Preferential Interaction of Sentrin with a Ubiquitin-conjugating Enzyme, Ubc9

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001–14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A β-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway. Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001–14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A β-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway. Sentrin is a 101-amino acid ubiquitin-like protein that interacts with the death domains of Fas and TNFR1 1The abbreviations used are: TNFR1, tumor necrosis factor receptor 1; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PCR, polymerase chain reaction. (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar), with PML, a tumor suppressor implicated in the pathogenesis of promyelocytic leukemia (2Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google Scholar), with Rad51 and Rad52, proteins that are involved in repairing double-stranded DNA breaks (3Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 36: 271-279Crossref PubMed Scopus (183) Google Scholar), and with RanGAP1, a GTPase-activating protein that is critically involved in nuclear protein transport (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar,5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (957) Google Scholar). Using the COS cell expression system, we have shown that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (6Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Moreover, sentrinized proteins appear to reside in the nucleus. We have also shown that the C-terminal four amino acids of sentrin, His-Ser-Thr-Val, were efficiently cleaved to allow the conjugation of sentrin to other proteins via the conserved Gly97 residue. Thus, the biochemistry of sentrinization pathway appears to be remarkably similar to that of the ubiquitination pathway. Conjugation of ubiquitin to its target protein requires the initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1 (7Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-233Crossref PubMed Scopus (784) Google Scholar, 8Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 9Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1205) Google Scholar, 10Wilkinson K.D. Ann. Rev. Nutr. 1995; 15: 161-189Crossref PubMed Scopus (133) Google Scholar, 11Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2234) Google Scholar). An intermediate, ubiquitin adenylate, is formed by displacement of PPi from ATP and ubiquitin adenylate is then transferred to a thiol site in E1 with release of AMP. Through transacylation, ubiquitin is transferred to a ubiquitin-conjugating enzyme, E2, to form another thiol ester bond. Finally, ubiquitin is transferred from E2 to its target protein through an isopeptide linkage with the ε-amino group of the Lys residue of the target protein. The transfer of ubiquitin from E2 to the target protein requires the participation of a ligase, E3, in many instances. In recent years, ubiquitination has been shown to play a critical role in antigen processing, in the regulation of cell cycle, in receptor endocytosis, and in signal transduction (12Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 13Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2196) Google Scholar, 14Murray A. Cell. 1995; 81: 149-152Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 15Hopkin K. J. Natl. Inst. Health Res. 1997; 9: 36-42Google Scholar, 16Cenciaelli C. Wilhelm K.G. Guo A. Weissman A.M. J. Biol. Chem. 1995; 271: 8709-8713Abstract Full Text Full Text PDF Scopus (72) Google Scholar, 17Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (616) Google Scholar, 18Kim T.K. Maniatis T. Science. 1996; 273: 1717-1720Crossref PubMed Scopus (361) Google Scholar). The biological specificity of the ubiquitination pathway appears to be regulated by a selective combination of E2 and E3 proteins (15Hopkin K. J. Natl. Inst. Health Res. 1997; 9: 36-42Google Scholar). Thus far, there are more than 30 E2s identified with the conserved UBC domain that contain a cysteine residue at the active site (7Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-233Crossref PubMed Scopus (784) Google Scholar, 8Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 9Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1205) Google Scholar). However, the molecular basis of this specificity has not been completely elucidated. Since the sentrinization pathway shares many similarities with the ubiquitination pathway, it is of interest to identify specific E2 or E3 proteins that may be involved