatant For lentiviral transfection, human HEK 293T cells were transiently transfected with lentiviral plasmids containing cDNAs coding for mouse Bmf S , Bmf CUG , Bmf S L/A mutant, Bmf CUG L/A mutant or eGFP as control, along with the packaging plasmids pSPAX and pVSV-G. After 48h and 72h lentiviral supernatant was collected and sterile filtered (0.2µM), supplemented with Polybrene to a final concentration of 4µg/ml and added to the target cells overnight.

For retroviral transduction of MEF, Lipofectamine (Invitrogen) was used to transiently transfect Phoenix packaging cells with constructs encoding for Bmf s , Bmf CUG , both isoforms, or empty control subcloned in the retroviral pBABE-puro backbone. After 48h, supernatant was collected and 0.20µm filter-sterilized. Polybrene was then added to reach a final concentration of 4µg/ml. The viral supernatant was added to MEF lacking Bax or Bak by spin infection. The following morning, fresh medium was added to the infected cells. After 48h, medium was replaced by medium containing 2µg/ml puromycin. Selection was performed for one week where medium was changed twice.

For assessment of colony formation, cells were washed once with PBS and incubated for 10 minutes in 0.5ml of crystal violet staining solution (0.2% crystal violet in 50% methanol).

Plates were then rinsed 3 times with bi-distilled water, air-dried and stored in the dark until photographic documentation.

Statistical Analysis.

Statistical analysis was performed using the unpaired Student t test and a Stat- Stable knock-down clones were selected using puromycin and used for survival analysis.

Cells were deprived of serum or exposed to the indicated drugs. Apoptosis was quantified by sub-G1 staining of cells followed by flow-cytometric analysis. Bars represent means ± SE of three independent experiments performed in duplicates using two independent sh_clones. [*]

indicates statistically significant differences between parental, sh_control and sh_bmf expressing cells and [ §] indicates significant differences between parental and sh_bmf expressing cells (p< 0.037 for -FCS; p< 0.002 for Cyt. D; p< 0.0019 for LY294002; p< 0.05 for rapamycin).

  experimental system, the same 258bp fragment (TSS-ATG2), conferring expression of FF luciferase in our dual-luciferase experiments was subcloned in between the open reading frames of two fluorescent proteins, ECFP and Venus (sFig. 3a). Fluorescence microscopy of 293T cells transfected with constructs allowing either expression of both fluorescence proteins as a single fusion protein or both ORFs separately, due to insertion of the TSS-ATG2 element preceded by a STOP-codon after ECFP, confirmed that both fluorescent proteins are translated individually in cells transfected with the latter construct (sFig. 3b). However, since we could not rule out completely that ribosomal slipping may contribute to Venus expression (or expression of the second luciferase gene in Fig.7a), we investigated if blocking CAPdependent translation by addition of rapamycin or the CAP-translation inhibitor 4EGI-1 favored the expression of the second fluorescent protein, Venus, over ECFP. Consistent with our idea that the 5´end of the bmf mRNA contains features of an IRES-element, we observed a clear shift towards the expression of the second fluorescence under these conditions (sFig.

