Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m703911200
ISSN1083-351X
AutoresMatthew D. Petroski, Xiulan Zhou, Guoqiang Dong, Sarkiz Daniel-Issakani, Donald G. Payan, Jianing Huang,
Tópico(s)Cancer-related Molecular Pathways
ResumoProtein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys63 of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys63-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys63-linked ubiquitin chain synthesis mechanism. Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys63 of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys63-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys63-linked ubiquitin chain synthesis mechanism. The post-translational modification of proteins through the covalent addition of the 76-amino acid protein ubiquitin has emerged as a critical mechanism for regulating many aspects of cellular physiology (1Pickart C.M. Fushman D. Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (835) Google Scholar). The role of ubiquitin in modulating protein activity is defined in part by the extent and type of modification. Single ubiquitin molecule attachments (mono-ubiquitination) as well as chains of ubiquitin molecules generated through isopeptide linkages between the primary amine of a lysine side chain of one molecule and the C terminus of another encode signals that are deciphered to trigger distinct cellular responses. Chains of specific ubiquitin-ubiquitin linkages result in distinct protein fates. For example, a lysine 48-linked chain of at least four ubiquitin molecules is the minimum signal for efficient degradation of a protein by the 26 S proteasome in vitro (2Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1314) Google Scholar). Emerging evidence suggests, in contrast, that Lys63-linked ubiquitin chains attached onto a protein serve in non-proteolytic functions such as regulating intracellular signaling (3Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1022) Google Scholar, 4Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). Other types of ubiquitin chains also exist including those containing mixed linkages and branched chains (5Ben-Saadon R. Zaaroor D. Ziv T. Ciechanover A. Mol. Cell. 2006; 24: 701-711Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 6Kirkpatrick D.S. Hathaway N.A. Hanna J. Elsasser S. Rush J. Finley D. King R.W. Gygi S.P. Nat. Cell Biol. 2006; 8: 700-710Crossref PubMed Scopus (350) Google Scholar). However, these have been primarily observed as products of in vitro reactions and their physiological significance is unclear. Perhaps the most well understood mechanism for generating specific ubiquitin linkages is through the activity of the ubiquitin-conjugating enzyme (E2) 2The abbreviations used are: E2, ubiquitin-conjugating enzyme; E1, ubiquitin-activating enzyme; Ni-NTA, nickel-nitrilotriacetic acid; NEM, N-ethylmaleimide; Ub, ubiquitin. UBC13. UBC13 functions as a heterodimer by binding through a large hydrophobic interface with the UEV (ubiquitin-conjugating enzyme variant) family of which MMS2 and UEV1A are representative members (4Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Pastushok L. Moraes T.F. Ellison M.J. Xiao W. J. Biol. Chem. 2005; 280: 17891-17900Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). UEVs contain the conserved enzymatic core of ubiquitin-conjugating enzymes, but lack a catalytic cysteine. By binding a ubiquitin molecule and positioning its Lys63 near the active site cysteine of UBC13, UEVs promote Lys63-linked ubiquitin chain synthesis (8VanDemark A.P. Hofmann R.M. Tsui C. Pickart C.M. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Structural and biophysical studies have uncovered the molecular basis of how UBC13-UEV generates Lys63-linked ubiquitin chains (4Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 7Pastushok L. Moraes T.F. Ellison M.J. Xiao W. J. Biol. Chem. 2005; 280: 17891-17900Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8VanDemark A.P. Hofmann R.M. Tsui C. Pickart C.M. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 9Eddins M.J. Carlile C.M. Gomez K.M. Pickart C.M. Wolberger C. Nat. Struct. Mol. Biol. 2006; 13: 915-920Crossref PubMed Scopus (273) Google Scholar, 10McKenna S. Hu J. Moraes T. Xiao W. Ellison M.J. Spyracopoulos L. Biochemistry. 2003; 42: 7922-7930Crossref PubMed Scopus (42) Google Scholar, 11McKenna S. Moraes T. Pastushok L. Ptak C. Xiao W. Spyracopoulos L. Ellison M.J. J. Biol. Chem. 2003; 278: 13151-13158Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12McKenna S. Spyracopoulos L. Moraes T. Pastushok L. Ptak C. Xiao W. Ellison M.J. J. Biol. Chem. 2001; 276: 40120-40126Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), yet little is known about how this ubiquitin-conjugating enzyme functions with its cognate ubiquitin ligases to modify protein substrates. To study this process, we have focused on reconstituting the modification of TRAF6 by UBC13-UEV1A in vitro with purified proteins. TRAF6 functions downstream of specific members of the tumor necrosis factor receptor family and upon receptor stimulation promotes NF-κB activation (13Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1122) Google Scholar, 14Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Fractionation experiments initially identified UBC13 as a TRAF6 interacting protein and a variety of cell-based and in vitro experiments suggest that TRAF6 modification with Lys63-linked ubiquitin chains has a non-proteolytic function in NF-κB activation through the kinase TAK1 (15Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 16Sun L. Deng L. Ea C.K. Xia Z.P. Chen Z.J. Mol. Cell. 2004; 14: 289-301Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 17Wooff J. Pastushok L. Hanna M. Fu Y. Xiao W. FEBS Lett. 2004; 566: 229-233Crossref PubMed Scopus (46) Google Scholar). In this case, TRAF6 appears to function both as a ubiquitin ligase (it contains a RING motif and interacts with its ubiquitin-conjugating enzyme UBC13-UEV1A) and substrate for its own modification (it is subjected to “auto-ubiquitination”). Here, we examine how UBC13-UEV1A functions with this ubiquitin ligase to promote TRAF6 auto-ubiquitination and uncover that the activity provided by UEV1A primarily promotes Lys63-linked ubiquitin chain synthesis that effectively reduces TRAF6 modification. Based on these observations, we propose a model for how substrate modification with Lys63-linked ubiquitin chains occurs through the activity of this ubiquitin-conjugating enzyme. Ubiquitin-activating Enzyme (E1)—Human E1 containing an N-terminal hexahistidine tag was expressed in Sf9 insect cells from recombinant baculovirus and purified from lysates by Ni-NTA chromatography, followed by Mono Q-Sepharose separation. Fractions containing E1 were pooled and dialyzed into 20 mm Tris-HCl, pH 8.0, at 25 °C, 100 mm NaCl, 0.5 mm EDTA, 0.5 mm Tris(2-carboxyethyl)phosphine, and 10% glycerol. UBC13-UEV1A and UbcH5c—The subunits of human UBC13-UEV1A were expressed individually as hexahistidine-tagged proteins in Escherichia coli and purified by Ni-NTA chromatography. UBC13 contains an N-terminal hexahistidine tag and UEV1A contains a C-terminal hexahistidine tag. For experiments using the intact complex, both proteins were initially mixed together at equal concentrations. See Fig. 1A for a Coomassie-stained gel of purified UBC13 (lane 3) and UEV1A (lane 4). UbcH5c was also expressed in E. coli and purified by Ni-NTA chromatography via an N-terminal hexahistidine tag. Protein concentration was measured using a commercial protein concentration kit (Bio-Rad Protein Assay) using bovine γ-globulin as a standard as per the manufacturer's instructions and verified by SDS-PAGE and Coomassie staining. To compare the relative amount of active UBC13 and UbcH5c in these preparations, we examined the ability of these enzymes (5 μm) to be charged with 40 μm ubiquitin