Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila
2004; Springer Nature; Volume: 23; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7600356
ISSN1460-2075
AutoresMarek Jindra, Ivana Gažiová, Mirka Uhlířová, Masataka Okabe, Yasushi Hiromi, Susumu Hirose,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoArticle12 August 2004free access Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila Marek Jindra Marek Jindra Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Search for more papers by this author Ivana Gaziova Ivana Gaziova Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Search for more papers by this author Mirka Uhlirova Mirka Uhlirova Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Search for more papers by this author Masataka Okabe Masataka Okabe Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Search for more papers by this author Yasushi Hiromi Yasushi Hiromi Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan Search for more papers by this author Susumu Hirose Corresponding Author Susumu Hirose Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan Search for more papers by this author Marek Jindra Marek Jindra Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Search for more papers by this author Ivana Gaziova Ivana Gaziova Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Search for more papers by this author Mirka Uhlirova Mirka Uhlirova Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic Search for more papers by this author Masataka Okabe Masataka Okabe Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Search for more papers by this author Yasushi Hiromi Yasushi Hiromi Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan Search for more papers by this author Susumu Hirose Corresponding Author Susumu Hirose Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan Search for more papers by this author Author Information Marek Jindra1,2, Ivana Gaziova1, Mirka Uhlirova1, Masataka Okabe2, Yasushi Hiromi2,3 and Susumu Hirose 2,3 1Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic 2Department of Developmental Genetics, National Institute of Genetics, Mishima, Japan 3Department of Genetics, SOKENDAI, Mishima, Japan *Corresponding author. Department of Developmental Genetics, National Institute of Genetics, 1111, Yata, Mishima, Shizuoka-ken 411-8540, Japan. Tel.: +81 559 816771; Fax: +81 559 816776; E-mail: [email protected] The EMBO Journal (2004)23:3538-3547https://doi.org/10.1038/sj.emboj.7600356 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. We show that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. We conclude that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress. Introduction Organisms from the most primitive prokaryotes to mammals have evolved a number of mechanisms to maintain cellular redox balance and thus evade oxidative stress caused by naturally arising reactive oxygen species (ROS). These mechanisms include low-molecular radical scavengers and antioxidant enzymes. Studies in yeast and mammalian cell lines have identified regulatory pathways of antioxidant defense. These involve protein kinases, such as JNK and ERK, that activate transcription factors, which turn on stress response genes (Davis, 2000). JNK signaling is required for oxidative stress defense also in the fruit fly Drosophila melanogaster, which serves as a model to characterize it genetically (Stronach and Perrimon, 1999; Wang et al, 2003). Among the key transcription factors activated by the JNK and ERK kinases are the basic region leucine zipper (bZIP) proteins of the Jun and Fos families (Karin, 1995). Related bZIP proteins combine through their leucine zippers to yield an array of DNA-binding dimers, known as AP-1 (activator protein-1). AP-1 activity can be induced by signals as diverse as growth factors, peptidic hormones and neurotransmitters, microbial infections and physical and chemical stresses. In response, AP-1 triggers a spectrum of cellular processes such as apoptosis or proliferation, differentiation and mobilization of defense against stress (Shaulian and Karin, 2002). Jun and Fos not only activate the cellular defense against oxidative challenge, but also sense redox imbalances: their activity depends upon their redox state. This appears to be an important and universal principle, since also other transcription regulators, such as NF-κB or p53, are sensitive to oxidation (Marshall et al, 2000). The sensitivity is conferred by reactive sulfhydryl groups of cysteine residues. Abate et al (1990) have shown that specific cysteines within the basic regions of c-Jun