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• W2079180838 abstract "The forkhead transcription factor, DAF-16, a downstream target of the insulin/IGF-I signaling pathway in C. elegans, is indispensable both for lifespan regulation and stress resistance. The molecular mechanisms involved in regulating DAF-16 transcriptional activation remain undefined. Here, we have identified an E3 ubiquitin ligase, RLE-1 (regulation of longevity by E3), which regulates aging in C. elegans. Disruption of RLE-1 expression in C. elegans increases lifespan; this extension of lifespan is due to elevated DAF-16 protein but not to changes of daf-16 mRNA levels. We have also found that RLE-1 catalyzes DAF-16 ubiquitination, leading to degradation by the proteasome. Elimination of RLE-1 expression in C. elegans causes increased transcriptional activation and sustained nuclear localization of DAF-16. Overexpression of DAF-16 in rle-1 mutants increases worm lifespan, while disruption of DAF-16 expression in rle-1 mutants reverses their longevity. Thus, RLE-1 is an E3 ubiquitin ligase of DAF-16 that regulates C. elegans aging. The forkhead transcription factor, DAF-16, a downstream target of the insulin/IGF-I signaling pathway in C. elegans, is indispensable both for lifespan regulation and stress resistance. The molecular mechanisms involved in regulating DAF-16 transcriptional activation remain undefined. Here, we have identified an E3 ubiquitin ligase, RLE-1 (regulation of longevity by E3), which regulates aging in C. elegans. Disruption of RLE-1 expression in C. elegans increases lifespan; this extension of lifespan is due to elevated DAF-16 protein but not to changes of daf-16 mRNA levels. We have also found that RLE-1 catalyzes DAF-16 ubiquitination, leading to degradation by the proteasome. Elimination of RLE-1 expression in C. elegans causes increased transcriptional activation and sustained nuclear localization of DAF-16. Overexpression of DAF-16 in rle-1 mutants increases worm lifespan, while disruption of DAF-16 expression in rle-1 mutants reverses their longevity. Thus, RLE-1 is an E3 ubiquitin ligase of DAF-16 that regulates C. elegans aging. The insulin/IGF-1 (insulin-like growth factor-1) pathway, which includes the DAF-2 transmembrane receptor, a series of intracellular kinases, and the DAF-16 forkhead-family transcription factor, is involved in aging in C. elegans (reviewed by Gami and Wolkow, 2006Gami M.S. Wolkow C.A. Studies of Caenorhabditis elegans DAF-2/insulin signaling reveal targets for pharmacological manipulation of lifespan.Aging Cell. 2006; 5: 31-37Crossref PubMed Scopus (79) Google Scholar). Animals with weak daf-2 mutations age more slowly than wild-type animals and live much longer (Riddle, 1977Riddle D.L. A genetic pathway for dauer larva formation in C. elegans.Stadler Genet. Symp. 1977; 9: 101-120Google Scholar, Kimura et al., 1997Kimura K.D. Tissenbaum H.A. Liu Y. Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans.Science. 1997; 277: 942-946Crossref PubMed Scopus (1615) Google Scholar, Tissenbaum and Ruvkun, 1998Tissenbaum H.A. Ruvkun G. An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.Genetics. 1998; 148: 703-717PubMed Google Scholar). Furthermore, mutation of the downstream age-1 gene, which encodes a protein similar to the mammalian p110 catalytic subunit of PI3K, leads to a 65% increase in mean lifespan (Friedman and Johnson, 1988Friedman D.B. Johnson T.E. A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility.Genetics. 1988; 118: 75-86Crossref PubMed Google Scholar, Paradis et al., 1999Paradis S. Ailion M. Toker A. Thomas J.H. Ruvkun G. A PDK1 homolog is necessary and sufficient to transduce AGE-1 PI3 kinase signals that regulate diapause in Caenorhabditis elegans.Genes Dev. 1999; 13: 1438-1452Crossref PubMed Scopus (314) Google Scholar). These effects have been shown to depend on the activity of DAF-16, which has similarity to a family of mammalian forkhead transcription factors (Lin et al., 1997Lin K. Dorman J.B. Rodan A. Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans.Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1155) Google Scholar). All known daf-2 mutant phenotypes are completely dependent upon DAF-16 (Kenyon et al., 1993Kenyon C. Chang J. Gensch E. Rudner A. Tabtiang R. A C. elegans mutant that lives twice as long as wild type.Nature. 1993; 366: 461-464Crossref PubMed Scopus (2298) Google Scholar, Gottlieb and Ruvkun, 1994Gottlieb S. Ruvkun G. daf-2, daf-16 and daf-23: genetically interacting genes controlling Dauer formation in Caenorhabditis elegans.Genetics. 1994; 137: 107-120Crossref PubMed Google Scholar, Larsen et al., 1995Larsen P.L. Albert P.S. Riddle D.L. Genes that regulate both development and longevity in Caenorhabditis elegans.Genetics. 1995; 139: 1567-1583Crossref PubMed Google Scholar, Dorman et al., 1995Dorman J.B. Albinder B. Shroyer T. Kenyon C. The age-1 and daf-2 genes function in a common pathway to control the lifespan of Caenorhabditis elegans.Genetics. 1995; 141: 1399-1406Crossref PubMed Google Scholar, Tissenbaum and Ruvkun, 1998Tissenbaum H.A. Ruvkun G. An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.Genetics. 1998; 148: 703-717PubMed Google Scholar, Lee et al., 2001Lee R.Y. Hench J. Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway.Curr. Biol. 2001; 11: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). A null mutation in daf-16 suppresses the phenotypes of daf-2 and age-1, indicating that the insulin/IGF-I receptor-like signaling pathway regulates aging by modulating gene expression (Hsu et al., 2003Hsu A.L. Murphy C.T. Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor.Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1033) Google Scholar, Lee et al., 2003Lee S.S. Kennedy S. Tolonen A.C. Ruvkun G. DAF-16 target genes that control C. elegans life-span and metabolism.Science. 2003; 300: 644-647Crossref PubMed Scopus (493) Google Scholar). The function of DAF-16, a transcriptional activator, is regulated by the insulin/IGF pathway. Activation of AKT by insulin/IGF results in the phosphorylation of DAF-16, and this phosphorylation event likely inhibits the nuclear translocation and transcriptional activity of DAF-16 in a DAF-2-dependent manner (Henderson and Johnson, 2001Henderson S.T. Johnson T.E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.Curr. Biol. 2001; 11: 1975-1980Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, Lee et al., 2001Lee R.Y. Hench J. Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway.Curr. Biol. 2001; 11: 1950-1957Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, Lee et al., 2003Lee S.S. Kennedy S. Tolonen A.C. Ruvkun G. DAF-16 target genes that control C. elegans life-span and metabolism.Science. 2003; 300: 644-647Crossref PubMed Scopus (493) Google Scholar, Lin et al., 2001Lin K. Hsin H. Libina N. Kenyon C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling.Nat. Genet. 2001; 28: 139-145Crossref PubMed Scopus (693) Google Scholar, Kops et al., 2002Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress.Nature. 2002; 419: 316-321Crossref PubMed Scopus (1190) Google Scholar, Hsu et al., 2003Hsu A.L. Murphy C.T. Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor.Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1033) Google Scholar). Motta et al. reported that the mammalian NAD-dependent deacetylase, SIRT1, deacetylates and represses the activity of the forkhead transcription factor Foxo3a, the mammalian homologue of DAF-16; this result suggests a similar mechanism may exist in C. elegans (Motta et al., 2004Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Mammalian SIRT1 represses forkhead transcription factors.Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). Moreover, the c-Jun N-terminal kinase (JNK) is a positive regulator of DAF-16, both for lifespan regulation and stress resistance (Oh et al., 2005Oh S.W. Mukhopadhyay A. Svrzikapa N. Jiang F. Davis R.J. Tissenbaum H.A. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.Proc. Natl. Acad. Sci. USA. 2005; 102: 4494-4499Crossref PubMed Scopus (391) Google Scholar). Thus, multiple protein kinases of the DAF-2 signaling pathway converge to negatively regulate DAF-16 activity by changing its localization within the cell. However, the exact mechanisms involved in regulating the cytoplasmic-nuclear shuttling of DAF-16 are relatively unknown. Protein ubiquitination has emerged as one of the key posttranslational regulatory mechanisms affecting many aspects of biological function, including cell-cycle control, immune response, apoptosis, and neuronal development (Nakata et al., 2005Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, Zhong et al., 2005Zhong Q. Gao W. Du F. Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis.Cell. 2005; 121: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Ubiquitin is a highly conserved 76 amino acid polypeptide that can be covalently conjugated to the lysine sites of a target protein. Ubiquitin conjugation to a substrate requires a cascade of at least three different enzymatic reactions. First, ubiquitin is activated by E1, the ubiquitin-activating enzyme. Next, ubiquitin is transiently transferred to E2, the ubiquitin-conjugating enzyme. Finally, an E3 ubiquitin ligase transfers the activated ubiquitin molecule from the E2 enzyme to a lysine residue on the substrate (reviewed by Hershko and Ciechanover, 1998Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6475) Google Scholar). Ubiquitin conjugation generally mediates protein degradation via the 26S proteasome (Pickart, 2001Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2757) Google Scholar). Recently, the systemic analysis by RNA interference of genes involved in longevity in C. elegans revealed that many genes in the ubiquitin pathway are involved in the regulation of lifespan (Hamilton et al., 2005Hamilton B. Dong Y. Shindo M. Liu W. Odell I. Ruvkun G. Lee S.S. A systematic RNAi screen for longevity genes in C. elegans.Genes Dev. 2005; 19: 1544-1555Crossref PubMed Scopus (385) Google Scholar). Here, we have identified an E3 ubiquitin ligase, RLE-1, that functions as an E3 ubiquitin ligase for DAF-16. Overexpression of RLE-1 promotes DAF-16 ubiquitination. Disruption of the rle-1 gene causes increased longevity in C. elegans, with elevated DAF-16 expression and increased DAF-16 transcriptional activity. Overexpression of DAF-16 in rle-1 mutants further increases C. elegans lifespan, while daf-16;rle-1 double mutants show a reversal of this increased longevity. Our findings indicate that RLE-1 regulates C. elegans lifespan by controlling the transcriptional activation of DAF-16 via the ubiquitin pathway. Our laboratory is currently interested in studying an E3 ubiquitin ligase, Roquin, which has been demonstrated to play important roles in autoimmunity in the mouse (Vinuesa et al., 2005Vinuesa C.G. Cook M.C. Angelucci C. Athanasopoulos V. Rui L. Hill K.M. Yu D. Domaschenz H. Whittle B. Lambe T. et al.A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity.Nature. 2005; 435: 452-458Crossref PubMed Scopus (669) Google Scholar). To investigate the functional roles of Roquin family proteins, we obtained a genetically modified C. elegans allele, cxTi510, which was generated by the Segalat laboratory (University of Lyon, France) by using the gene trap approach with the Tc5 vector (http://www.wormbase.org/db/gene/variation?name=cxTi510;class=Variation). We back crossed the rle-1 (cxTi510) animals six times with wild-type (N2) animals to eliminate any additional copies of Tc5, confirmed the genotype of the rle-1 mutants by PCR, and named this strain KB6. The exact position of the insertion of the Tc5 vector was determined (Figure S1A, see the Supplemental Data available with this article online), with DNA sequencing revealing the Tc5 transposon is inserted into the fourth exon of the rle-1 gene (Figure S1B). PCR using primers corresponding to genomic DNA from each side of the Tc5 insertion failed