  Transient transfections, immunoblotting, immunoprecipitation and phosphatase treatment 293T human embryonic kidney cells or NIH3T3 fibroblasts were transiently transfected in 6well plates using 3µl of Lipofectamine 2000 reagent (Invitrogen) and a total of 1µg of plasmid DNA. Cells were incubated in Optimem (Gibco) with the transfection mix for 6h before replacing the medium. After 48h cells were harvested and lysed in ONYX buffer (20mM TrisHCl pH 7.5, 135mM NaCl, 1.5 mM MgCL 2 , 1mM EGTA, 1mM EDTA, 1% TritonX-100, 10% glycerol) for 30 min on ice. Insoluble debris was cleared by centrifugaiton and protein concentration was quantified using the Bradford assay. Usually, 75-100µg of protein separated by SDS-PAGE using 4-20% PAGE Novex-ready gels (Invitrogen). After transfer, membranes were probed with rat monoclonal antibodies specific for Bmf (clone 9G10, 17A9 or 12E10) or specific for the FLAG epitope (mouse anti-FLAG M2, Sigma). Equal loading of proteins was confirmed by probing filters with antibodies specific for α-tubulin (DM1A, Santa Cruz Biotechnologies), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 71.1, Sigma-Aldrich), or rabbit anti CoxIV polyclonal (Abcam) for mitochondrial fractions. Horseradish peroxidase (HRP)-conjugated sheep-anti rat Ig antibodies (Silenus) served as secondary reagents and the enhanced chemiluminescence (ECL) system was used for detection. Immunoprecipitation was performed using Protein G-sepharose (GE Healthcare). For dephosphorylation, single cell suspensions were prepared from thymus or spleen from wt mice. Resuspended cells were washed twice in PBS then lysed 30 min at 4°C in Chaps buffer (20 mM Tris-HCl, 5 mM MgCl2, 137 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5% Chaps pH 7.5) supplemented with (Complete TM , Roche). Cellular debris were removed by centrifugation at 10.000xg for 20 min at 4°C. Protein extract was dephosphorylated with lambda protein phosphatase (7u/µg of protein extract) (BioLabs) and 2 mM MnCl2 2h at 4 or 30°C. The reaction was stopped by addition of SDS-PAGE loading buffer.

containing mitochondria was subdivided into 3

 3 aliquotes and centrifuged at 10000g, for 10min at 4°C. The pellet resulting from one aliquot was resuspended in 15µl of MB-EGTA buffer containing 1% Triton and used for protein quantification by Bradford. The two other mitochondrial pellets were subjected to alkali extraction. Pellets were resuspended in 125µl of H 2 O and 125µl of NaCl (2M) or Na 2 CO 3 (2M). Samples were incubated on ice for 30 minutes mixed by vortexing every 10 minutes followed by centrifuged at 100000g for 30 minutes at 4°C. Alkaline-sensitive fractions (supernatant) were precipitated adding 12% TCA (final concentration) and then resuspended in 15µl of 2x Laemmli buffer prior boiling. The alkalineresistant pellet fraction was boiled in the same volume. Finally 15µl of each fraction were loaded on a 4-20% Tris-HCl gel (Biorad) and separated by SDS gel electrophoresis.Viral Transduction & Colony Formation AssaysU2OS cells and NIH3T3 cells were transduced with lentiviruses encoding different isoforms of Bmf, MEF lacking Bax or Bak were transduced retrovirally.For overexpression of the various Bmf isoforms, the corresponding cDNAs were PCR amplified and recombined into pDONR-207 (Invitrogen) using Invitrogen's B/P recombination kit. Sequence verified clones were used for L/R recombination with pHR-tetCMV-dest[START_REF] Ploner | The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia[END_REF] thereby generating the lentiviral plasmids pHR-tetCMV-Bmf S , pHR-tetCMV-Bmf CUG , pHR-tetCMV-Bmf S L/A, pHR-tetCMV-Bmf CUG L/A and pHR-tetCMV-eGFP.Generation of lentiviral plasmid for constitutive knock-down of human BMF were described before oligonucleotides were phosphorylated, annealed and cloned into the BglII-HindIII site of pENTR-THT[START_REF] Ploner | The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia[END_REF]. Thereafter, sequence verified clones were used for L/R recombination into the final lentiviral destination vectors pHR-dest-SFFV-eGFP and pHRdest-SFFV-Puro, thereby generating pHR-THT-mBmf shRNA-SFFV-eGFP and pHR-THT-mBmf shRNA-SFFV-Puro.

view 4. 1 softwareSuppl. Figure 4 :

 14 Abbreviation List: Bcl-2-modifying factor (Bmf); Bcl-2 homology domain (BH); in vitro

Figure 1 :

 1 Figure 1: Characterization of Bmf isoforms

Figure 2 :

 2 Figure 2: The non-conventional CUG-translation initiation site of Bmf is conserved

Figure 3 :Figure 4 :

 34 Figure 3: Bmf S and Bmf CUG show the same interaction pattern with anti-apoptotic Bcl-2

Figure 5 :