and c-Fos are responsible for oxidative inhibition of AP-1 DNA-binding capacity in vitro, which can be restored with reducing agents. In contrast, Jun and Fos mutants with serine replacing the critical cysteine residues bind DNA regardless of redox conditions. Thus the hypersensitive cysteine introduces a new element of regulation into AP-1. That such regulation is indeed necessary for normal cell functioning is obvious from consequences of the critical cysteine mutation into serine. Exactly such substitution is found in the viral transforming protein v-Jun (Bohmann et al, 1987; Maki et al, 1987), where it contributes to the oncogenic activity synergistically with other mutations (Morgan et al, 1994). Similarly, the corresponding cysteine-to-serine substitution leads to a transforming activity of Fos (Okuno et al, 1993). In both cases, the mutant AP-1 forms apparently escape the redox regulation. Paradoxically, while AP-1 mobilizes antioxidant defense, it is at the same time sensitive to oxidation. This suggests that during redox imbalances in vivo, something must protect Jun and Fos from oxidative damage. A nuclear protein Ref-1 (redox factor 1), also implicated in DNA repair, has been shown to reactivate AP-1 by a thioredoxin-dependent reduction of the critical cysteine residues (Xanthoudakis and Curran, 1992; Xanthoudakis et al, 1992; Hirota et al, 1997; Ordway et al, 2003). While Ref-1 mediates a reparative reduction of once inactivated nuclear AP-1, it seems logical that some factor should also prevent oxidation of newly synthesized Jun and Fos, particularly during oxidative stress. A good candidate to perform this protective role is the multiprotein bridging factor 1 (MBF1). MBF1, also known as endothelial differentiation-related factor 1 (EDF1), primarily resides in the cytoplasm and can relocate to the nucleus upon external stimuli (Mariotti et al, 2000). MBF1 acts as a coactivator of the bZIP protein GCN4, the closest relative of Jun in the budding yeast. MBF1 interacts directly with both GCN4 and the TATA-binding protein (TBP), implying that it interconnects the bZIP factor with the basal transcription machinery (Takemaru et al, 1998). MBF1 has also been shown to bind human c-Jun (Kabe et al, 1999) and stimulate c-Jun-dependent transcription (Busk et al, 2003). Unlike other AP-1 interacting proteins, MBF1 is unique in that it binds to the basic region where the redox-sensitive cysteines are located. We have chosen the fruit fly Drosophila to examine the relationships between AP-1 and MBF1. Compared to four Fos and three Jun paralogs in mammals, Drosophila is a simple model with only one D-Fos and one D-Jun protein (Kockel et al, 2001). In this study, we show that through a direct interaction with D-Jun, MBF1 protects the critical cysteine residue from oxidation and stimulates AP-1 binding to DNA. A mutation removing mbf1 causes sensitivity to oxidative stress in vivo and compromises an AP-1-dependent process of epithelial tissue closure. Studies of MBF1 therefore open an avenue to learn more about AP-1 regulation and function. Results Critical cysteine residues confer D-Jun and D-Fos sensitivity to oxidation Drosophila D-Jun and D-Fos proteins have cysteine residues in the same positions as the subunits of human AP-1, which rapidly loses DNA-binding activity upon oxidation (Figure 1A). To see whether Drosophila AP-1 also undergoes oxidative inactivation, we tested the binding of bacterially expressed D-Jun and D-Fos bZIP domains to an AP-1 site using electrophoresis mobility shift assays under disparate redox conditions. Both D-Jun and D-Fos were truncated such that each protein harbored a single cysteine within the basic region, and were designated J and F, respectively (Figure 1B). Their DNA-binding properties were compared with those of point mutants, JS and FS, in which the critical cysteine residues had been replaced with serine (Figure 1A). Like their human orthologs, J and F proteins were unable to bind DNA in the absence of dithiothreitol (DTT; Figure 2A, lane 1). Because neither protein bound to DNA alone in our conditions, no binding occurred also when the cysteine was mutated in either D-Fos (Figure 2A, lane 4) or D-Jun (not shown). Only when both proteins were mutated, their complex with the AP-1 site could be detected in the absence of DTT (lane 7). Addition of 1 mM DTT allowed weak binding of the J/F dimer (lane 2); the binding of the mutant proteins under the same conditions was stronger (lanes 5 and 8). Increasing DTT concentration to 10 mM enhanced the binding of dimers in which at least one protein contained