by 150 nm E1 in the presence of 1 mm ATP. At the indicated times, reaction aliquots were removed and added to non-reducing sample buffer prior to analysis by SDS-PAGE and Coomassie staining (Fig. 4C).FIGURE 4UBC13-UEV1A predominantly synthesizes ubiquitin chains in the presence of TRAF6.A, ubiquitin incorporation; and B, TRAF6 modification were analyzed in reactions containing UBC13-UEV1A (lanes 1–6), UbcH5c (lanes 7–12), and UBC13 (lanes 13–18) at the time points shown. Reaction aliquots were removed at the indicated times and added to an equal volume of reducing sample buffer prior to analysis by SDS-PAGE and immunoblotting with anti TRAF6 and anti-ubiquitin antisera. The bottom panels in B are longer exposures of the top panels. The relative positions of ubiquitin (Ub), ubiquitin chains, TRAF6, and ubiquitinated TRAF6 (TRAF6-Ubn) are indicated. C, the ability of UBC13 (5 μm, lanes 1–6) and UbcH5c (5 μm, lanes 7–12) to be charged with ubiquitin (12.5 μm) was assessed in the presence of 150 nm E1 and 1 mm ATP. Reaction aliquots were removed at the indicated times and analyzed under non-reducing conditions by SDS-PAGE followed by Coomassie staining. The relative positions of free ubiquitin, unmodified UbcH5c (UbcH5c), unmodified UBC13 (UBC13), UbcH5c charged with ubiquitin (UbcH5c-Ub), UBC13 charged with ubiquitin (UBC13-Ub) and E1 are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TRAF6—The mouse TRAF6 open reading frame (amino acid sequence is >80% identical to human TRAF6) was cloned into pFastBac1 containing sequences encoding N-terminal streptavidin-binding peptide and calmodulin-binding peptide. High titer stocks of recombinant baculovirus were generated as per the manufacturer's instructions (Invitrogen). For TRAF6 expression, Sf9 cells (107 cells/10 cm2 plate) were infected at a multiplicity of infection of 10 for ∼42 h. Lysates (1 ml of lysis buffer/10-cm2 plate) were prepared in Lysis Buffer containing 50 mm HEPES, pH 7.4, 300 mm NaCl, 0.2% Triton X-100, 10% glycerol, and 1 mm dithiothreitol. Clarified lysates were added to streptavidin-agarose (1 ml of lysate to 50 μl of streptavidin-agarose) and incubated for 30 min at 4 °C with mixing. After washing 4 times with Lysis Buffer and twice with Lysis Buffer without Triton X-100, bound material was eluted three times with 2 bead volumes of Lysis Buffer without Triton X-100 containing 2.5 mm d-desthiobiotin. Protein concentration was measured using Bio-Rad Protein Assay reagent using γ-globulin as a standard and purity was assessed by SDS-PAGE followed by Coomassie staining (see Fig. 1A, lane 5). Ubiquitin and Ubiquitin Derivatives—Ubiquitin, K0 ubiquitin, Lys63 only ubiquitin, and K63R ubiquitin were purchased from Boston Biochem and resuspended in water. Ubiquitin stocks were prepared at 25 mg/ml and the various ubiquitin derivatives were prepared at 5 mg/ml. FLAG-ubiquitin (ubiquitin containing an N-terminal FLAG epitope) was expressed in E. coli and purified by acid elution from anti-FLAG-Sepharose. Eluted material was dialyzed into 20 mm Tris-HCl, pH 8.0, at 25 °C, 100 mm NaCl, 0.5 mm EDTA, 0.5 mm Tris(2-carboxyethyl) phosphine, and 10% glycerol. Protein concentration was measured using Bio-Rad Protein Assay reagent as per the manufacturer's instructions and verified by SDS-PAGE and Coomassie staining. Reactions were performed in ubiquitination reaction buffer containing 50 mm HEPES, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 1 mm ATP, and 0.5 mm dithiothreitol. The indicated proteins were assembled on ice and the reaction was initiated by the addition of TRAF6 (where added) prior to incubation for 30 min at 37 °C. Reactions (10 μl) contained the following protein concentrations: 150 nm E1, 156 μm ubiquitin or various ubiquitin derivatives, 5 μm UBC13, 5 μm UEV1A, 5 μm UbcH5c, and 300 nm TRAF6. Under these conditions, TRAF6 is limiting relative to E2 based on our titration experiments examining ubiquitin discharge