 5 Figure 5: Bmf S and Bmf CUG reduce clonal survival in U2OS cells and can engage either

Figure 6 :

 6 Figure 6: Bmf isoforms localize preferentially to the outer mitochondrial membrane

Figure 7 :

 7 Figure 7: Bmf isoforms can be translated in an IRES-dependent manner

Figure 8 :

 8 Figure 8: Bmf accumulates prior cell death in NIH3T3 cells

Figure 9 :

 9 Figure 9: Bmf-deficiency delays apoptosis by cell stress associated with CAP-

Acknowledgments

We thank Profs A Strasser, C Borner, M Schuler and W Doppler for cells and reagents. S Kiessling and F Müllauer for first experiments initiating this work, W Chmelewskij for help with MEF experiments, B Tomaselli for help with hypoxia experiments, Irene Gaggl for expert technical assistance, as well as all our colleagues in the lab for insightful discussions. This work was supported by grants from the AICR, St. Andrews, UK (#06-0440), the Graduate School for Molecular Biology and Oncology (MCBO) and SFB021, both funded by the Austrian Science Fund (FWF).

Online supplemental material: Suppl. Figure 1: Bmf expression in lymphocytes and HC11 mammary epithelial cells (A) Splenic extracts were subjected to dephosphorylation treatment for one hour using increasing doses of phosphatase. (B) Protein lysates from thymus and spleen of vav-bcl-2 transgenic animals were subjected to IP-analysis using an anti-Bcl-2 specific mAb. Immune complexes were separated by SDS-PAGE and immunoblotted using anti-Bmf specific mAb (17A9). (C) WEHI231 pre-B cells were treated with solvent, cycloheximide (10µg/ml) or cycloheximide plus the proteasome inhibitor MG-132 (10µg/ml). Cells were harvested after 4h and 8h, lysed and protein extracts separated by SDS-PAGE and immunoblotting was performed using anti-Bmf specific mAb (17A9). (D) HC11 cells were deprived of serum or incubated with indicated compounds over time. Cells were analyzed as in (C). Membranes were reprobed with an anti-Tubulin mAb to compare protein loading.

Suppl. Figure 2: Cell death analysis in BaF3 cells

Suppl. Figure 3: Fluorescence detection of IRES-mediated expression of Venus

(A) 293T HEK cells cultured on glass cover slips were transfected with plasmids encoding either ECFP and Venus as a fusion protein, or ECFP followed by a STOP-codon and the Apoptosis of MEF was assessed by sub-G1 staining and flow cytometric analysis. Bars represent means ± SE of three independent experiments. Experiments using three independent MEF clones of each genotype yielded comparable results and were therefore pooled (STS=staurosporine). Asterices * indicate statistically significant differences between wt and bmf -/-MEF (p< 0.006 for LY294002; p< 0.001 for rapamycin). (D) Wt and Bmf-deficient mice were injected i.p. with a single dose of polyIC (100µg/mouse), or saline. After 20h, mice were sacrificed and the percentage of naïve CD8 + CD44 -T cells quantified by flow cytometric analysis. Bars represent means of ± SEM of three animals per genotype and group. * indicates statistically significant differences between wt and bmf -/-(p<0.003).


Julien Fouquau

Esc Rouen

Leo

Threshold effects in Okun's Law: a panel data analysis

Our approach involves the use of switching regime models, to take account of the structural asymmetry and time instability of Okun's coefficient. More precisely, we apply the non-dynamic panel transition regression model introduced by Hansen (1999) to a panel of 20 OECD countries over the last three decades. With all specifications applied, the tests lead to the rejection of the null hypothesis of a linear relationship between cyclical output and cyclical unemployment. The asymmetry implies the existence of four regimes. For lower or higher values of cyclical unemployment, it follows that there is a relatively strong negative correlation between unemployment rate and output. However, when unemployment stands at intermediate levels, this relationship tends to weaken.

We wish to thank C. Hurlin and A. Olosutean for their helpful comments and suggestions.