cysteine (lanes 3 and 6). These results show that Drosophila Jun and Fos are readily inactivated by oxidation of the critical cysteine residues, and suggest existence of factor(s) that maintain the activity of these proteins under oxidative conditions. Figure 1.Recombinant D-Fos, D-Jun and MBF1 proteins used in this study. (A) Drosophila Fos and Jun truncated proteins, aligned with human c-Fos and c-Jun. Conserved basic regions and leucine residues of the bZIP domains are shaded; the critical cysteines are in black boxes. These cysteines were mutated to serine to produce redox-insensitive D-Fos and D-Jun forms (FS and JS). The cysteines C-terminal to the leucine zipper were replaced with stop codons in all D-Fos and D-Jun constructs. (B) His-tagged D-Fos (F), D-Jun (J) and MBF1 (M) were expressed in E. coli either separately or coexpressed from bicistronic plasmid constructs (FexM and JexM). rbs, ribosome-binding site. (C) AP-1 and MBF1 proteins were expressed from constructs shown in (B), affinity purified using the His tag, separated on a reducing SDS–polyacrylamide gel and stained with Coomassie blue. Download figure Download PowerPoint Figure 2.Effects of oxidation and MBF1 on the binding of D-Fos and D-Jun to an AP-1 site. Gel retardation assays were performed with Jun and Fos, each containing the single critical cysteine (J, F), and with the serine mutants (JS, FS). MBF1 was added separately (J+F+M), coexpressed with Jun (JexM) or Fos (FexM) from a bicistronic plasmid, or copurified from mixed E. coli cultures each expressing one protein (JcoM). The arrow shows the AP-1/DNA complex. (A) Binding of freshly purified proteins under oxidative (no DTT) or reducing conditions revealed that Drosophila AP-1 activity depends on the redox state of the critical cysteine residues in its DNA-binding domain. (B) Binding of freshly purified proteins in the presence of 1 mM DTT showed that weak AP-1 activity (lane 5) was greatly enhanced by coexpression of Jun with MBF1 (lane 10). Both Jun and Fos were required for the binding. (C) The assay conditions were as in (B) except that the proteins were aged for 5 days in solution at 4°C. No binding was observed unless MBF1 had been coexpressed with Jun. Download figure Download PowerPoint MBF1 ensures AP-1 binding to DNA through interaction with D-Jun To study whether Drosophila MBF1 supports AP-1 activity, we first examined the effect of MBF1 on the DNA-binding activity of AP-1 (Figure 2). When MBF1 was added to the electrophoresis mobility shift assay with bacterially produced J and F, it only mildly stimulated DNA binding (Figure 2B, compare lanes 5 and 7). MBF1 exerted a stronger effect when copurified with J from pooled bacterial cultures, each expressing only one protein (lane 9), and the strongest effect was observed when MBF1 was coexpressed with J in Escherichia coli using a bicistronic construct (lane 10). The increased DNA-binding activity was not due to a higher yield of the coexpressed proteins (Figure 1C). Thus, MBF1 had to be in contact with D-Jun already within the E. coli cells or at least during the purification steps in order to ensure robust AP-1 activity; later addition of MBF1 was not sufficient. MBF1 could also stimulate the DNA-binding activity of the mutant JS/FS complex (Figure 2B, lanes 6 and 11), which is only partially sensitive to oxidative condition (Figure 2A, lanes 7 and 8). This result suggests that MBF1 has a more general positive effect on D-Jun that is not entirely mediated by keeping the cysteines reduced. To further test the function of MBF1 in preserving AP-1 activity, we artificially ‘aged’ purified proteins for several days at 4°C. Upon such treatment, AP-1 completely lost its DNA-binding activity, suggesting inactivation due to oxidation and/or denaturation (compare Figure 2B, lane 5, with Figure 2C, lane 1). However, when D-Jun was coexpressed with MBF1, it was able to form active AP-1 even after the aging treatment (Figure 2C, lane 3). Once lost, the AP-1 activity could not be restored by subsequent addition of MBF1 (Figure 2C, lane 2). MBF1 showed its protective effect only on D-Jun; aged AP-1 proteins failed to bind DNA when MBF1 was either added or coexpressed with D-Fos (Figure 2C, lanes 2, 4 and 5). However, D-Fos was still functional, because no DNA binding occurred without it in our assays (Figure 2B, lanes 2–4). These results indicate that MBF1 prevents the deterioration of AP-1 activity specifically through acting on D-Jun. MBF1 binds the basic region of D-Jun and protects the critical cysteine from oxidation The enhanced DNA-binding