from UBC13 (Fig. 7). For reactions lacking a particular reaction component as indicated, an equivalent volume of ubiquitination reaction buffer was added. At the end of the incubation, an equal volume of denaturing sample buffer was added and the reactions were heated to 70 °C for 5 min prior to analysis by SDS-PAGE and immunoblotting with anti-ubiquitin (P4D1, Santa Cruz Biotechnology) and anti-TRAF6 (H-274, Santa Cruz Biotechnology) antisera. For time course experiments (Fig. 4), 10-μl aliquots of a 70-μl reaction (conditions as described above) were removed at the indicated times, added to reducing sample buffer, heated to 70 °C for 5 min and analyzed with anti-ubiquitin and anti-TRAF6 immunoblotting. To analyze TRAF6 modification (Fig. 5), TRAF6 was bound to streptavidin-Sepharose (10-μl bead volume with 250 μl of TRAF6 baculovirus-infected insect cell lysate). After washing the Sepharose 3 times with Lysis Buffer (described above) and 2 times with ubiquitination reaction buffer, the indicated E2 subunits (5 μm) were added to reactions containing a final concentration of 150 nm E1 and 100 μm ubiquitin in a 20-μl total volume. After 20 min at room temperature with mixing, non-reducing sample buffer was added directly to 1 set of reactions (“soluble” reactions). The Sepharose for the other set of reactions (“bound” reactions) were washed in Lysis Buffer. TRAF6 was subsequently eluted from the streptavidin-Sepharose by a volume of non-reducing sample buffer equivalent to the total volume of the soluble reactions. Soluble and bound reactions were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin and anti-calmodulin-binding peptide (Upstate). UBC13 (2 μm final concentration in 10 μl of charging reaction) was added to reactions containing ubiquitin (156 μm) and 150 nm E1 and incubated for 20 min at room temperature in ubiquitination reaction buffer (described above). The charging reaction was terminated by the addition of N-ethylmaleimide (NEM) (5 mm final concentration) and EDTA (10 mm final concentration), followed by a 15-min incubation at room temperature. Discharge reactions containing the various indicated proteins and ubiquitin (312 μm in final discharge reaction) were set up on ice in a ubiquitin reaction buffer without ATP. Reactions were initiated by the addition of an equal volume of charged and treated UBC13 (20 μl total volume), rapidly mixed, and incubated at 37 °C. Aliquots were removed at the indicated times and added to an equal volume of non-reducing SDS-PAGE sample buffer. UBC13-Ub thioester, UBC13, and TRAF6 were analyzed by SDS-PAGE and immunoblotting with anti-UBC13 (Invitrogen) and anti-TRAF6 (Santa Cruz Biotechnology) antisera. The final concentrations of the proteins used in the reactions shown in Fig. 6 were 1 μm UBC13, 300 nm TRAF6, and 300 nm UEV1A. Fig. 7 utilized UBC13 at 500 nm and variable concentrations of either UEV1A or TRAF6, 200 nm, 500 nm, 1.25 mm, or 2.5 mm, to give the indicated molar ratios. To examine potential downstream effects of NEM in chase reactions, UBC13 (2 μm) was charged with 3.75 μm ubiquitin containing an N-terminal FLAG epitope (Fig. 6, C and D). Reactions were either untreated (-NEM) or treated with NEM (+NEM, 5 mm final concentration) and EDTA (10 mm final concentration) for 15 min prior to adding to chase reactions containing 300 nm UEV1A and 300 nm TRAF6 and ubiquitin (312 μm). The final concentration of UBC13 in the chase reaction was 1 μm. Reaction aliquots were removed at the indicated times and analyzed under non-reducing conditions by immunoblotting with anti-FLAG horseradish peroxidase (Sigma) to detect UBC13-Ub thioester. The x-ray structures for UBC13-MMS2 (Protein Data Bank 1JAT) and MMS2-ubiquitin (PDB 1ZGU) were structurally superimposed on MMS2 using PyMol (DeLano Scientific, San Carlos, CA) to generate a model of UBC13-MMS2-ubiquitin (root mean square 0.817). The distance from the amino group of the lysine 63 side