Introduction

Since Okun's seminal paper (1962), there have been numerous studies which provide evidence of the correlation between variations in unemployment and real output over the business cycle. Nevertheless, this interest has lately been enhanced by the fact that economic growth has been productive of fewer "jobs" in recent years than had been the case in the past in some OECD countries. As an illustration of this, an increase in French gross domestic product (GDP) of 2.5 % led to the creation of 39,000 jobs in 2004, whereas in 2001, 246,000 jobs were created despite less dynamic economic growth of 2.1 %.

However, most of the existing literature dealing with Okun's law tends to focus on the lack of robustness of the Okun's coefficient, without questioning the linear nature of the relationship. The exceptions are recent papers by [START_REF] Lee | The robustness of Okun's law: evidence from OECD countries[END_REF], [START_REF] Virèn | The okun curve is non-linear[END_REF] and [START_REF] Harris | Testing for asymmetry in okun's law: a cross-country comparison[END_REF]. Responding to this challenge, our approach involves the use of switching regime models, to take account of the structural asymmetry and time instability of Okun's coefficient.

The innovation in the present paper is the application of this approach to a panel of 20 OECD countries over the last three decades. To do this, we have used the non-dynamic panel transition regression model with fixed individual effects introduced by [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF]. Focusing on a panel of countries rather than on a single country allows us to learn about the behavior of individuals by observing the behavior of the others and allows us to derive results for a wide geographical area. Moreover, it highlights non-linear behavior that could not be taken into account by time series, due to the insufficient number of points in each regime.

Applying all specifications, the tests lead to rejection of the null hypothesis of a linear relationship between cyclical output and cyclical unemployment. To be more precise, the asymmetry entails the existence of four regimes. For lower or higher values of cyclical unemployment, a relatively strong negative correlation is observed between unemployment rate and output. However, when unemployment stands at intermediate levels, this relationship tends to weaken. Our results also characterize individual heterogeneity, making it possible for countries not to belong simultaneously to the same regime.

The outline of this paper is the following: the next section describes Okun's law. The third introduces the model specification and the methods of estimation. The fourth presents the data and the empirical results. The final section concludes with the threshold effects on Okun's law.

The model specification

Okun's law refers to the empirical regularity that seems to hold between cyclical unemployment and cyclical output. A version of this relationship may be represented by the following set of equations given by [START_REF] Weber | Cyclical output, cyclical unemployment, and Okun's coefficient : a new approach[END_REF]:

y c it ≡ y it -y n it u c it ≡ u it -u n it u c it = αy c it + ǫ it α < 0 (1)
Where y c it 1 captures the cyclical level of output (output gap), y it is the logarithm of the actual output and y n it represents the potential or trend level of the output. Similarly, u c it captures the cyclical unemployment rate (unemployment gap), u it is the observed unemployment rate, u n it represents the natural unemployment rate and ǫ it is a stochastic error term. The parameter α is commonly known as the Okun coefficient. Initially, Okun (1962) found a significant negative correlation (-0.3) between unemployment and growth for US quarterly data over the period of 1947-1960. Recently, the results of various studies have confirmed the negative relationship for USA and other developed countries [START_REF] Döpke | The "employment intensity"of growth in Europe[END_REF]. However, [START_REF] Lee | The robustness of Okun's law: evidence from OECD countries[END_REF] argues that the coefficient has substantial sensitivity to the choice of method for extraction of the cyclical component. This step is complicated by the presence of a unit root in the GDP and in the unemployment rate. For this reason, we apply four different methods in succession in order to assess the robustness of our result. Firstly, we use a difference model where the output and the unemployment variable are expressed in first differences. However, as suggested by [START_REF] Attfiled | Okun's law, cointegration and gap variables[END_REF], this method suffers from a major drawback. This is so because if the series are not only individually integrated as I (1) but are also cointegrated together, then the model is misspecified. A second solution involves considering Okun's law from the standpoint of the notion of the "gap" between actual and equilibrium output and between actual and equilibrium unemployment. To that end, we apply three filters 2 in succession: [START_REF] Hodrick | Postwar U.S. business cycles: An empirical investigation[END_REF] filter, the [START_REF] Baxter | Measuring business cycles: approximate band-pass filters for economic time series[END_REF] filter and the Beveridge & Nelson (1991) (filters HP, BK and BN in what follows).