activity of AP-1 in the presence of MBF1 suggests that MBF1 may protect AP-1 from oxidation through a direct contact. To test for interaction between MBF1 and AP-1 proteins, we performed GST pull-down assays with the hexahistidine-tagged D-Jun and D-Fos bZIP domains (Figure 1) and a GST-MBF1 fusion. Figure 3A shows that MBF1 specifically bound the D-Jun but not the D-Fos bZIP region. The failure to bind D-Fos likely was not due to D-Fos deterioration, since this protein was active in our electrophoresis mobility shift assays. Although we have not mapped the exact amino acids required for MBF1 binding in the D-Jun bZIP peptide, we surmise that they include the basic residues near the critical cysteine (Figure 3B), because these basic residues in GCN4 are required for yeast MBF1 binding (Takemaru et al, 1998). A very similar basic motif in the nuclear receptor Ftz-F1 (Figure 3B) is necessary for the binding of insect MBF1 (Takemaru et al, 1997). Figure 3.MBF1 binds a conserved basic motif. (A) GST pull-down assay showed a direct interaction of a GST-MBF1 fusion protein with the His-tagged D-Jun bZIP domain (J) but not with the same region of D-Fos (F). (B) Alignment of basic regions in the yeast bZIP factor GCN4, a nuclear receptor Ftz-F1, and the human and Drosophila AP-1 family members. All proteins except D-Fos have been shown to bind MBF1. The black boxes indicate arginine residues in GCN4 and Ftz-F1, known to be required for MBF1 binding; the corresponding basic residues in Fos and Jun are shaded. The arrow points to the critical redox-sensitive cysteine in AP-1 proteins. Download figure Download PowerPoint To test whether MBF1 prevents oxidation of the redox-sensitive cysteine of D-Jun, the D-Jun bZIP domain expressed in E. coli either alone or with MBF1 (Figure 1C) was subjected to MALDI-TOF mass analysis. Figure 4 shows that D-Jun coexpressed with MBF1 remained in the reduced state. In contrast, when expressed alone, a majority of D-Jun increased its mass by 222.6 Da, an increment corresponding to S-cystenyl cystenylation (i.e. cystenyl cyteine attached to D-Jun via a mixed disulfide bond). Consistently, the modified form retained a single reactive SH group per molecule as revealed by monoalkylation with iodoacetamide (data not shown). To confirm that S-cystenyl cystenylation indeed concerned the critical cysteine residue, we show that no such modification occurred in the D-Jun bZIP domain harboring the cysteine-to-serine substitution (JS), expressed in the absence of MBF1 (Figure 4). MBF1 therefore functions to protect the critical cysteine from oxidative modification. Figure 4.MBF1 prevents an oxidative modification of D-Jun. The His-tagged bZIP domain of D-Jun (J) was expressed in E. coli either alone (top) or coexpressed with MBF1 (JexM); its cysteine-to-serine mutant (JS) was expressed alone (bottom). The purified proteins (see Figure 1C) were subjected to MALDI-TOF mass analysis. D-Jun expressed alone shows a mass increment of 222.6 Da, corresponding to S-cystenyl cystenylation. The mass of His-tagged MBF1 is around 18 kDa. Download figure Download PowerPoint MBF1 expression and nuclear translocation with D-Jun Since oxidative stress can occur at any time, MBF1 should be expressed constantly in order to prevent AP-1 oxidation. We determined the developmental profile of MBF1 expression using a specific polyclonal antibody that detected a single band of the expected size (16 kDa) on Western blots. The presence of the MBF1 protein started in the embryo with a strong maternal contribution and was maintained throughout embryogenesis, with zygotic translation ensuing 5–7 h after egg laying (Figure 5A). Expression continued for the entire postembryonic life without temporal fluctuations (Figure 5B). Among tissues exhibiting high MBF1 levels were the central nervous system, imaginal discs and gonads, but not the fat body (Figure 5C–E). Figure 5.Expression pattern of MBF1 in Drosophila. (A) Western blot showing constitutive MBF1 expression during embryogenesis. Embryos from mbf1 mutant mothers were devoid of all maternal MBF1 protein. Zygotic expression from the paternal wild-type chromosome began between 5 and 7 h after egg laying (right panel). (B) Western blot showing MBF1 expression throughout the postembryonic life. W-1 and W-2, wandering stages of larvae. (C–E) Sites of high MBF1 expression during larval life were the central nervous system (C, second instar), imaginal discs (D, late third instar) and the testis (E, center), but not the fat body (E, surrounding tissue). MBF1 was detected with a specific polyclonal antibody, and