to detect the bands in the rle-1 mutant that are present in wildtype DNA (Figure S1A). This insertion apparently disrupts the expression of the C-terminal proline-rich region of the RLE-1 protein. We further confirmed the lack of expression of RLE-1 in rle-1 mutants by western blotting with an anti-RLE-1 antibody that we generated (Figures S2A and S2B). A 130 kDa band, which corresponds to the predicted molecular weight of the RLE-1 protein, was detected in wild-type but not in rle-1 animals (Figure S2C). These data indicate that the KB6 strain is a homozygous rle-1 mutant (rle-1−/−). Next, we analyzed the lifespan of the rle-1 mutant. Lifespans were determined at 25°C, with the day of hatching (first larval stage, L1) used as the first time point. Under the same conditions, the rle-1 mutants live much longer than wild-type (N2) animals. The average lifespan of rle-1 worms is 23.9 ± 1.2 days, while N2s have an average lifespan of 19.1 ± 1.1 days (p < 0.001) (Figure 1A and Table 1). When lifespan was analyzed beginning from the L4 larval stage, rle-1 animals still live significantly longer than N2 animals (p < 0.01) (Figure S3). These results indicate that loss of RLE-1 increases C. elegans lifespan.Table 1The Lifespan of AnimalsStrainsGenotypeNumber of Worms TestedMean Lifespan ± SEM (days)p Value against N2p Value against Specified GroupN2wild-type18319.1 ± 1.1KB6rle-1−/−16823.9 ± 1.2<0.0010.16aTJ356.TJ356daf-16::gfp11623.1 ± 0.8<0.0050.02bKB7.KB7rle-1−/−;daf-16::gfp13725.4 ± 1.6<0.001N2wild-type18319.1 ± 1.1KB6rle-1−/−16823.9 ± 1.2<0.001<0.001cCF1038.CF1038daf-16−/−10714.3 ± 0.5<0.0010.08dKB9.KB8rle-1-;daf-16−/−12515.7 ± 0.9<0.001N2wild-type7820.5 ± 1.2KB6rle-1 mutant9125.1 ± 1.4<0.001CF1308daf-16−/−8713.5 ± 0.6<0.001CF1308-HZ∗daf-16+/−5819.1 ± 0.80.13KB9rle-1−/−;daf-16+/−6322. 7 ± 0.9<0.01<0.005eCF1038-HZ.p values represent the probability that the estimated survival function of the experimental group of animals is equal to that of the control group. p values are determined by using the logrank (Mantel-Cox) statistics. p values less than 0.05 are considered statistically significant, demonstrating that the two survival functions are different. ∗CF1308-HZ, CF1308-heterozygous.a TJ356.b KB7.c CF1038.d KB9.e CF1038-HZ. Open table in a new tab p values represent the probability that the estimated survival function of the experimental group of animals is equal to that of the control group. p values are determined by using the logrank (Mantel-Cox) statistics. p values less than 0.05 are considered statistically significant, demonstrating that the two survival functions are different. ∗CF1308-HZ, CF1308-heterozygous. We also determined that rle-1 mutants have fewer progeny over a longer period of time when compared to N2s. As shown in Figure 1B, there is a 2 day delay in producing embryos for the rle-1 worms. In addition, the total numbers of embryos produced by the rle-1 mutants are reduced about 40%–50% when compared to those produced by wild-type animals. Disruption of RLE-1 expression also resulted in partial (about 20%–30%) embryonic lethality (Figure 1C). Moreover, the rle-1 mutants grow more slowly and even as adults are thinner and slightly shorter than N2s (Figure 1D). These phenotypes are similar to those observed in daf-16::gfp transgenic animals (Henderson and Johnson, 2001Henderson S.T. Johnson T.E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.Curr. Biol. 2001; 11: 1975-1980Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar), in which DAF-16 is overexpressed, suggesting that RLE-1 could regulate this signaling pathway. Previous studies have demonstrated a correlation between increased longevity and stress resistance (Kenyon et al., 1993Kenyon C. Chang J. Gensch E. Rudner A. Tabtiang R. A C. elegans mutant that lives twice as long as wild type.Nature. 1993; 366: 461-464Crossref PubMed Scopus (2298) Google Scholar, Tissenbaum and Ruvkun, 1998Tissenbaum H.A. Ruvkun G. An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.Genetics. 