A Functional Interaction between Dorsal and Components of the Smt3 Conjugation Machinery
2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês
10.1074/jbc.275.6.4033
ISSN1083-351X
AutoresVinay Bhaskar, Scott A. Valentine, Albert J. Courey,
Tópico(s)Genomics and Chromatin Dynamics
ResumoTo identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodesDrosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation. To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodesDrosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation. polymerase chain reaction glutathione S-transferase open reading frame green fluorescent protein expressed sequence tag polyacrylamide gel electrophoresis hemagglutinin ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase Dorsoventral patterning of the Drosophila embryo is initiated by Dorsal, a member of the Rel family of transcription factors. This maternally expressed factor is distributed in a nuclear concentration gradient in the blastoderm embryo (1.Drier E.A. Steward R. Semin. Cancer Biol. 1997; 8: 83-92Crossref PubMed Scopus (47) Google Scholar) and functions as an activator and repressor of transcription to establish multiple domains of zygotic gene activity, thereby subdividing the embryo into several discrete domain along its dorsoventral axis (2.Courey A.J. Huang J.D. Biochim. Biophys. Acta. 1995; 1261: 1-18Crossref PubMed Scopus (36) Google Scholar, 3.Ray R.P. Arora K. Nüsslein-Volhard C. Gelbart W.M. Development. 1991; 113: 35-54Crossref PubMed Google Scholar).Dorsal-binding proteins direct the spatially regulated nuclear import of Dorsal via an evolutionarily conserved signal transduction pathway, which targets Dorsal and its cytoplasmic inhibitor Cactus (4.Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar, 5.Govind S. Steward R. Trends Genet. 1991; 7: 119-125Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 6.Yang J. Steward R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14524-14529Crossref PubMed Scopus (32) Google Scholar). Prior to activation of the pathway, an interaction between Cactus and Dorsal serves to retain Dorsal in the cytoplasm. Activation of the pathway by a signal originating in the ventral extraembryonic space results in phosphorylation of Cactus and Dorsal. The phosphorylation of Cactus triggers its degradation via the ubiquitin-proteasome pathway, freeing Dorsal to enter the nucleus (7.Reach M. Galindo R.L. Towb P. Allen J.L. Karin M. Wasserman S.A. Dev. Biol. 1996; 180: 353-364Crossref PubMed Scopus (100) Google Scholar), whereas the phosphorylation of Dorsal is required to render the factor competent for efficient nuclear uptake (8.Drier E.A. Huang L.H. Steward R. Genes Dev. 1999; 13: 556-568Crossref PubMed Scopus (79) Google Scholar).A very similar pathway involving the vertebrate homolog of Cactus, IκB is involved in the regulated nuclear import of vertebrate Rel family proteins, such as NFκB (9.Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Crossref PubMed Scopus (902) Google Scholar, 10.Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4570) Google Scholar). In addition, recent studies suggest that the nuclear import of NFκB may be influenced by the Smt3 conjugation pathway (11.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). Smt3 is a small ubiquitin-like protein that can be enzymatically conjugated to various protein substrates via an amide linkage between the C-terminal carboxyl group of Smt3 and a lysine ε-amino group on the target protein. Conjugation of mammalian SMT3C to IκB is thought to stabilize IκB by blocking ubiquitylation and therefore subsequent proteasomal degradation. By stabilizing IκB, the vertebrate Smt3 conjugation pathway results in the down-regulation of NFκB activity.In an effort to illuminate further the mechanisms by which Dorsal activity is regulated, we sought to identify novel Dorsal interacting proteins via a yeast two-hybrid screen. One of the proteins identified in this screen was Drosophila Ubc9 (DmUbc9) (12.Joanisse D.R. Inaguma Y. Tanguay R.M. Biochem. Biophys. Res. Commun. 1998; 244: 102-109Crossref PubMed Scopus (44) Google Scholar). This protein is homologous to yeast and mammalian Ubc9, which are thought to function as Smt3-conjugating enzymes (13.