Moreover, regarding data decomposition procedures, a major issue for Okun's law is that this relationship generally leaves out of account asymmetry phenomena in the employment dynamic. However, [START_REF] Harris | Testing for asymmetry in okun's law: a cross-country comparison[END_REF] point to four elements to justify 1 The subscript t denotes the time period and i the country.

2 Three methods are used to assess the robustness of our result. However, the HP filter and BK filter could overestimate the importance of transitory shocks in the presence of unit root [START_REF] Murray | Cyclical properties of baxter-king filtered time series[END_REF]. On the other hand, the BN decomposition allows the decomposition of an I(1) series into a stochastic trend and a cyclical component. But, this procedure needs to identify an ARIM A(p, 1, q), and this step is not straightforward in time series. To achieve this goal, we use the methodology of the Extended Sample Autocorrelation Function and the Smallest CANonical correlation [START_REF] Tsay | Consistent estimates of autoregressive parameters and Extended Sample Autocorrelation Function for stationary and nonstationary ARMA[END_REF], [START_REF] Tsay | Use of canonical analysis in time series model identification[END_REF]).

them: capacity constraints, signal extraction, costly adjustment and downward nominal wage rigidity. For this reason, we apply a class of panel threshold models developed by [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] to characterize the relationship between cyclical unemployment and cyclical output. The corresponding model with fixed effects α i is then defined as follows:

U c it = µ i + β ′ 0 y c it I(q it ≤ c) + β ′ 1 y c it I(q it > c) + ǫ it (2)
where q it is the threshold variable3 and c the threshold parameter. The transition function is an indicator function I(.) which equals 1 when the threshold condition in brackets is satisfied and 0 otherwise. In this model, the observations are divided into two regimes depending on whether the threshold variable q it is smaller or greater than the threshold parameter c. The regimes are distinguished by different regression slopes, β 1 and β 2 . A first advantage of this model is that it allows parameters to vary across individuals (heterogeneity issue), but also with time (stability issue) depending on the number of regimes. For our specific purpose, the PTR model has a second advantage in investigating the asymmetries in the employment dynamics.

There is no reason to limit our analysis to just two regimes. Such an assumption reduces the possibility of heterogeneity and may be unrealistic even for OECD countries which have different labor market flexibilities. Moreover, the estimation approach proposed by [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] allows a more general specification with r thresholds (i.e. r + 1 regimes), which take the form of:

U c it = µ i + β 0 y c it I(q it ≤ c 1 ) + β 1 y c it I(c 1 < q it ≤ c 2 ) + ...β r y c it I(c r-1 < q it ) + ǫ it (3)
where the threshold parameters c j are sorted in ascending order, c 1 < ... < c r .

Estimation and tests

Estimation of the PTR model involves several stages. First, estimation of the parameters model requires eliminating the individual effects α i by removing individual-specific means and then applying the least squares sequential procedure(see [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] or [START_REF] Hurlin | Network effects of the productivity of infrastructure in developing countries[END_REF] for more details). For example, if we consider a single threshold model, for a given value of the threshold parameter c, the slope coefficients β 1 and β 2 can be estimated by OLS. Thus, we can compute the sum of squared errors, denoted S 1 (c):

S 1 (c) = N i=1 T t=1 ǫ2 it (4)
The threshold parameter c is then estimated by minimizing S 1 (c).

ĉ = ArgMin c S 1 (ĉ) (5)
As [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] emphasizes, the minimization problem can be reduced to a search over the values of c equal to the distinct values of q it in the sample. However, it is pointless to select a threshold c, which leads to fewer observations in one regime or another. For this reason, we impose a supplementary constraint: there should be at least T /2 observations in a given regime. 4 .