DAPI was used for DNA staining in (E). Magenta is used as colorblind friendly (http://jfly.iam.u-tokyo.ac.jp/color/text.html). Download figure Download PowerPoint Although MBF1 interacts with nuclear partners, previous data (Kabe et al, 1999; Mariotti et al, 2000; Liu et al, 2003) have shown that MBF1 is primarily a cytoplasmic protein, suggesting that MBF1 may cotransport with interacting transcription factors to the nucleus. We have tested whether MBF1 translocates to the nucleus with D-Jun in Drosophila cells. As shown in Figure 6, the MBF1 protein resides predominantly in the cytoplasm of both embryonic (S2) and imaginal disc (Cl.8+) cells, cultured under control conditions. In contrast, MBF1 moved to the nucleus in S2 cells that had been transfected with a plasmid expressing D-Jun-His, the entire D-Jun protein with a C-terminal hexahistidine tag (Figure 6A). As revealed with an antibody against His tag, the D-Jun-His protein also accumulated in the nucleus. Co-immunoprecipitation of MBF1 and D-Jun-His from these transfected cells with the His-tag antibody (Figure 6B) suggested that MBF1 translocated to the nucleus upon interaction with D-Jun. Such an interaction likely occurs through MBF1 binding to the bZIP domain of D-Jun, demonstrated in the GST pull-down assay (Figure 3). Transfection of Cl.8+ (Figure 6C) or S2 cells (not shown) with only the His-tagged bZIP domain of D-Jun confirmed that this domain alone was sufficient for the nuclear translocation of MBF1. These data suggest that MBF1 migrates to the nucleus in complex with the newly synthesized D-Jun protein. Figure 6.MBF1 and D-Jun form a complex and cotranslocate to the nucleus in Drosophila cells. (A) Drosophila S2 cells showed translocation of the endogenous cytoplasmic MBF1 protein (control) to the nucleus upon misexpression of the whole His-tagged D-Jun protein (right column). (B) Upon cotransfection of S2 cells with MBF1 and D-Jun-His (but not GFP), MBF1 was recovered from the cell lysate together with the D-Jun-His protein by using an anti-His-tag monoclonal antibody. The Western blot was first probed with anti-MBF1, then stripped and re-probed with a D-Jun antiserum (bottom). (C) Expression of a His-tagged bZIP domain of D-Jun in Cl.8+ cells was sufficient for nuclear translocation of the MBF1 protein (left panel); expression of nuclear GFP had no effect on MBF1 localization. Cells were fixed and stained 36 h after transfection. Staining with anti-His tag was visualized with DTAF (FITC)-conjugated secondary antibody. Anti-MBF1 was detected with a Cy3-conjugated secondary antibody, shown as magenta that is friendly to colorblind people (http://jfly.iam.u-tokyo.ac.jp/color/). GFP was visualized with direct fluorescence. Download figure Download PowerPoint Null mbf1 mutants are sensitive to oxidative stress To study the role of MBF1 in vivo, we generated deletions in the Drosophila mbf1 gene through P-element transposon insertion and its subsequent imprecise excision (Figure 7A). The molecular lesions of four deletion alleles (mbf11 through mbf14) spanned from the original P-element insertion site toward the coding region, affecting mbf1 but no other gene (Figure 7A and B). Except for the mbf11 allele that had a shortened mRNA, all alleles failed to produce the mbf1 transcript (Figure 7C). All four alleles, either homozygous or hemizygous over a Df(3L)st7P deficiency that includes mbf1, were totally devoid of the MBF1 protein (e.g. mbf12; Figure 7D). Despite the complete absence of the MBF1 protein, all four alleles were viable and fertile under standard laboratory conditions. We used mbf12, which had the longest deletion of 2082 bp, in all experiments described hereafter. A rescue construct that includes a 4.6 kb genomic mbf1 fragment in a P-element vector restored production of the MBF1 protein at all stages examined (Figure 7D and data not shown). Figure 7.mbf1 mutant flies are molecularly null. (A) Map of the mbf1 gene located on chromosome 3L with a P-element insertion (P) 21 bp upstream of the first exon and four imprecise P excisions (1–4). Black boxes denote coding exons of mbf1, used as a hybridization probe in (B, C); untranslated regions are open. The dotted lines represent DNA deleted in mbf1 alleles mbf11 to mbf14 (1–4). Centromere is to the right. (B) Southern blot of genomic DNA from wild-type (wt) and homozygous mbf1 mutant flies shows that mbf12and mbf13 lack the coding region of mbf1. (C) Northern hybridization of mRNA shows the complete loss of both transcripts (1.6 and 1.1 kb) in mbf12 to mbf14 homozygotes. (D) Western blot analysis of the