the sentrinization pathway. Using sentrin as a bait in the yeast two-hybrid screen, we identified a ubiquitin-conjugating enzyme, Ubc9, which binds to sentrin with high affinity. The interaction between sentrin and Ubc9 is much stronger than that between ubiquitin and Ubc9 or between sentrin and three other E2s. Furthermore, the conserved C-terminal Gly-Gly residues of sentrin are required for the high affinity interaction, suggesting that sentrin could form a thiol ester bond with Ubc9. This is further substantiated by an in vitro binding assay in which a β-mercaptoethanol-sensitive sentrin-Ubc9 conjugate was observed. Thus, Ubc9 appears to be a key conjugating enzyme for the sentrinization pathway. Yeast strains, SFY526 and HF7c, and the shuttle vectors pGBT9 and pGAD424 were purchased fromCLONTECH. The bait plasmid pGBT9-sentrin was constructed by inserting the full-length sentrin cDNA in-frame in the BamHI site of the pGBT9 vector. Deletion mutants were produced by a polymerase chain reaction-based strategy with appropriate primer sets. These inserts contained a BamHI linker at the 5′-end and a stop codon and a PstI linker at the 3′-end. The PCR products were digested with both BamHI andPstI and then were ligated into the yeast two-hybrid vectors that had been predigested with the same enzymes. The full-length or truncated human Ubc9 constructs were made by PCR amplification with appropriate primers from a plasmid containing the UBC9cDNA and subcloned into pGAD424. The E2 cDNAs were amplified by PCR from a human placenta cDNA library. The substitution mutant of Ubc9(C93S) was generated using QuikChange site-directed mutagenesis kit (Stratagene). The sequences of all constructs were confirmed by automated sequencing (Applied Biosystems Inc.). Yeast two-hybrid library screening and analysis were performed essentially as described previously (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar). Briefly, the yeast strain HF7c containing the bait plasmid pGBT9-sentrin was transformed with a human placenta cDNA library (from CLONTECH) using the lithium acetate method as described in the CLONTECH's Matchmaker Library Protocol. Approximately 5 × 106 transformants were selected for growth on plates lacking histidine, leucine, and tryptophan. His+ colonies were subsequently analyzed for β-galactosidase activity and retested for their specificity by using a panel of plasmids encoding different polypeptides fused to the Gal4 DNA binding domain (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar). The full-length cDNA of UBC9 was amplified by PCR from a human placenta cDNA library. The PCR product was subcloned into pGEX-2T (Pharmacia Biotech Inc.) using BamHI andEcoRI restriction sites to generate pGEX-UBC9.Escherichia coli BL21 cells carrying pGEX-UBC9 or pGEX-2T were grown to saturation in 10 ml of LB containing 50 μg/ml ampicillin, transferred to 500 ml of LB broth for expansion to an absorbance (600 nm) of 0.8. After the addition of isopropyl-β-d-thiogalactopyranoside (final concentration: 0.1 mm), the culture was incubated at room temperature for 3 h. The cells were sonicated in lysis buffer (20 mmTris, pH 8.0, 1 mm EDTA, 100 mm NaCl, 1% IGEPAL CA-630, 100 μg/ml egg white lysozyme), and bacterial debris was removed by centrifugation. The sonicated lysates were incubated at 4 °C for 1 h with 500 μl of a 50% slurry of glutathione-Sepharose 4B beads. After centrifugation, GST-fusion protein bound beads were washed three times with 10 ml of TENI buffer (20 mm Tris, pH 8.0, 1 mm EDTA, 100 mm NaCl, and 1% IGEPAL CA-630). For in vitroexpression of sentrin protein, the pBS-sentrin plasmid, containing the sentrin cDNA insert, was linearized with XbaI. Sentrin protein was labeled with 35SMet by using an in vitro transcription and translation kit (TNT T7-coupled rabbit reticulocyte lysates, Promega) according to the supplier's instruction. 