chain to the sulfur of the active site cysteine of UBC13 was estimated from this model (see Fig. 8B). Ubiquitin Chain Synthesis by UBC13-UEV1A Is Stimulated by TRAF6—The modification of TRAF6 with Lys63-linked ubiquitin chains by UBC13-UEV1A promotes the activation of the kinase TAK1, which leads to the activation of NF-κB gene expression (15Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 16Sun L. Deng L. Ea C.K. Xia Z.P. Chen Z.J. Mol. Cell. 2004; 14: 289-301Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Unlike ubiquitin ligases that recognize specific substrates that are targets for ubiquitin modification, TRAF6 functions both as a ubiquitin ligase and its own substrate. To study the molecular mechanisms underlying this process, we sought to recapitulate TRAF6 modification with Lys63-linked ubiquitin chains in vitro using purified recombinant proteins (Fig. 1A). We performed experiments to compare the activity of UBC13-UEV1A and TRAF6 separately to the complete reaction. Reaction products were subsequently analyzed for ubiquitin chain synthesis with an anti-ubiquitin antibody (Fig. 1B) and TRAF6 modification with anti-TRAF6 (Fig. 1C). Whereas UBC13-UEV1A synthesizes detectable amounts of ubiquitin chains after 30 min in the absence of a ubiquitin ligase, the addition of TRAF6 robustly stimulates this activity (Fig. 1B, compare lanes 1 and 3). As expected, TRAF6 had no detectable activity in the presence of E1, ATP, and ubiquitin (Fig. 1B, lane 2). Despite TRAF6 efficiently stimulating the activity of UBC13-UEV1A to synthesize ubiquitin chains, we did not observe TRAF6 ubiquitination in comparing reactions performed with TRAF6 alone to reactions containing TRAF6 and UBC13-UEV1A (Fig. 1C, compare lanes 2 and 3). To rule out the possibility that our antibody cannot efficiently recognize ubiquitinated TRAF6 (generated against amino acids 1–274), we analyzed TRAF6 in these reactions with other antibodies generated against TRAF6 as well as one of the epitope tags present on this form of TRAF6 (calmodulin binding peptide) and obtained similar results (data not shown). A Promiscuous E2, UbcH5c, Efficiently Ubiquitinates TRAF6—Our observation that TRAF6 is not efficiently ubiquitinated in our in vitro system suggests that TRAF6 may not properly present an appropriate nucleophile (i.e. the amino group of a lysine residue side chain) for modification. Several recent reports have proposed that TRAF6 must oligomerize to form an active ubiquitin ligase complex and various proteins downstream of specific classes of tumor necrosis factor receptors (TIFA, MALT1, BCL10, and others) may facilitate this process (16Sun L. Deng L. Ea C.K. Xia Z.P. Chen Z.J. Mol. Cell. 2004; 14: 289-301Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 18Dong W. Liu Y. Peng J. Chen L. Zou T. Xiao H. Liu Z. Li W. Bu Y. Qi Y. J. Biol. Chem. 2006; 281: 26029-26040Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19Ea C.K. Sun L. Inoue J. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15318-15323Crossref PubMed Scopus (103) Google Scholar). Thus, it is possible the deficiency of TRAF6 ubiquitination in our in vitro system may simply arise from an absence of these factors. To explore this possibility, we tested UbcH5c, a promiscuous ubiquitin-conjugating enzyme (20Brzovic P.S. Klevit R.E. Cell Cycle. 2006; 5: 2867-2873Crossref PubMed Scopus (68) Google Scholar), at an identical concentration to UBC13-UEV1A and under identical reaction conditions to determine whether its activity could be stimulated by TRAF6 (Fig. 2). We reasoned if TRAF6 oligomerization through accessory protein function is required for proper assembly into a ubiquitin ligase and substrate, reactions utilizing UbcH5c should have a defect similar to those using UBC13-UEV1A. Moreover, if TRAF6 is unable to present an appropriate nucleophile for modification, this would likely be reflected in reactions with UbcH5c due to the highly conserved