Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3
2009; Springer Nature; Volume: 28; Issue: 4 Linguagem: Inglês
10.1038/emboj.2008.289
ISSN1460-2075
AutoresSokol V. Todi, Brett J Winborn, K. Matthew Scaglione, Jessica R. Blount, Sue M. Travis, Henry L. Paulson,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle15 January 2009free access Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3 Sokol V Todi Sokol V Todi Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Brett J Winborn Brett J Winborn Department of Neurology, University of Michigan, Ann Arbor, MI, USA Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author K Matthew Scaglione K Matthew Scaglione Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Jessica R Blount Jessica R Blount Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Sue M Travis Sue M Travis Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author Henry L Paulson Corresponding Author Henry L Paulson Department of Neurology, University of Michigan, Ann Arbor, MI, USA Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author Sokol V Todi Sokol V Todi Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Brett J Winborn Brett J Winborn Department of Neurology, University of Michigan, Ann Arbor, MI, USA Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author K Matthew Scaglione K Matthew Scaglione Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Jessica R Blount Jessica R Blount Department of Neurology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Sue M Travis Sue M Travis Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author Henry L Paulson Corresponding Author Henry L Paulson Department of Neurology, University of Michigan, Ann Arbor, MI, USA Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA Search for more papers by this author Author Information Sokol V Todi1, Brett J Winborn1,2, K Matthew Scaglione1, Jessica R Blount1, Sue M Travis3 and Henry L Paulson 1,2 1Department of Neurology, University of Michigan, Ann Arbor, MI, USA 2Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA 3Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA *Corresponding author. Department of Neurology, University of Michigan, 109 Zina Pitcher Place, BSRB Room 4188, Ann Arbor, MI 48109, USA. Tel.: +3194002008; Fax: +7346155655; E-mail: [email protected] The EMBO Journal (2009)28:372-382https://doi.org/10.1038/emboj.2008.289 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin-3, but does not alter its preference for K63-linked Ub chains. In cells, ubiquitination of endogenous ataxin-3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin-3 in protein quality control are modulated through ubiquitination. Ataxin-3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes. Introduction Post-translational modification of proteins by ubiquitin (Ub) regulates many cellular events. When conjugated to a protein, Ub targets it to specific subcellular compartments or to macromolecular complexes, such as the proteasome, achieving this essentially by altering protein–protein interactions. An 8 kDa globular protein, Ub, is covalently attached to proteins through the concerted action of three proteins (E1–E2–E3). The Ub-activating enzyme (E1) transfers Ub to an E2-conjugating enzyme. Through a Ub ligase (E3), Ub is ultimately transferred to a lysine (K) residue of either a target protein or another Ub, forming an isopeptide bond. Proteins can be mono-ubiquitinated (one Ub linked to a target protein), multi-mono-ubiquitinated (two or more single Ub linked to different lysine residues of a protein) or poly-ubiquitinated (one or more Ub chains linked to a protein). As Ub itself contains seven lysines, different chain linkages can be formed by the direct attachment of one Ub to another. The best studied among these are K48-linked chains, which target proteins for proteasomal degradation (Thrower et al, 2000). Other linkages, for example, K63-linked Ub, are implicated in other processes, including DNA repair, the NFκB