1998; 148: 703-717PubMed Google Scholar, Murakami and Johnson, 2001Murakami S. Johnson T.E. The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans.Curr. Biol. 2001; 11: 1517-1523Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, Lithgow and Walker, 2002Lithgow G.J. Walker G.A. Stress resistance as a determinate of C. elegans lifespan.Mech. Ageing Dev. 2002; 123: 765-771Crossref PubMed Scopus (149) Google Scholar). Because a loss of RLE-1 expression increases the lifespan of C. elegans, we hypothesized that RLE-1 might be involved in stress resistance. We therefore conducted physiological tests to compare the responses of the rle-1 mutants to those of wild-type animals when challenged with heat shock, ultraviolet (UV) damage, and pathogens. The rle-1 mutation results in significantly increased resistance both to heat shock (Figure S4A) and to UV damage (Figure S4B). RLE-1 is the homolog of the mammalian Roquin, which is involved in regulating the immune response and in autoimmunity (Vinuesa et al., 2005Vinuesa C.G. Cook M.C. Angelucci C. Athanasopoulos V. Rui L. Hill K.M. Yu D. Domaschenz H. Whittle B. Lambe T. et al.A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity.Nature. 2005; 435: 452-458Crossref PubMed Scopus (669) Google Scholar). It is therefore possible that RLE-1 is involved in the innate immune response of C. elegans. To support this hypothesis, we found rle-1 mutants survive significantly longer when exposed to the pathogenic bacterium P. aeruginosa (Figure S4C). In addition, disruption of RLE-1 expression results in partial dauer formation when these animals are grown at 25°C (Figure S4D). Thus, the physiological evidence supports an involvement of RLE-1 in multiple responses to stress in C. elegans. Mutation of DAF-2, the sole insulin/IGF-1 receptor, negatively regulates DAF-16 transcriptional activation and causes longevity and stress resistance (Riddle, 1977Riddle D.L. A genetic pathway for dauer larva formation in C. elegans.Stadler Genet. Symp. 1977; 9: 101-120Google Scholar, Kenyon et al., 1993Kenyon C. Chang J. Gensch E. Rudner A. Tabtiang R. A C. elegans mutant that lives twice as long as wild type.Nature. 1993; 366: 461-464Crossref PubMed Scopus (2298) Google Scholar, Murakami and Johnson, 2001Murakami S. Johnson T.E. The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans.Curr. Biol. 2001; 11: 1517-1523Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Collectively, our results suggest that RLE-1 may be involved in crosstalk with the insulin/IGF pathway in regulating lifespan and stress resistance in C. elegans. The forkhead family transcription factor DAF-16 plays important roles in regulating lifespan in C. elegans. Overexpression of a fusion protein of DAF-16 tagged with GFP increases the lifespan of transgenic animals (Henderson and Johnson, 2001Henderson S.T. Johnson T.E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.Curr. Biol. 2001; 11: 1975-1980Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). We thus asked whether rle-1 worms have elevated DAF-16 expression. As shown in Figure 2A, more DAF-16 was detected in the rle-1 mutant (lanes 2 and 4) than in N2 wild-type lysates (lanes 1 and 3). As a loading control, the protein levels of Actin were comparable in these lysates. Quantification of the densities of DAF-16 bands from three independent experiments indicated that the protein level of DAF-16 in rle-1 mutants was significantly higher than that in N2 animals (Figure 2B), while the mRNA levels of daf-16 were comparable between rle-1 mutants and wild-type animals, as determined both by RT-PCR (Figure 2C) and real-time quantification PCR (Figure 2D). These results indicate that a mutation in the gene that encodes RLE-1 causes increased protein but not increased mRNA for DAF-16, suggesting that RLE-1 is involved in the longevity of C. elegans through regulation of DAF-16 expression at the posttranscriptional level. To determine the physiological function of RLE-1, we cloned