enzymatic core of the ubiquitin-conjugating enzyme family (21Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar). In contrast to UBC13-UEV1A, UbcH5c did not detectably generate ubiquitin chains in the absence of TRAF6 (Fig. 2A, lane 1). However, its ability to synthesize ubiquitin chains was stimulated by TRAF6, similar to UBC13-UEV1A (Fig. 2A, lane 3). In examining these reactions for TRAF6 ubiquitination, we observed efficient modification of TRAF6 by UbcH5c such that the majority of TRAF6 was converted into a high molecular mass form (Fig. 2B, lane 3). Taken together, our results suggest that TRAF6 assembles into an active ubiquitin ligase complex in the absence of accessory factors in vitro. Furthermore, TRAF6 is not defective in nucleophile presentation and can serve as a substrate for modification, at least in conjunction with UbcH5c. UBC13 Modifies TRAF6 in the Absence of UEV1A—Our experiments comparing the ubiquitin ligase activity of TRAF6 with UBC13-UEV1A or UbcH5c revealed a critical difference between these E2s. Whereas UbcH5c efficiently modifies TRAF6 and generates ubiquitin conjugates in the presence of TRAF6, it has no detectable ubiquitin-conjugating activity in its absence. UBC13-UEV1A, in contrast, generates detectable amounts of ubiquitin chains even in the absence of TRAF6 (Fig. 1B, lane 1). The presence of TRAF6 stimulates ubiquitin chain synthesis, yet very little modification of TRAF6 occurs (Fig. 1B, lane 3). As the UEV1A subunit binds a ubiquitin molecule to position Lys63 of ubiquitin near the active site cysteine of UBC13, we hypothesized that its presence may drive ubiquitin chain synthesis that in turn may suppress TRAF6 modification. To test this hypothesis, we performed experiments in which we tested the individual subunits of the ubiquitin-conjugating enzyme in the presence of TRAF6 with either wild-type ubiquitin (Ub) or a form of ubiquitin in which all lysine residues are mutated to arginine and therefore cannot synthesize ubiquitin chains (K0 Ub). In examining ubiquitin chain synthesis in these reactions (Fig. 3A), we detected ubiquitin chain synthesis only in the presence of Ub and both subunits of the ubiquitin-conjugating enzyme (Fig. 3A, lane 3). In contrast, however, we observed TRAF6 modification by UBC13 alone in reactions with both Ub and K0 Ub (Fig. 3B, lanes 1 and 4) and with intact UBC13-UEV1A in the presence of K0 Ub (Fig. 3B, lane 6). As the pattern of ubiquitin conjugation on TRAF6 is identical in all of these reactions, regardless of using Ub or K0 Ub, this modification is consistent with single ubiquitin attachments (i.e. “monoubiquitination”) and not ubiquitin chains. This observation is particularly striking when the ability of UbcH5c to generate high molecular weight conjugates on TRAF6 is considered (Fig. 2B, lane 3) as it suggests UBC13 has a very specific function in substrate modification with individual ubiquitin molecules. Furthermore, these experiments suggest that a single site on TRAF6 may be the predominant site of ubiquitin attachment by UBC13, supporting recent observations by Lamonthe et al. (22Lamothe B. Besse A. Campos A.D. Webster W.K. Wu H. Darnay B.G. J. Biol. Chem. 2007; 282: 4102-4112Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) that identified a single lysine residue as important for TRAF6 function upstream of NF-κb activation. Lys63 of Ubiquitin and UEV1A Stimulate Ubiquitin Chain Synthesis by UBC13 and Their Absence Promotes Substrate Modification—To test how the presence of Lys63 of ubiquitin affects substrate modification, we performed similar ubiquitination reactions with ubiquitin derivatives, focusing on intact (Lys63 only ubiquitin, with all other lysine residues mutated to arginine) or mutated Lys63 (K63R ubiquitin). Ubiquitin chain synthesis, as expected, requires the presence of both UEV1A and Lys63 of ubiquitin to occur (Fig. 3C, lanes 2 and 8), supporting the hypothesis that the positioning