pathway, Lewy body and aggresome formation, and autophagy (Arnason and Ellison, 1994; Hofmann and Pickart, 1999; Deng et al, 2000; Pickart and Fushman, 2004; Chen, 2005; Lim et al, 2005; Tan et al, 2007). Protein ubiquitination is reversed by deubiquitinating enzymes (DUBs). Nearly 100 DUBs are encoded by the human genome (Nijman et al, 2005). Although relatively little is known about most DUBs, they clearly function as more than Ub recyclers. DUBs have important functions in numerous cellular pathways: from DNA transcription to protein degradation, from cell division to death. DUBs perform their functions by cleaving Ub from proteins, altering the length or type of Ub chains, or disassembling untethered Ub chains (reviewed by Amerik and Hochstrasser, 2004; Nijman et al, 2005; Ventii and Wilkinson, 2008). DUB activity can be regulated at the level of transcription, degradation, complex formation, or phosphorylation (Amerik and Hochstrasser, 2004; Nijman et al, 2005; Ventii and Wilkinson, 2008; Yao et al, 2008). Several DUBs have been reported to be ubiquitinated (Shen et al, 2005; Wada and Kamitani, 2006; Fernandez-Montalvan et al, 2007; Meray and Lansbury, 2007; Todi et al, 2007a), but whether ubiquitination of DUBs directly regulates their activity has not been reported. The possibility that DUB activity could be regulated by ubiquitination is suggested by studies of other enzymes involved in Ub or Ub-like pathways. For example, the activity of cullin-based Ub ligases is enhanced through their modification by the Ub-like protein Nedd8 (Parry and Estelle, 2004; Duda et al, 2008). Ubiquitination of the E2 Cdc34 inhibits its activity (Scaglione et al, 2007). Substrate discrimination for Ubc9, the E2 for the Ub-like modifier SUMO, is regulated by its own SUMOylation (Knipscheer et al, 2008). Finally, SUMOylation of the DUB USP25 reduces its enzymatic activity by interfering with the ability of USP25 to interact with Ub (Meulmeester et al, 2008). Here, we report that the catalytic activity of a DUB, ataxin-3, is directly enhanced by ubiquitination. Ataxin-3 (AT3; Figure 1A) is a DUB implicated in Ub-dependent protein quality control (Wang et al, 2000, 2006; Burnett et al, 2003; Doss-Pepe et al, 2003; Chai et al, 2004; Mao et al, 2005; Nicastro et al, 2005; Warrick et al, 2005; Zhong and Pittman, 2006). An important feature of AT3 is its polyglutamine tract, which when expanded causes the neurodegenerative disorder Spinocerebellar Ataxia Type 3/Machado–Joseph disease (SCA3/MJD) (Kawaguchi et al, 1994; Stevanin et al, 1995a, 1995b). Through its multiple Ub-interacting motifs (UIMs; Figure 1A), AT3 binds both K48- and K63-linked Ub chains, yet preferentially cleaves K63-linked Ub chains in vitro, and is efficient at cleaving K63 linkages within short, mixed-linkage chains (Winborn et al, 2008). The UIMs are required both for this cleavage preference and for the ability to bind Ub chains (Winborn et al, 2008). Figure 1.Endogenous AT3 is ubiquitinated under basal conditions, and its ubiquitination is enhanced during proteasome inhibition. (A) Schematic of AT3 showing the N-terminal catalytic (Josephin) domain and the C-terminal Ub-interacting motifs (UIMs) flanking the polyglutamine tract. (B) AT3 is ubiquitinated in cells. FLAG–AT3 and HA–Ub were coexpressed in Cos7 cells. Lysates were subjected to stringent immunopurification with anti-FLAG antibody, then probed with anti-AT3 and anti-HA antibodies. Several high molecular weight ubiquitinated AT3 (AT3-Ub) bands are recognized by both anti-AT3 (1H9) and anti-HA antibodies. (C) Endogenous AT3 is ubiquitinated. Left: M17 cells were treated with or without the proteasome inhibitor lactacystin. Several higher molecular weight AT3-Ub species present in untreated cells are enriched when the proteasome is inhibited. Right: AT3 immunoprecipitated with polyclonal anti-AT3 antibody (MJD) from cells treated with lactacystin was probed with monoclonal anti-AT3 (1H9) and anti-Ub (P4D1) antibodies, confirming