The next step is to determine whether the threshold effect is statistically significant relative to a linear specification. The null hypothesis in this case describes the simple linear specification and can be expressed as: H 0 : β 1 = β 2 . This hypothesis could be tested by a likelihood ratio test:

F 1 = S 0 -S 1 (ĉ) σ2 (6)
where S 0 is the sum of the squared residuals of the linear model, S 1 the sum of the squared residuals of the one-threshold model and σ2 = S 1 (ĉ) n(T -1) . Unfortunately, the distribution of this test is non-standard since the PTR model contains unidentified nuisance parameters c under H 0 [START_REF] Davies | Hypothesis testing when a nuisance parameter is present only under the alternative[END_REF]. A solution consists of simulating by Bootstrap the asymptotic distribution of the statistic F 1 . When the threshold effect is proved, the same procedure can be applied to general models (equations 3) in order to determine the number of thresholds required to capture the whole non-linearity. The new null hypothesis consists of testing a specification with r regimes versus a specification with r + 1 regimes. The procedure starts by testing one threshold versus two, and then two versus three, and so on. The procedure stops when the null hypothesis is not rejected.

Data and results

The present study focus on a selection of 20 OECD countries: Australia, Belgium, Canada, Denmark, Finland, France, Germany, Ireland, Iceland, Italy, Japan, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom and USA. Our data were extracted from the OECD database (Economic Outlook N 75.). As recommended by [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF], we consider only balanced panels. We have therefore used quarterly data over the period of 1970-2004.

For the preliminary results, we were interested in studying the stationary of the GDP and unemployment rate series. It will be recalled that the data decomposition procedures are more complex in the presence of unit roots and the BN method is more appropriate. With this in mind, our econometric methodology is based on second-generation panel data integration tests proposed by [START_REF] Choi | Combination unit root tests for cross-sectionally correlated panels[END_REF] and [START_REF] Pesaran | A simple panel unit root test in the presence of cross section dependence[END_REF]. These tests allow the assumption of the cross-sectional dependence among panel units. The results reported in Table 1 indicate essentially that the null hypothesis of unit-root in the series cannot be rejected by the two tests at the five percent level. There is only an exception for the statistics without trend by [START_REF] Choi | Combination unit root tests for cross-sectionally correlated panels[END_REF] in the unemployment case. This rejection of the null hypothesis implies that at least one country is stationary, the others probably not being so. As a result, we can reach a no-stationary conclusion on the variables.

In the estimation phase of the PTR, determining the threshold variable is an issue. We consider each of two potential "candidates" in turn: cyclical unemployment and output gap lagged by one period5 . These variables were selected for two reasons. Firstly, it appears logical that past cyclical unemployment influences Okun's law. Secondly, [START_REF] Lee | The robustness of Okun's law: evidence from OECD countries[END_REF] stresses the non-stability and asymmetry related to this variable. However, it is possible that the transition is induced by the cyclical output: a higher output gap level implies a different impact on cyclical unemployment than a lower level. We discriminate among these "candidates" according to two criteria: we select the threshold variable which minimizes the sum of squared residuals [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] and which leads to the strongest rejection of the linearity hypothesis6 .

For each threshold variable and data decomposition procedure, the first step is to test the linear specification and, possibly, to determine the number of thresholds. The results of the linearity test and the determination of the number of thresholds are reported in table 2. The linearity tests (F 1 ) clearly lead to the rejection of the null hypothesis of linearity of the Okun's law, whatever the threshold variable chosen and the filter considered. Moreover, the presence of strong threshold effects detected in the cases of the two selected threshold variables. The likelihood ratio tests F 2 and F 3 are also significant at a level of 10% for the two variables. This means that there are at least four regimes. According to Hansen's procedure, it would be necessary to estimate and test four thresholds, five thresholds and so on, until the corresponding F-test is statistically non-significant. However, we have limited our analysis to a model with at most three threshold parameters (i.e. four regimes). This choice can be justified on two grounds [START_REF] Hurlin | Network effects of the productivity of infrastructure in developing countries[END_REF]: firstly, computational costs and, secondly, an extra regime will not affect (or only slightly affect) the estimates of the other threshold parameters and the estimates of the slope parameters in the existing regimes. Looking at table 2, we can also determine the "optimal" variable. For all the data decomposition procedures, this is cyclical unemployment lagged by one period. Given this, we continue our analysis using this threshold variable and four regime models.