of Lys63 of ubiquitin by UEV1A near the active site cysteine of UBC13 is part of the molecular basis of ubiquitin linkage specificity and our in vitro system recapitulates this specificity. In examining these reactions for TRAF6 modification, we observed that UBC13 alone can modify TRAF6 with a single predominant site of ubiquitin attachment with 2–4 total sites detectable for all ubiquitin derivatives tested (Fig. 3D, lanes 1, 3, 5, and 7), consistent with multiple monoubiquitination events. Similar to our results with K0 Ub (Fig. 3B), TRAF6 modification occurs in the presence of UEV1A only in the absence of Lys63 (Fig. 3D, lanes 4 and 6), which correlates to an observed lack of ubiquitin chain synthesis (Fig. 3C, lanes 4 and 6). Our observation that K63R ubiquitin bound to UEV1A did not block TRAF6 modification suggests that these proteins do not simply mask sites of TRAF6 modification. Instead our data are consistent with the hypothesis that ubiquitin chain synthesis may dominate our in vitro system due to a preference for ubiquitin-ubiquitin discharge by UBC13 involving Lys63 of ubiquitin provided by the presence of UEV1A over TRAF6 modification. Ubiquitin Chains Are the Major in Vitro Product Synthesized by UBC13-UEV1A in the Presence of TRAF6—To further explore the role of TRAF6 as both a ubiquitin ligase and substrate, we performed experiments examining the rate of ubiquitin chain synthesis and TRAF6 modification by UBC13-UEV1A, UbcH5c, and UBC13 alone (Fig. 4). As expected, UBC13 alone did not generate detectable ubiquitin chains in the presence of TRAF6 over the time course examined (Fig. 4A, lanes 13–18). UBC13-UEV1A (lanes 1–6) and UbcH5c (lanes 7–12), in contrast, synthesized high molecular weight ubiquitin conjugates with qualitatively similar rates of appearance. These reactions were also analyzed for TRAF6 modification (Fig. 4B). UbcH5c modified TRAF6 such that the majority of the input substrate was converted into a high molecular weight form at the end of the time course analyzed (lanes 7–12). UBC13 alone (lanes 13–18), in contrast, converted TRAF6 considerably slower to a ubiquitinated form. The pattern of ubiquitin incorporation onto TRAF6 by UBC13 at 20 min (lane 18) resembled the pattern generated by UbcH5c at 1 min (lane 8), suggesting that features within the largely conserved catalytic core of these E2s may contribute to their distinct ability to modify TRAF6. Whereas the molecular basis of this difference remains to be further explored, it is unlikely due to differences in the amount of active E2 in the enzyme preparations or their ability to be charged with ubiquitin as both are charged with ubiquitin by E1 with similar rates when tested at similar concentrations (Fig. 4C, compare lanes 1–6 to lanes 7–12). As expected, UBC13-UEV1A did not efficiently modify TRAF6 when compared with either UbcH5c or UBC13 (lanes 1–6). However, longer exposures of the data (bottom panels of Fig. 4B) revealed the appearance of faint higher molecular weight species in the presence of UBC13-UEV1A after 5 min (compare lane 1 to lanes 4–6), consistent with ubiquitinated TRAF6. TRAF6 Is Modified with Individual Ubiquitin Molecules by UBC13 and Lys63-linked Ubiquitin Chains by UBC13-UEV1A—Our data investigating ubiquitin chain synthesis and TRAF6 modification suggest a small percentage of TRAF6 is modified by UBC13-UEV1A and ubiquitin chains are the major in vitro reaction products. To directly examine if TRAF6 is modified by UBC13-UEV1A with ubiquitin chains, we performed in vitro experiments using TRAF6 immobilized on streptavidin-Sepharose. By extensively washing Sepharose-bound material after our in vitro reactions, we could directly examine the extent of TRAF6 modification (Fig. 5). In the absence of E2 or with UEV1A alone, we observed no modification of TRAF6 (Fig. 5A, lanes 1, 3, 5, and 7) and no detectable ubiquitin chain synthesis (Fig. 5B, lanes 1, 3, 5, and 7). As e
Referência(s)