that the higher molecular weight forms of AT3 are ubiquitinated. Asterisks: mono-ubiquitinated AT3 exists in cells under basal conditions. AT3 doublets probably reflect allelic differences in the CAG/polyQ repeat in M17 cells. Ctrl: polyclonal, anti-HA antibody. Download figure Download PowerPoint AT3 is a putative neuroprotective protein that functions in protein quality control pathways. AT3 rescues neurodegeneration caused by expanded polyQ proteins in Drosophila, doing so in an activity-dependent manner (Warrick et al, 2005). AT3 has also been implicated in ER-associated degradation (ERAD), where it assists in targeting proteins to the proteasome (Wang et al, 2006; Zhong and Pittman, 2006). When coexpressed with Ub, AT3 becomes ubiquitinated in cells (Shoesmith Berke et al, 2005; Todi et al, 2007a), but the functional consequences of this ubiquitination are unknown. Here, we show that ubiquitination of AT3 enhances cleavage of Ub chains by this DUB and that levels of ubiquitinated AT3 rise with certain cellular stressors. These results show protein ubiquitination as a direct enhancer of enzymatic activity, and suggest that the functions of AT3 in protein quality control are modulated through ubiquitination. Results Endogenous AT3 is ubiquitinated FLAG–AT3 becomes ubiquitinated when coexpressed with Ub in mammalian cells (Figure 1B). To determine whether endogenous AT3 is also ubiquitinated, we conducted stringent denature/renature immunopurification of AT3 from cells (Figure 1C). In neural M17 cells, inhibition of the proteasome with lactacystin led to the accumulation of higher molecular weight AT3-immunoreactive bands (Figure 1C). Immunopurification confirmed that these bands are ubiquitinated forms of AT3 (AT3-Ub), including mono-ubiquitinated AT3 (Figure 1C). Ubiquitinated AT3 is present also under non-stressed conditions, although at lower levels. Thus, a fraction of AT3 is ubiquitinated in unperturbed cells under steady-state conditions, and proteasome inhibition increases this fraction. Ubiquitinated AT3 cleaves Ub chains more quickly than unmodified AT3 We hypothesized that AT3 ubiquitination might regulate its enzymatic activity. To investigate this, we purified unmodified or ubiquitinated AT3 from transfected cells and tested their activity in vitro (Figure 2A). Immunopurified AT3-Ub showed markedly increased DUB activity towards K63-linked hexa-Ub chains (K63-Ub6; Figure 2B). AT3-Ub also rapidly and quantitatively cleaved higher molecular weight Ub chain complexes (HMW; Figure 2B) that most likely represent longer polymers of Ub6 chains (Winborn et al, 2008). Mutating the catalytic cysteine at position 14 of AT3 to alanine renders the enzyme inactive (Burnett et al, 2003; Mao et al, 2005; Nicastro et al, 2005). Catalytically inactive, ubiquitinated AT3 (AT3(C14A)-Ub) showed no activity towards Ub chains (Figure 2B), indicating that the increased activity of AT3-Ub is not due to the spurious co-purification of another DUB. The major AT3-Ub species present in our immunopurifications is mono-ubiquitinated AT3, suggesting that a single conjugated Ub is sufficient to activate AT3 (Figure 2B). Figure 2.Ubiquitinated AT3 shows enhanced catalytic activity. (A) Diagram of preparation and immunoprecipitation of unmodified and ubiquitinated FLAG–AT3 from Cos7 cells transfected as indicated. (B) Ubiquitinated AT3 immunopurified from cells cleaves K63-Ub6 chains more rapidly than does unmodified AT3. AT3(WT), AT3(WT)-Ub, or catalytically inactive AT3-Ub (AT3(C14A)-Ub) (50 nM) were incubated with K63-Ub6 chains (250 nM). HMW: high molecular weight Ub species. Bottom: membrane was stripped and probed with anti-AT3 antibody. (C) AT3 ubiquitinated in vitro cleaves K63-Ub6 chains more quickly than does unmodified AT3. Anti-AT3 blot shows GST–AT3 species used in reactions. Download figure Download PowerPoint To exclude the possibility that AT3-Ub isolated from cells co-purifies with modulators of its enzymatic activity, we performed similar