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14.Schwarz S.E. Matuschewski K. Liakopoulos D. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (188) Google Scholar, 15.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar). Experiments examining the effect of DmUbc9 on Dorsal nuclear uptake and Dorsal-mediated transcriptional activation demonstrate that the effect of the Smt3 conjugation pathway on Dorsal activity is opposite to the effect of this pathway on the activity of the vertebrate Rel family protein NFκB. In particular, we find that DmUbc9 conjugatesDrosophila Smt3 (DmSmt3) to Dorsal and overcomes Cactus-dependent sequestration of Dorsal in the cytoplasm. Furthermore, we find that the effects of DmUbc9 on Dorsal activity are enhanced by overexpression of DmSmt3 and a DmSmt3-activating enzyme. Thus, the Smt3 conjugation pathway enhances Dorsal nuclear translocation.DISCUSSIONIn this study, we demonstrate that DmUbc9, an Smt3-conjugating enzyme (13.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14.Schwarz S.E. Matuschewski K. Liakopoulos D. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (188) Google Scholar, 15.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar), is a Dorsal-interacting protein. We show that DmUbc9 catalyzes the conjugation of DmSmt3 to Dorsal and opposes the inhibitory effect of Cactus on Dorsal nuclear uptake. We also report the identification of DmSAE1/DmSAE2, the Drosophila homolog of the Smt3-activating enzyme. We further show that DmSmt3, DmSAE1/DmSAE2, and DmUbc9 function synergistically to stimulate the activity of a Dorsal-responsive reporter gene. These findings suggest that the Smt3 conjugation pathway enhances Dorsal activity by enhancing its nuclear uptake. Although Ubc9 family proteins have been found to interact with a variety of transcription factors (33.Tashiro K. Pando M.P. Kanegae Y. Wamsley P.M. Inoue S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7862-7867Crossref PubMed Scopus (44) Google Scholar, 38.Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 39.Loveys D.A. Streiff M.B. Schaefer T.S. Kato G.J. Gene (Amst .). 1997; 201: 169-177Crossref PubMed Scopus (18) Google Scholar), to our knowledge, this is the first study to show that Ubc9 can actually conjugate Smt3 to a sequence-specific transcription factor and enhance its activity. Furthermore, our finding that the effects of DmUbc9 on Dorsal activity are potentiated by DmSmt3 and a DmSmt3-activating enzyme provides some of the best evidence to date that Smt3 conjugation is directly involved in regulating transcription factor activity.Smt3 Conjugation as an Evolutionarily Conserved Pathway for the Regulation of Subcellular LocalizationSmt3 homologs have been cloned from eukaryotes as diverse as yeast, Arabidopsis, and humans (16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar, 35.Huang H.W. Tsoi S.C. Sun Y.H. Li S.S. Biochem. Mol. Biol. Int. 1998; 46: 775-785PubMed Google Scholar, 40.Mannen H. Tseng H.M. Cho C.L. Li S.S. Biochem. Biophys. Res. Commun. 1996; 222: 178-180Crossref PubMed Scopus (50) Google Scholar, 41.Qi F. Ridpath J.F. Berry E.S. Virus Res. 1998; 57: 1-9Crossref PubMed Scopus (47) Google Scholar). These proteins in general display greater than 50% identity with one another but also roughly 20% identity with ubiquitin. The identification of the components of the Smt3 conjugation pathway in yeast, humans, and now Drosophila has revealed that Smt3 conjugation and ubiquitin conjugation proceed by similar pathways (16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar, 37.Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 42.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Both pathways require an activating enzyme, or E1 protein, which becomes covalently attached to ubiquitin or Smt3 via a high energy thioester bond, and a conjugating enzyme, or E2 protein, which accepts ubiquitin or Smt3 from the E1 protein forming a second thioester-linked covalent complex. Ubiquitin or Smt3 is then transferred to an ε-amino group on a final protein substrate. The transfer of ubiquitin from the E2 protein to the final substrate often requires a ubiquitin ligase, or E3 protein. In contrast, an E3-type protein is apparently not required for Smt3 conjugation.Although ubiquitin conjugation targets proteins for proteasomal degradation (42.