MBF1 protein from adult flies confirms that mbf12 is a null allele. A transgenic construct P(mbf1+) restores the production of the MBF1 protein. TM3 is a third chromosome balancer; Df(3L)st7P is a deficiency including mbf1. Download figure Download PowerPoint The ability of MBF1 to prevent oxidation of D-Jun suggests that the mbf1 gene might have an important function during stress, when AP-1 triggers various stress responses. To test the possibility that the loss of mbf1 affects oxidant tolerance in Drosophila, we compared the survival of the mbf12 mutants with mbf1+ animals (P(w+mbf1+)/P(w+,mbf1+); mbf12) in the presence of hydrogen peroxide (H2O2) as a source of oxidative stress. When placed on diet containing 0.1 or 0.3% H2O2 as first instar larvae, mbf12 animals reached adulthood about 3.5 times less frequently than the mbf1+ strain (Figure 8A). To test oxidative stress tolerance in adults, we exposed males of equal size and age to 0.5% H2O2 and percent surviving was scored at regular intervals (Figure 8B). The median survival time of the mbf12 homozygotes was 67 h, compared to 93 h for the mbf1+ strain. mbf1− hemizygotes obtained from Df(3L)st7P/+ mothers, crossed with mbf12 males, lived on average 64 h on 0.5% H2O2. The sensitivity was therefore not caused by another mutation on the mbf1− chromosome or by a maternal effect. Flies possessing four doses of mbf1+ (two endogenous and two transgenic) were more resistant to H2O2 than animals with two copies (Figure 8B). When catalase activity was inhibited prior to H2O2 treatment by feeding flies with 5 mM aminotriazole, the effect of H2O2 was greatly enhanced; the lifespan of mbf1 mutants was less than 60% that of the rescued flies (Figure 8B). A similar enhancement was observed when flies were pretreated with 5 mM buthionine-sulfoximine, a drug that reduces the free radical scavenging capacity by depleting the endogenous pool of glutathione (data not shown). Together, these results show that the loss of MBF1 renders animals sensitive to ROS. Figure 8.MBF1-deficient mutants are sensitive to oxidative stress. (A) Equal numbers of first instar larvae of the mbf1 mutant and rescued lines were placed on diet containing indicated concentrations of H2O2 and numbers of emerging adults were counted. Values above bars indicate the numbers of larvae. (B) Adult males (30 per vial) were exposed to H2O2 either directly (solid lines) or after reducing their catalase activity by feeding 5 mM aminotriazole for 60 h (broken lines), and their lifetime was recorded. The open symbols denote flies possessing at least one copy of mbf1+. Line P(mbf1+)/P(mbf1+); +/+ contains four copies of mbf1+. The numbers of flies tested per genotype ranged between 120 and 540, and the resistance of each genotype was tested at least three times. Download figure Download PowerPoint An AP-1-dependent developmental process becomes sensitive to oxidative stress in mbf1 mutant background In addition to triggering antioxidant responses, AP-1 has various developmental functions including formation of the adult thorax (reviewed by Kockel et al, 2001). D-Fos is required for fusion of the wing imaginal discs at the dorsal midline (Riesgo-Escovar and Hafen, 1997a; Zeitlinger and Bohmann, 1999). To see whether also D-Jun is required for thorax closure, we induced an RNA interference (RNAi) knockdown of D-Jun in the dorsal epithelium using the pnr-Gal4 driver (Heitzler et al, 1996). This resulted in mild to severe defects of thorax fusion in 23% of the UAS-D-JunRNAi/+; pnr-Gal4/+ flies (Figure 9C). Figure 9.Genetic interaction between MBF1 and AP-1 during thorax closure. (A) The JNK cascade is required for Drosophila thorax closure (after Kockel et al, 2001). Hemipterous (Hep) phosphorylates a Jun N-terminal kinase Basket (Bsk). The redox-sensitive JNK substrates (AP-1) are in green. A JNK phosphatase Puckered (Puc) and a TGFβ signal Dpp (Decapentaplegic) are putative targets of AP-1. Incomplete function of Hep or D-Fos causes cleft adult thorax; Puckered is a negative regulator of the thorax closure. (B, C) RNAi knockdown of D-Jun, targeted to the dorsal epidermis using pnr-Gal4, prevents complete fusion of the thorax; a control expressing the driver alone has normal thorax (B). (D–G) Animals of indicated genotypes were challenged with H2O2 at the onset of metamorphosis. Emerging adults doubly mutant for mbf1 and either D-jun (E) or D-fos (F,G) occasionally displayed partially cleft thorax with naked cuticle (arrows), sometimes accompanied with necrosis (arrowhead). Download
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