10 μl of in vitro translated sentrin was incubated with 50 μl of glutathione-Sepharose beads containing approximately 1 μg of GST or GST-fusion proteins for 12 h at 4 °C in the binding buffer described previously (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar). After washing five times in binding buffer, the samples were divided into two equal aliquots. One aliquot was treated with SDS-loading buffer without β-mercaptoethanol, another was treated with SDS-loading buffer containing 5% β-mercaptoethanol. Finally, the samples were loaded onto a 15% SDS-polyacrylamide gel and visualized by autoradiography. We have shown previously that sentrin can be conjugated to a number of cellular proteins in a manner analogous to ubiquitination (6Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It is of interest to determine whether sentrin utilizes a similar set of enzymes in the processing of its C terminus, in the activation of the Gly residue, and in conjugation to other proteins. For this purpose, the yeast two-hybrid system was used to identify cDNA clones that encode polypeptides that are able to interact with sentrin. Yeast strain HF7c, which contains two Gal4-inducible reporter genes,HIS3 and lacZ, was initially transformed with the bait plasmid pGBT9-sentrin and then the resulting transformant was used as host for transformation with the placenta cDNA library. Approximately 0.5 × 107 primary library transformants were plated onto plates lacking histidine, leucine, and tryptophan. A total of 104 colonies appeared on the histidine dropout plates, 16 of which stained positive when tested for expression of β-galactosidase. To test whether sentrin was required for interaction with the products of the isolated cDNAs, all of these plasmids were retransformed into the yeast strain SYP526 together with plasmids encoding other Gal4 DNA binding domain fusion proteins. All hybrid proteins were found to interact only with Gal4-sentrin. Subsequent analyses indicated that one of these colonies contained an approximately 1.8-kilobase pair insertion. Analysis of the cDNA sequence showed a single open reading frame of 158 amino acids. Comparison of the deduced amino acid sequence with GenBank™ revealed that the protein encoded by the newly identified cDNA is Ubc9, a human ubiquitin-conjugating enzyme that is a structural and functional homologue of ScUBC9 (19Yasugi T. Howley P. Nucleic Acids Res. 1996; 24: 2005-2010Crossref PubMed Scopus (82) Google Scholar, 20Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar). The yeast two-hybrid system was used to further assess the structural requirement of sentrin and Ubc9 interaction. Various deletion mutants of either sentrin or Ubc9 were constructed and tested in the yeast two-hybrid assay. As shown in Fig.1 A, wild type sentrin interacted strongly with Ubc9, as evidenced by a detectable color change within 45 min. This is comparable with the interaction between SV40 large T and p53 and is stronger than the FADD/MORT1 and Fas interaction (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar). Deletion of the four C-terminal amino acids had minimal effect on the interaction (3+). Deletion of the N-terminal 23 amino acids, sentrin (24–101), reduced the interaction from 4+ to 2+. Deletion of both the N and C termini, sentrin (24–97), reduced the interaction to barely detectable. Deletion of the C-terminal 31 amino acids, sentrin (1–70), completely abolished the interaction. The N-terminal 23 amino acids also could not interact with Ubc9. Thus, the ubiquitin domain of sentrin is required for the interaction. Since the ubiquitin domain of sentrin is required for the interaction, the interaction between ubiquitin and Ubc9 was also tested. As shown in Fig. 1 B, ubiquitin did interact with Ubc9, but the interaction was much weaker than the interaction between sentrin and Ubc9. Deletion of the C-terminal 50 amino acids or the N-terminal 30 amino acids of Ubc9 also abolished these interaction. These results suggest that the N-terminal 21 amino acids of sentrin enhances the interaction of sentrin with Ubc9. It should be emphasized that the yeast two-hybrid results should not be taken to imply that Ubc9 prefers sentrin over ubiquitin. The process of ubiquitination and sentrinization could be influenced by the presence of additional proteins in vivo. However, these results clearly demonstrate that the interaction between sentrin and Ubc9 is highly specific and thereby biologically Ubc9 to a of ubiquitin-conjugating proteins S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). All E2 enzymes have a conserved domain of approximately 16 the UBC This domain is at to all E2s and a cysteine residue for thiol ester formation S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). We have four other E2s by using PCR amplification from a human placenta cDNA library. These E2s were tested for their to interact with sentrin. M. Weissman A.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google had a interaction with sentrin. P. I. L. S. D. Natl. S. A. 88: PubMed Scopus Google Scholar), UbcH6 S. P. R. M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and Z. A.