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar), Smt3 conjugation appears to serve other purposes. Originally identified in yeast as an enzyme required for proper cell cycle progression, Ubc9 has been found to physically interact with a diverse array of proteins including RanGAP1, PML (promyelocytic leukemia protein), bleomycin hydrolase, E2A, androgen receptor, and c-Rel (11.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar, 33.Tashiro K. Pando M.P. Kanegae Y. Wamsley P.M. Inoue S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7862-7867Crossref PubMed Scopus (44) Google Scholar, 38.Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 39.Loveys D.A. Streiff M.B. Schaefer T.S. Kato G.J. Gene (Amst .). 1997; 201: 169-177Crossref PubMed Scopus (18) Google Scholar,43.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 44.Müller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar). Association of human Ubc9 with RanGAP1 results in the conjugation of RanGAP1 to the Smt3 homolog SMT3C/SUMO-1 (small ubiquitin-related modifier), allowing it to bind RanBP2 at the nuclear periphery. This allows RanGAP1 to stimulate GTP hydrolysis by Ran. Only SUMO-1-conjugated RanGAP1 binds to RanBP2, implying that SMT3C and Ubc9 are required for nuclear import. In the case of PML, interaction with Ubc9 and subsequent SUMO-1 conjugation is essential for targeting PML to discreet subnuclear structures known as PML-bodies or nuclear dots. In acute promyelocytic leukemia cells, the subnuclear localization of PML is altered, suggesting that improper SUMO-1 conjugation may trigger oncogenesis. These studies argue that one function of Smt3 conjugation is to regulate the subcellular localization of proteins.Roles for Smt3 Conjugation in the Regulation of Rel Family Protein ActivityAlthough we have found a possible role for Smt3 conjugation in regulating Dorsal activity, a number of reports have implicated Ubc9 in the modulation of transcriptional activation by other Rel family proteins. For example, a recent study showed that SUMO-1-conjugated IκB is resistant to degradation and, accordingly, that SUMO-1 and Ubc9 work together to inhibit activation of an NFκB-dependent reporter (11.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). This contrasts with our findings showing that the Smt3 conjugation pathway activates Dorsal-dependent reporters. This difference could relate to inherent differences between the NFκB/IκB and Dorsal/Cactus pathways. However, an earlier report (33.Tashiro K. Pando M.P. Kanegae Y. Wamsley P.M. Inoue S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7862-7867Crossref PubMed Scopus (44) Google Scholar) suggests that mammalian Ubc9 can enhance Rel protein function via an interaction with NFκB and/or IκB. Thus, an alternative explanation for the different effects of Smt3 conjugation on Rel protein activity could be that different Smt3 family proteins have different functions. An alignment of DmSmt3 with the three members of the human SMT3 family (Fig. 5 A) reveals that DmSmt3 displays significantly higher homology to SMT3A and SMT3B (77 and 75%, respectively) than to SMT3C/SUMO-1 (55%). Thus, DmSmt3, SMT3A, and SMT3B appear to define an Smt3 subfamily that is distinct from SMT3C/SUMO-1. Perhaps SMT3C/SUMO-1 antagonizes transcriptional activation by Rel proteins, whereas SMT3A/B-like proteins (such as DmSmt3) enhance Rel protein function.The Smt3 conjugation system may also function at other levels in the regulation of Rel family protein activity. For example, Ubc9 has been shown to associate with the type I TNFα receptor and MEKK1 and to synergize with MEKK1 to activate an NFκB-dependent reporter (45.Saltzman A. Searfoss G. Marcireau C. Stone M. Ressner R. Munro R. Franks C. D'Alonzo J. Tocque B. Jaye M. Ivashchenko Y. FEBS Lett. 1998; 425: 431-435Crossref PubMed Scopus (21) Google Scholar).Although we were able to detect a DmSmt3-Dorsal conjugate in cells that were simultaneously co-transfected