reactions with recombinant AT3-Ub generated in vitro using the E2 UbcH5c and the E3 CHIP. Recombinant AT3-Ub also showed increased DUB activity (Figure 2C). Thus, ubiquitination enhances AT3 activity independent of potential cofactors/interactors or other types of post-translational modification. Whereas AT3 does not cleave homotypic tetra-Ub (Ub4) chains efficiently, it does cleave mixed-linkage Ub4 chains (Winborn et al, 2008). Similar to unmodified AT3, AT3-Ub does not cleave K63-Ub4 chains but shows increased activity towards mixed-linkage Ub4 chains (Supplementary Figure 1). Therefore, ubiquitination enhances the activity of AT3 without altering its preference for K63-Ub6 or mixed-linkage Ub4 chains. We next compared the kinetics of DUB activity of AT3-Ub with unmodified AT3. AT3-Ub prepared in vitro cleaved K63-Ub6 chains much more rapidly than unmodified AT3: reaction products were detectable within 10 min and went to completion by 6 h (Figure 3). The major reaction products were tetra-, tri-, and di-Ub (Figure 3) even in reactions extending for 20 h (Figure 2). The fact that both AT3 and AT3-Ub cleave longer chains into smaller chains rather than into mono-Ub (Figures 2 and 3) suggests that AT3 ubiquitination enhances activity towards K63-Ub6 chains without affecting the manner in which it cleaves. Figure 3.Accelerated cleavage of K63-Ub6 chains by AT3-Ub. Left: untagged AT3 or AT3-Ub (ubiquitinated in vitro) was incubated with K63-Ub6 chains for the indicated times. Anti-AT3 blot shows AT3 species used in reactions. Right: time-course of Ub reaction product appearance, determined by semi-quantification of western blots as shown on the left. Means±standard deviations (s.d.), N=5. Asterisks: statistically significant difference at P<0.001. Download figure Download PowerPoint AT3-Ub prepared in vitro comprises a ladder of ubiquitinated species (Figures 2C and 3), suggesting that AT3 mono-ubiquitination may not be the only activating post-translational event. Therefore, we compared the activity of AT3-Ub prepared with wild-type UbcH5c, which can form poly-Ub chains, with AT3-Ub prepared with UbcH5c(S22R), which only mono-ubiquitinates substrates because it cannot extend Ub chains (Brzovic et al, 2006). AT3-Ub prepared either way was more active than unmodified AT3 (Supplementary Figure 2). Together, these data show that AT3 activity is enhanced by ubiquitination and that mono-ubiquitination of AT3 is sufficient for activation. UIMs are not necessary for increased AT3 activity by ubiquitination The UIMs of AT3 serve at least two functions: they mediate high-affinity binding to Ub chains and restrict the types of chains that can be cleaved by AT3. Whereas normal AT3 prefers cleaving K63 linkages, AT3 with mutated UIMs cleaves K63 and K48 linkages approximately equally well (Winborn et al, 2008). Considering this important function of UIMs in modulating the ability of AT3 to bind and cleave Ub chains, we hypothesized that an intramolecular interaction between UIMs and Ub conjugated to AT3 might underlie AT3 activation by ubiquitination. We investigated this possibility with AT3 mutated at conserved residues in each UIM (A → G/S → D; denoted as AT3(UIM*)). These mutations eliminate the ability of AT3 to bind Ub chains with high affinity (Supplementary Figure 3). The extent of activation of AT3(UIM*)-Ub towards K63 chains was less robust than that observed with AT3(WT)-Ub (Figure 4A). In contrast, AT3(UIM*)-Ub showed strong enhancement of activity towards penta-Ub K48 chains (K48-Ub5; Figure 4B). Intriguingly, the pattern of activation towards K63 versus K48 chains is reversed for AT3(WT)-Ub and AT3(UIM*)-Ub: AT3(WT)-Ub shows greater enhancement of K63 chain cleavage, whereas AT3(UIM*)-Ub shows greater enhancement of K48 chain cleavage (Supplementary Figure 4). Thus, although the UIMs are required for preferential cleavage of homotypic K63 chains by AT3 (Winborn et al, 2008), they are not necessary for activation of AT3 by ubiquitination. Figure 