The following remarks relate solely to Beveridge Nelson decomposition, but we will see later that the other filters largely confirm these conclusions. The estimates of the parameters of the PTR models with four regimes and the corresponding t-statistics based on standard errors corrected for heteroskedasticity are reported in Table 3, together with the threshold values. These parameters are important because they show when the transition between the two regimes occurs. For instance, if cyclical unemployment is greater than -0.0180 and less than -0.0123, the country concerned switches to the second regime. In light of these results, we note that the relationship between cyclical unemployment and output gap (-0.611 and -0.354) is the strongest in the two lower regimes and subsequently in the higher regime (-0.211). Conversely, the link is extremely weak in the higher intermediate regime, even if the coefficient is significantly different from zero. Okun's law may apply in extreme regimes, like Purchasing Power Parity.

According to the estimated threshold values, we can deduce the distribution of the countries among the different regimes (table 4) and plot this transition, taking into consideration time and countries7 (figure 1). We observe that the majority of observations are in the third regime, which corresponds to a weak Okun's law. The results for Japan are aligned with those generally admitted in time series analysis. This country is placed solely in the third regime and consequently confirms Lee's explanations of high rigidity in the labor market. Conversely, observations for the United Kingdom and USA are often in extreme regimes. This point highlights a degree of flexibility and confirms the high coefficients generally admitted in time series. France and Germany are in intermediate positions, but belong largely in the third regime. As a final comment on this table, we may note that the panel data investigation has revealed threshold effects that it would not have been possible to obtain in times series covering too few observations.

To check the robustness of our results, it is however useful to analyze not only the results using Beveridge Nelson data decomposition but also those of the other methods used. Looking at table 3, we once again see the four significant regimes including three extreme regimes with high coefficients compared to the remaining one, which encompasses a lower central regime. The only difference between these decompositions is to be found in the location of this regime in the HP filter (lower intermediate regime). However, the majority of the observations are located in this regime. Like [START_REF] Lee | The robustness of Okun's law: evidence from OECD countries[END_REF], our results are qualitatively similar, but differ in quantitative terms. Moreover, there is a gap between the values of the coefficients in different data decomposition methods. However, the economics are intuitively the same. For more precision, we have plotted the transition for time and countries in figure 2. We may observe once again that this threshold allows heterogeneity and time instability to be taken into account. Japan is still in the lower regime of Okun's law, and the US and United Kingdom are often outside this regime.

Conclusion

The purpose of this study was twofold: to prove that the law of Okun is not linear, and then represent it with threshold panel data models. Firstly, the methodology of [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] allows us to show the presence of threshold effects in Okun's law, irrespective of the data decomposition method or the threshold variable chosen. These results demonstrate the existence of an asymmetry in the relationship between cyclical unemployment and output gap. Secondly, this study offers an original approach by clustering countries with same dynamic and thus solving the heterogeneity problem and by introducing an Okun law which may vary in time. 7.96

tables and figures

(0,00) 1, 31 -0,09 Z 1, 6 (0,94) 0, 36 (0,64)
-6, 06

-0, 96 -0,17

L ⋆ 1, 48 (0,93) (0; 56) (0,57) -6, 37 (0,00)
-0, 78 -0,21

Pesaran (2003) CIP S 1 -1, 95 (0,27) -2, 03 (0,93) -2, 01 (0,20)
-2, 27 (0,00) (0,00) (0,00) (0,00) (0,00) (0,00) (0,00) (0,00) (0,00) (0,08) (0.04) (0.08) (0,00) (0,00) (0,00) (0,00) Note: F 1 , F 2 F 3 are the likelihood ratio statistics. pvalues are obtained with 300 simulations [START_REF] Hansen | Threshold effects in non-dynamic panels: estimation, testing, and inference[END_REF] and with 200 simulations for the models with four regimes because computational costs. RSS: Residuals Sum of Squared.

(0,64) CIP S 2 -1, 89 (0,34) -1, 89 (0,98) -1, 9 (0,33) -2, 27 (0,65

  p-value are bracketed. All specifications are estimated with fixed effects.