with Dorsal, DmUbc9, and DmSmt3, the level of conjugation was low: no more than about 10% of the Dorsal protein was found in the DmSmt3-conjugated form. This contrasts with the results of our experiments looking at the localization of a Dorsal-GFP fusion protein, in which we found that DmUbc9 was able to direct the relocalization of a large fraction of the Dorsal-GFP in these cells from the cytoplasm to the nucleus. This suggests that the conjugation of DmSmt3 to Dorsal may be transient. Perhaps Dorsal and DmSmt3 are deconjugated as soon as Dorsal enters the nucleus. In accord with this idea, recent observations suggest that a dynamic equilibrium may exist between Smt3-conjugated and unconjugated protein species. In yeast, the vast majority of cellular Smt3p is conjugated to other proteins, although the population of proteins that is covalently modified changes during the cell cycle. Furthermore, a yeast enzyme capable of catalyzing the deconjugation reaction has been identified, and homologs of this enzyme appear to exist in many other eukaryotic species (46.Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (601) Google Scholar).A genetically defined locus, termed semushi was recently found to be identical with DmUbc9. Experiments employing thesemushi allele suggest that DmUbc9 may be necessary for the nuclear import of the anteroposterior patterning morphogen Bicoid (24.Epps J.L. Tanda S. Curr. Biol. 1998; 8: 1277-1280Abstract Full Text Full Text PDF PubMed Google Scholar). This study was silent about potential roles of the Smt3 conjugation pathway in other developmental processes. However, a recent preliminary analysis of embryos lacking maternally supplied DmUbc9 indicates the presence of multiple patterning defects of varying penetrance. 2V. Bhaskar and M. Smith, unpublished data. Because of the complex nature of these defects, their characterization will require extensive phenotypic analysis and the generation of additional DmUbc9 alleles. The possibility that DmUbc9 has pleiotropic developmental roles is not surprising given increasing evidence for wide spread roles of Smt3 conjugation in transcription factor function and in the targeting of proteins to their proper subcellular locales.Note Added in ProofSince the submission of this paper, Smt3/SUMO-1 modification has been reported to activate the transcriptional response of p53 (Gostissa, M., Hengstermann, A., Fogal, V., Sandy, P., Schwarz, S. E., Scheffner, M., and Del Sal, G. (1999)EMBO J. 18, 6462–6471; Rodriguez, M. S., Desterro, J. M., Lain, S., Midgley, C. A., Lane, D. P., and Hay, R. T. (1999)EMBO J. 18, 6455–6461). Dorsoventral patterning of the Drosophila embryo is initiated by Dorsal, a member of the Rel family of transcription factors. This maternally expressed factor is distributed in a nuclear concentration gradient in the blastoderm embryo (1.Drier E.A. Steward R. Semin. Cancer Biol. 1997; 8: 83-92Crossref PubMed Scopus (47) Google Scholar) and functions as an activator and repressor of transcription to establish multiple domains of zygotic gene activity, thereby subdividing the embryo into several discrete domain along its dorsoventral axis (2.Courey A.J. Huang J.D. Biochim. Biophys. Acta. 1995; 1261: 1-18Crossref PubMed Scopus (36) Google Scholar, 3.Ray R.P. Arora K. Nüsslein-Volhard C. Gelbart W.M. Development. 1991; 113: 35-54Crossref PubMed Google Scholar). Dorsal-binding proteins direct the spatially regulated nuclear import of Dorsal via an evolutionarily conserved signal transduction pathway, which targets Dorsal and its cytoplasmic inhibitor Cactus (4.Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar, 5.Govind S. Steward R. Trends Genet. 