RLE-1 from C. elegans by RT-PCR. The full-length rle-1 cDNA encodes a 1,014 amino acid protein (Figure S5A). The RLE-1 protein contains an N-terminal RING finger, indicative of an E3 ubiquitin ligase. RLE-1 also has an ROQ domain, which has high homology with the mammalian Roquin (Figure S5). The functions of the ROQ domain are not known; however, it is predicted to mediate protein-protein interactions. The C terminus of RLE-1 contains a long proline-rich region, a motif which has also been demonstrated to mediate protein-protein interactions. Moreover, RLE-1 contains two coiled-coil domains, motifs which often function as protein-protein interaction domains (Newman and Keating, 2003Newman J.R. Keating A.E. Comprehensive identification of human bZIP interactions with coiled-coil arrays.Science. 2003; 300: 2097-2101Crossref PubMed Scopus (368) Google Scholar). To investigate the physiological roles of RLE-1 in regulating DAF-16 expression, we examined whether RLE-1 interacts with DAF-16. DAF-16 and Flag-tagged RLE-1 expression plasmids were cotransfected into HEK293 cells. RLE-1 was detected by coimmunoprecipitation and western blotting when it was coexpressed with DAF-16 but not in the control lane of RLE-1 alone, suggesting that DAF-16 can interact with RLE-1 (Figure 3A). More interestingly, RLE-1 was detected in the anti-DAF-16 immunoprecipitate when lysates of wild-type worms but not of rle-1 mutants were used (Figure 3B). These results indicate that RLE-1 interacts with DAF-16 in vivo. To support this conclusion, we further demonstrated by confocal microscopy that RLE-1 colocalizes with DAF-16 in C. elegans (Figure S6A) as well as in HEK293 cells (Figure S6B). Since the rle-1 mutation likely results in a deletion of the C-terminal 491 amino acids of RLE-1, we hypothesized that the C-terminal region of RLE-1 is important for its interaction with DAF-16. Analysis of the cDNA sequence of rle-1 revealed a convenient restriction enzyme site, Kpn I, by which a truncated RLE-1 was generated that removed the C-terminal 473 amino acids (RLE-1/ΔC). RLE-1/ΔC is similar in length (586 aa) to the size of the truncated RLE-1 protein that could be produced in the rle-1 mutants (558 aa). As shown in Figure 3C, deletion of the C terminus of RLE-1 completely abolished its interaction with DAF-16, indicating the RLE-1 C terminus is required for the interaction of RLE-1 with DAF-16. Since RLE-1 contains a RING finger, which has been demonstrated as an E3 ubiquitin ligase domain (Zheng et al., 2000Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar), we proposed that RLE-1 regulates DAF-16 expression via its E3 activity. To test this hypothesis, an in vivo ubiquitination assay was performed (Gao et al., 2006Gao B. Lee S.M. Fang D. The tyrosine kinase c-Abl protects c-Jun from ubiquitination-mediated degradation in T cells.J. Biol. Chem. 2006; 281: 29711-29718Crossref PubMed Scopus (39) Google Scholar). When DAF-16 was coexpressed with HA-Ub and RLE-1, higher molecular weight ladders were detected in the anti-DAF-16 immunoprecipitates, which indicates that DAF-16 is modified by ubiquitin (Figure 4A, lane 2). Treatment of these transfected HEK293 cells with the proteasome inhibitor, MG132 (Saito et al., 1992Saito Y. Tsubuki S. Ito H. Ohmi-Imajo S. Kawashima S. Possible involvement of clathrin in neuritogenesis induced by a protease inhibitor (benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) in PC12 cells.J. Biochem. (Tokyo). 