Figure 1 :

 1 Figure 1: Distribution of eight countries among the different regimes

Figure 2 :

 2 Figure 2: Distribution of the countries applying other decomposition procedures

Table 1 :

 1 Unit root tests in panel data

			GDP	Unemployment
		Statistic without trend trend without trend Trend
	Choi (2002)	P m	-2, 15	0, 93
			(0,98)	(0,17)

Table 2 :

 2 Linearity test and determination of regime number

					Threshold Variables
		B.N Filter	First Dif.	B.K Filter	H.P filter
		u t-1	y t-1	u t-1	y t-1	u t-1	y t-1	u t-1	y t-1
	Two Rgimes
	RSS	0.181 0.227 0.0253 0.0261 0,052 0,061 0.0714 0.0811
	F 1	1047	292	108	16.4	397	51,7	382	14.3
	p-value

Table 3 :

 3 Threshold model with four regimes

	Filter	BN	H.P	First Dif.	B.K
	Lower Threshold	-0.0180	-0,0075	-0,0047	-0,0093
	Middle Threshold	-0.0123	0,0031	-0,0028	-0,0065
	Higher Threshold	0.0067	0,0070	0,0076	0,0068
	RSS	0.1616	0,0609	0,0249	0.0441
	Lower regime Coef. β 0 -0.611 ⋆⋆⋆ (-34.0)	-0, 421 ⋆⋆⋆ (-30.5)	-0.264 ⋆⋆⋆ (-14.2)	-0.533 ⋆⋆⋆ (-28.4)
	Lower Middle Coef. β 1 -0.355 ⋆⋆⋆ (-8.69)	-0, 100 ⋆⋆⋆ (-13.5)	-0.160 ⋆⋆⋆ (-9.75)	-0.334 ⋆⋆⋆ (-15.6)
	Higher Middle Coef. β 2 -0.010 ⋆⋆⋆ (-3.22)	-0, 273 ⋆⋆⋆ (-15.6)	-0.066 ⋆⋆⋆ (-11.1)	-0.127 ⋆⋆⋆ (-17.0)
	Higher Coef. β 3	-0.211 ⋆⋆⋆ (-18.4)	-0, 554 ⋆⋆⋆ (-33.2)	-0.251 ⋆⋆⋆ (-6.90)	-0.572 ⋆⋆⋆ (-33.7)
	Note: Residuals Sum of Squared. t-statistics corrected for heteroskedasticity are
	bracketed.***: significance level at 1%, **: significance level at 5%,*: significance
	level at 10%.

Table 4 :

 4 Data distribution between regimes and countries

		Lower	Lower Middle	Upper Middle	Superior
	Australia	0	2	129	4
	Belgium	2	9	92	32
	Canada	0	0	126	9
	Denmark	0	2	124	9
	Finland	29	5	44	57
	France	0	0	129	6
	Germany	0	0	131	4
	Ireland	1	4	98	32
	Island	0	0	103	32
	Italy	0	0	135	0
	Japan	0	0	135	0
	Luxembourg	0	0	135	0
	Netherland	6	12	79	38
	Norway	0	0	135	0
	Portugal	3	5	98	29
	Spain	17	10	60	48
	Sweden	0	0	129	6
	Switzerland	0	0	134	1
	United Kingdom	27	11	59	38
	USA	2	5	110	18
	Note: Data decomposition procedure is Beveridge Nelson decompo-
	sition. The threshold variable is the cyclical unemployment lagged
	by one period.

No constraints are imposed on the choice of the threshold variable, except that it cannot be a contemporaneous endogenous variable and time independent. The choice of this threshold variable is discussed further in the next section.

The choice of this constraint is a guarantee that the influence of a given sector in the search of c is not neglected.

We have also estimated the lag between one and four. But we have not reported these results because they lead the same number of regimes and these threshold variables are statistically lower "optimal". However, the results are available upon request.

The "optimal" threshold variable in a panel smooth transition model corresponds to the variable which leads to the strongest rejection of the linearity hypothesis. We extend here this result to the PTR class of model.

We have reported only eight countries. Others are available on upon request.