1991; 7: 119-125Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 6.Yang J. Steward R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14524-14529Crossref PubMed Scopus (32) Google Scholar). Prior to activation of the pathway, an interaction between Cactus and Dorsal serves to retain Dorsal in the cytoplasm. Activation of the pathway by a signal originating in the ventral extraembryonic space results in phosphorylation of Cactus and Dorsal. The phosphorylation of Cactus triggers its degradation via the ubiquitin-proteasome pathway, freeing Dorsal to enter the nucleus (7.Reach M. Galindo R.L. Towb P. Allen J.L. Karin M. Wasserman S.A. Dev. Biol. 1996; 180: 353-364Crossref PubMed Scopus (100) Google Scholar), whereas the phosphorylation of Dorsal is required to render the factor competent for efficient nuclear uptake (8.Drier E.A. Huang L.H. Steward R. Genes Dev. 1999; 13: 556-568Crossref PubMed Scopus (79) Google Scholar). A very similar pathway involving the vertebrate homolog of Cactus, IκB is involved in the regulated nuclear import of vertebrate Rel family proteins, such as NFκB (9.Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Crossref PubMed Scopus (902) Google Scholar, 10.Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4570) Google Scholar). In addition, recent studies suggest that the nuclear import of NFκB may be influenced by the Smt3 conjugation pathway (11.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). Smt3 is a small ubiquitin-like protein that can be enzymatically conjugated to various protein substrates via an amide linkage between the C-terminal carboxyl group of Smt3 and a lysine ε-amino group on the target protein. Conjugation of mammalian SMT3C to IκB is thought to stabilize IκB by blocking ubiquitylation and therefore subsequent proteasomal degradation. By stabilizing IκB, the vertebrate Smt3 conjugation pathway results in the down-regulation of NFκB activity. In an effort to illuminate further the mechanisms by which Dorsal activity is regulated, we sought to identify novel Dorsal interacting proteins via a yeast two-hybrid screen. One of the proteins identified in this screen was Drosophila Ubc9 (DmUbc9) (12.Joanisse D.R. Inaguma Y. Tanguay R.M. Biochem. Biophys. Res. Commun. 1998; 244: 102-109Crossref PubMed Scopus (44) Google Scholar). This protein is homologous to yeast and mammalian Ubc9, which are thought to function as Smt3-conjugating enzymes (13.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14.Schwarz S.E. Matuschewski K. Liakopoulos D. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (188) Google Scholar, 15.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar). Experiments examining the effect of DmUbc9 on Dorsal nuclear uptake and Dorsal-mediated transcriptional activation demonstrate that the effect of the Smt3 conjugation pathway on Dorsal activity is opposite to the effect of this pathway on the activity of the vertebrate Rel family protein NFκB. In particular, we find that DmUbc9 conjugatesDrosophila Smt3 (DmSmt3) to Dorsal and overcomes Cactus-dependent sequestration of Dorsal in the cytoplasm. Furthermore, we find that the effects of DmUbc9 on Dorsal activity are enhanced by overexpression of DmSmt3 and a DmSmt3-activating enzyme. Thus, the Smt3 conjugation pathway enhances Dorsal nuclear translocation. DISCUSSIONIn this study, we demonstrate that DmUbc9, an Smt3-conjugating enzyme (13.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14.Schwarz S.E. Matuschewski K. Liakopoulos D. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (188) Google Scholar, 15.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar), is a Dorsal-interacting protein. We show that DmUbc9 catalyzes the conjugation of DmSmt3 to Dorsal and opposes the inhibitory effect of Cactus on Dorsal nuclear uptake. We also report the identification of DmSAE1/DmSAE2, the Drosophila homolog of the Smt3-activating enzyme. We further show that DmSmt3, DmSAE1/DmSAE2, and DmUbc9 function synergistically to stimulate the activity of a Dorsal-responsive reporter gene. These findings suggest that the Smt3 conjugation pathway enhances Dorsal activity by enhancing its nuclear uptake. Although Ubc9 family proteins have been found to interact with a variety of transcription factors (33.Tashiro K. Pando M.P. Kanegae Y. Wamsley P.M. Inoue S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7862-7867Crossref PubMed Scopus (44) Google Scholar, 38.Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 39.Loveys D.A. Streiff M.B. Schaefer T.S. Kato G.J. Gene (Amst .). 