4.The UIMs of AT3 are not necessary for activation by ubiquitination. (A) Ubiquitinated, UIM-mutated AT3 shows enhanced activity towards K63-Ub6 chains. Left: wild-type (AT3(WT)) or UIM-deficient AT3 (AT3(UIM*)) was ubiquitinated in vitro, then incubated with K63-Ub6 chains. Right: semi-quantitative representation of western blot results as on the left. Means±s.d.; N=6. (B) AT3(UIM*)-Ub cleaves K48-Ub5 chains more quickly than AT3(UIM*). Left: AT3 species were incubated with K48-Ub5 chains. Right: semi-quantitative representation of four independent experiments. Means±s.d. Anti-AT3 blots show GST–AT3 species used in reactions in (A) and (B). Download figure Download PowerPoint To examine which domains of AT3 are sufficient for ubiquitination-dependent activation, we studied the effect of ubiquitination on the activity of the isolated Josephin domain. The Josephin domain alone (denoted as AT3(J)) comprises amino acids 1–182 and excludes all UIMs. Although AT3(J)-Ub was not activated towards K63-Ub6 chains (Figure 5A), it cleaved K48-Ub5 chains more rapidly than unmodified Josephin domain (Figure 5B). These results support the conclusion that UIMs are not necessary for activation of AT3 by ubiquitination. Figure 5.Ubiquitination of the Josephin domain enhances cleavage of K48-linked Ub chains. (A) Ubiquitination of the Josephin domain does not enhance activity towards K63-Ub6 chains. Left: Josephin domain of AT3 (AT3(J)) was ubiquitinated in vitro and incubated with K63-Ub6 chains. Right: semi-quantitative representation of experiments conducted as on the left. Means±s.d.; N=3. (B) Left: ubiquitinated Josephin domain has enhanced activity towards K48-Ub5 chains. Right: semi-quantitative representation of experiments conducted as on the left. Means±s.d.; N=5. Asterisks: statistically significant difference at P<0.01. GST-tagged and untagged Josephin domain proteins yield similar results. (C) AT3 activation by ubiquitination does not occur in trans. AT3 species were prepared in vitro. Catalytically inactive AT3 (AT3(C14A)), either unmodified or ubiquitinated, was incubated with wild-type AT3 (AT3(WT)) and K63-Ub6 chains. Anti-AT3 blot shows GST–AT3 species used in reactions. Results representative of three independent experiments are shown. Download figure Download PowerPoint AT3 activation by ubiquitination does not occur in trans AT3 has been reported to self-associate in cells (Todi et al, 2007a) and in vitro (Ellisdon et al, 2006). It is thus possible that activation of AT3 by ubiquitination depends on homomeric interactions between AT3 proteins. To explore this possibility, we prepared unmodified and ubiquitinated forms of catalytically inactive AT3 (AT3(C14A)) to test in DUB assays with AT3(WT). AT3(C14A) or AT3(C14A)-Ub was mixed with unmodified, wild-type AT3 at equimolar concentrations, then incubated with K63-Ub6 chains. AT3(WT) activity towards K63-Ub6 was not enhanced by the presence of AT3(C14A)-Ub, indicating that activation most likely occurs in cis (Figure 5C). Moreover, as both AT3 and AT3-Ub fractionate identically by size-exclusion chromatography (Supplementary Figure 5), ubiquitination of AT3 does not appear to lead to a major change in AT3 tertiary structure. AT3 runs as an elliptical protein (Chow et al, 2006), enabling us to use size-exclusion chromatography to detect large conformational changes. This method, however, cannot detect conformational changes occurring at a domain or subdomain level. Activity of pathogenic AT3 is also enhanced by ubiquitination PolyQ expansion in AT3 causes the neurodegenerative disease SCA3/MJD. Disease protein context and the normal cellular functions of the implicated protein are important factors in the selective, progressive neurodegeneration seen in polyQ diseases (reviewed by Gatchel and Zoghbi, 2005; Todi et al, 2007b; Williams and Paulson, 2008). Therefore, understanding how AT3 function is affected by polyQ expansion may provide clues to pathogenesis. Expanded AT3 binds and cleaves