1992; 112: 448-455Crossref PubMed Scopus (6) Google Scholar), significantly increased the ubiquitination of DAF-16 (Figure 4A, lane 3). These results suggest that RLE-1 is an E3 ubiquitin ligase of DAF-16 and that the proteasome pathway is involved in DAF-16 degradation. A direct interaction is required for RLE-1-mediated DAF-16 ubiquitination because overexpression of RLE-1/ΔC failed to induce DAF-16 ubiquitination (Figure 4B). In addition, a functional RING finger, which binds to ubiquitin-carrying E2s (Zheng et al., 2000Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar), is also required for RLE-1-mediated ubiquitin conjugation of DAF-16. Mutation of the active cysteine site in the RING finger, RLE-1/C34A, essentially abolished its E3 ubiquitin ligase activity for DAF-16 ubiquitination (Figure 4C) without affecting the interaction of RLE-1 with DAF-16 (Figure 4D). These results collectively demonstrate that RLE-1 is an E3 ubiquitin ligase for the C. elegans DAF-16. The fact that the protein but not the mRNA level of daf-16 is increased in rle-1 mutants suggests that RLE-1 is involved in the protein degradation of DAF-16. To test this hypothesis, we found that coexpression of RLE-1 and DAF-16 in cultured cells dramatically accelerated the degradation of DAF-16 by shortening its half-life to about 1.2 hours, down from 2.3 hours for DAF-16 alone (Figure 5A). Overexpression of RLE-1 even reduces the levels of DAF-16 protein when less DAF-16 plasmid is cotransfected (Figure S7). These results indicate that RLE-1 induces DAF-16 degradation. Ubiquitination can mediate protein degradation via either the proteasome or the lysosome pathway, or both (reviewed by Hershko and Ciechanover, 1998Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6475) Google Scholar). Our findings that RLE-1-mediated ubiquitination of DAF-16 is increased in the presence of the proteasomal inhibitor, MG132 (Figure 4A), suggest that ubiquitination may induce DAF-16 degradation via the proteasome pathway. The protein stability of DAF-16 was thus analyzed in the presence of MG132. Treatment of cells with MG132 prevented RLE-1-mediated degradation of DAF-16 (Figure 5A, right panel). These results suggest that RLE-1 induces DAF-16 degradation via the proteasome pathway. We next examined how RLE-1-mediated ubiquitination regulates the transcriptional activation of DAF-16. To determine the physiological functions of RLE-1-mediated DAF-16 ubiquitination in C. elegans, we compared the transcriptional activity of DAF-16 between wild-type worms and rle-1 mutants. Several target genes of DAF-16 have been identified in C. elegans, including sod-3 and mlt-1 (Essers et al., 2005Essers M.A. de Vries-Smits L.M. Barker N. Polderman P.E. Burgering B.M. Korswagen H.C. Functional interaction between beta-catenin and FOXO in oxidative stress signaling.Science. 2005; 308: 1181-1184Crossref PubMed Scopus (560) Google Scholar). These target genes play important roles in regulating the lifespan of C. elegans. Under normal conditions with plenty of food, both sod-3 and mlt-1 are expressed at very low levels in N2s, as demonstrated by the weak bands corresponding to sod-3 and mlt-1 detected after 40 cycles of PCR amplification. In contrast, the mRNA levels of both sod-3 and mlt-1 were dramatically increased in rle-1 mutants (Figure 5B). As controls, the mRNA levels of both actin and daf-16 were comparable between N2 worms and rle-1 mutants (Figure 5B, bottom panel, Figure 2C, and Figure S2D). Results from the real-time PCR experiments indicated that sod-3 transcript levels are at least four-fold higher in rle-1 mutants than in wild-type (Figure 5C). These results indicate that in C. elegans RLE-1 functions as a negative regulator of DAF-16 transcriptional activation. In the AKT pathway, activation by growth factors induces DAF-16 phosphorylation and inhibits DAF-16 nuclear translocation, while stress or withdrawal of growth factors triggers the dephosphorylation and relocalization of DAF-16 to the nucleus (Lin et al., 1997Lin K. Dorman J.B. Rodan A. Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans.Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1155) Google Scholar). Some evidence has shown that nuclear DAF-16 is ex" @default.
• W2079180838 created "2016-06-24" @default.
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• W2079180838 title "RLE-1, an E3 Ubiquitin Ligase, Regulates C. elegans Aging by Catalyzing DAF-16 Polyubiquitination" @default.
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