1997; 201: 169-177Crossref PubMed Scopus (18) Google Scholar), to our knowledge, this is the first study to show that Ubc9 can actually conjugate Smt3 to a sequence-specific transcription factor and enhance its activity. Furthermore, our finding that the effects of DmUbc9 on Dorsal activity are potentiated by DmSmt3 and a DmSmt3-activating enzyme provides some of the best evidence to date that Smt3 conjugation is directly involved in regulating transcription factor activity.Smt3 Conjugation as an Evolutionarily Conserved Pathway for the Regulation of Subcellular LocalizationSmt3 homologs have been cloned from eukaryotes as diverse as yeast, Arabidopsis, and humans (16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar, 35.Huang H.W. Tsoi S.C. Sun Y.H. Li S.S. Biochem. Mol. Biol. Int. 1998; 46: 775-785PubMed Google Scholar, 40.Mannen H. Tseng H.M. Cho C.L. Li S.S. Biochem. Biophys. Res. Commun. 1996; 222: 178-180Crossref PubMed Scopus (50) Google Scholar, 41.Qi F. Ridpath J.F. Berry E.S. Virus Res. 1998; 57: 1-9Crossref PubMed Scopus (47) Google Scholar). These proteins in general display greater than 50% identity with one another but also roughly 20% identity with ubiquitin. The identification of the components of the Smt3 conjugation pathway in yeast, humans, and now Drosophila has revealed that Smt3 conjugation and ubiquitin conjugation proceed by similar pathways (16.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar, 37.Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 42.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Both pathways require an activating enzyme, or E1 protein, which becomes covalently attached to ubiquitin or Smt3 via a high energy thioester bond, and a conjugating enzyme, or E2 protein, which accepts ubiquitin or Smt3 from the E1 protein forming a second thioester-linked covalent complex. Ubiquitin or Smt3 is then transferred to an ε-amino group on a final protein substrate. The transfer of ubiquitin from the E2 protein to the final substrate often requires a ubiquitin ligase, or E3 protein. In contrast, an E3-type protein is apparently not required for Smt3 conjugation.Although ubiquitin conjugation targets proteins for proteasomal degradation (42.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar), Smt3 conjugation appears to serve other purposes. Originally identified in yeast as an enzyme required for proper cell cycle progression, Ubc9 has been found to physically interact with a diverse array of proteins including RanGAP1, PML (promyelocytic leukemia protein), bleomycin hydrolase, E2A, androgen receptor, and c-Rel (11.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar, 33.Tashiro K. Pando M.P. Kanegae Y. Wamsley P.M. Inoue S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7862-7867Crossref PubMed Scopus (44) Google Scholar, 38.Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 39.Loveys D.A. Streiff M.B. Schaefer T.S. Kato G.J. Gene (Amst .). 1997; 201: 169-177Crossref PubMed Scopus (18) Google Scholar,43.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 44.Müller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar). Association of human Ubc9 with RanGAP1 results in the conjugation of RanGAP1 to the Smt3 homolog SMT3C/SUMO-1 (small ubiquitin-related modifier), allowing it to bind RanBP2 at the nuclear periphery. This allows RanGAP1 to stimulate GTP hydrolysis by Ran. Only SUMO-1-conjugated RanGAP1 binds to RanBP2, implying that SMT3C and Ubc9 are required for nuclear import. In the case of PML, interaction with Ubc9 and subsequent SUMO-1 conjugation is essential for targeting PML to discreet subnuclear structures known as PML-bodies or nuclear dots. In acute promyelocytic leukemia cells, the subnuclear localization of PML is altered, suggesting that improper SUMO-1 conjugation may trigger oncogenesis. These studies argue that one function of Smt3 conjugation is to regulate the subcellular localization of proteins.Roles for Smt3 Conjugation in the Regulation of Rel Family Protein ActivityAlthough we have found a possible role for Smt3 conjugation in regulating Dorsal activity, a number of reports have implicated Ubc9 in the modulation of transcriptional activation by other Rel family proteins. For example, a recent study showed that SU
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