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google were to interact with sentrin. These further demonstrate the specificity of the interaction between sentrin and Ubc9 interaction. that the C of sentrin is efficiently and Gly97 is for sentrinization to (6Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A number of C-terminal deletion and substitution mutants were constructed and tested in the yeast two-hybrid assay. As shown in Fig. 1 deletion of or or substitution of for abolished the interaction of sentrin with Ubc9. These results are with that a critical role in the formation of sentrinized proteins. These results also suggest that a linkage of sentrin to Ubc9 for the strong interaction between these proteins in the yeast two-hybrid interaction. To further for the interaction between sentrin and Ubc9, a GST-Ubc9 fusion protein was and GST or GST-Ubc9 proteins were then used to in vitro and translated sentrin. As shown in Fig. GST-Ubc9 but not GST could in vitro translated sentrin. In a could also be This sentrin conjugated to GST-Ubc9 via a thiol ester because it when the was reduced with 5% In we have shown that other GST-fusion proteins could not sentrin not Thus, it appears that sentrin was by an E1 in the reticulocyte The sentrin then bound to GST-Ubc9 via a thiol ester To further this the active site cysteine residue in Ubc9, which is for thiol ester was to In Fig. in sentrin could be precipitated by GST-Ubc9 and However, the molecular was only in the precipitated by These results suggest that sentrin could form a thiol ester linkage with Ubc9 via the conserved cysteine vitro interaction of sentrin with Ubc9 and In vitro translated sentrin was precipitated by GST GST-Ubc9 and The of labeled sentrin and the conjugate are ScUBC9 was reported by and in W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar). showed that of Ubc9 cell at the or the of large cells with a single a and In mutants both an and an are W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar). In wild type cells the protein is the cell cycle, only at a specific cell there have been a number of the of Ubc9 with biologically proteins. G. S. Jentsch S. R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google showed that Ubc9 binds to the of protein. M. S. B. E. M. P. 1996; 61: PubMed Scopus Google that human Ubc9 interacts with and the and W. Genet. 1996; Google showed that interacts with of W. T. W. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google reported that human Ubc9 with the domain of the tumor product and Howley (19Yasugi T. Howley P. Nucleic Acids Res. 1996; 24: 2005-2010Crossref PubMed Scopus (82) Google also showed that human Ubc9 interacts with the human type 16 E1 protein. E. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google also reported that the Ubc9 interacts with the proteins. additional Futcher B. J. Biol. Chem. 1995; 271: Full Text Full Text PDF Scopus Google reported that human Ubc9 is with the form of human In of that Fas binds to sentrin (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google and sentrin binds to Ubc9, these results could that either Fas is a target for sentrinization or Fas is of a sentrinization that sentrin and Ubc9. The of both sentrin and Ubc9 with other proteins is not to and reported that human Ubc9 interacts with human Rad51 A. T. D. T. D. C. E. Natl. S. A. 1996; PubMed Scopus Google Scholar). Furthermore, H. R. M. M. Natl. S. A. 1997; PubMed Scopus Google reported that with Ubc9 and a form of RanGAP1. The interaction of sentrin Ubc9 with other proteins is in to It is not which protein in is sentrinized or Ubc9 as a specific is the only protein that is to be sentrinized M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (957) Google or (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar), were used by the which the of by an ubiquitin-like protein (2Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google or (3Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 36: 271-279Crossref PubMed Scopus (183) Google were used by two other to protein to sentrin. there is the We have to the sentrin in with two (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (957) Google Scholar, T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It is also that of these proteins could either interact with other sentrinized protein or as an E3 in ubiquitination or that have been shown to interact with either sentrin or Ubc9 or to interact of of tumor by by sentrin. in a In results suggest that Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway. additional conjugating as UbcH5B, may also play a role in the sentrinization pathway to be