Ub chains similarly to normal AT3 in vitro (Burnett et al, 2003; Chai et al, 2004; Winborn et al, 2008). In cells, however, expanded AT3 (AT3(Q80)) leads to accumulation of ubiquitinated proteins, probably through an indirect mechanism (Winborn et al, 2008). Given both increased AT3 activity when it is ubiquitinated and the fact that AT3(Q80) becomes ubiquitinated when coexpressed with Ub (Todi et al, 2007a; and data not shown), we investigated the ability of AT3(Q80)-Ub to cleave K63 and K48 chains. Expanded AT3-Ub prepared in vitro showed enhanced activity towards both K63 and K48 chains (Figure 6A). This enhancement did not differ from that of wild-type AT3-Ub (Figure 6B). Thus, polyQ expansion in AT3 does not appreciably alter activation by ubiquitination in vitro. Figure 6.Expanded (pathogenic) AT3 is also activated by ubiquitination. (A) Recombinant, expanded AT3 (AT3(Q80)) was ubiquitinated in vitro, then incubated with the indicated Ub chains. AT3(Q80)-Ub shows greater enhancement of activity towards K63-Ub6 chains. (B) Normal and expanded AT3 show similar enhancement in activity when ubiquitinated. Results in (A) and (B) are each representative of three independent experiments. Anti-AT3 blots show GST–AT3 species used in reactions. Download figure Download PowerPoint Ubiquitination of AT3 increases with certain stressors As AT3 is implicated in Ub-dependent protein quality control (Wang et al, 2000, 2006; Doss-Pepe et al, 2003; Chai et al, 2004; Warrick et al, 2005; Zhong and Pittman, 2006), we investigated whether perturbations in protein homoeostasis alter ubiquitination of endogenous AT3. In M17 (Figure 1C) and Cos7 cells (Figure 7A), levels of AT3-Ub increased during proteasome inhibition. Ub overexpression also led to higher levels of AT3-Ub (Figure 7A). Jointly inhibiting the proteasome and overexpressing Ub did not further increase AT3 ubiquitination, suggesting a saturation point for endogenous AT3 ubiquitination. Proteasome inhibition led to higher levels of conjugated Ub, whereas Ub overexpression increased levels of both conjugated and non-conjugated Ub (Figure 7A), suggesting that the ubiquitination status of AT3 functions as a feedback sensor of the overall levels of conjugated Ub in the cell. Figure 7.Levels of ubiquitinated endogenous AT3 are enhanced by certain stressors. (A) The fraction of endogenous AT3 ubiquitinated under basal conditions is enhanced by proteasome inhibition or by increasing Ub levels. Left: top blot shows Cos7 cells treated with lactacystin (10 μM; 10 h), or transfected with HA–Ub and treated as indicated. Bottom blot shows the same cell lysates loaded in 4–20% SDS–PAGE gel to probe for conjugated and non-conjugated Ub. All lanes are from the same exposure of the same blot. Right: AT3 was immunoprecipitated from Cos7 cells treated with lactacystin. Endogenous AT3-Ub bands are detected by both anti-AT3 and anti-Ub antibodies, confirming the identity of HMW AT3 species on the left as AT3-Ub. Representative results from four independent experiments are shown. (B) Induction of the unfolded protein response transiently leads to higher levels of ubiquitinated endogenous AT3. Treatment of Cos7 or 293 cells with the UPR inducer, DTT (5 μM), for the indicated times leads to the appearance of a higher molecular AT3 band consistent with ubiquitinated AT3. AT3 doublets in 293 cells most likely reflect allelic differences in CAG/polyQ repeat length. Representative results from at least three independent experiments are shown. (C) Heat shock does not alter the levels of ubiquitinated endogenous AT3. Cos7 cells were heat-shocked briefly (left) or for a prolonged time (right). Hsp70 levels confirmed induction of the heat shock response in treated cells. Representative results from at least three independent experiments are shown. Equal protein was loaded in (A–C). (D) Pathogenic AT3 is more heavily ubiquitinated than unexpanded AT3 in brain lysates from transgenic mice expressing normal (AT3(Q15)) or expanded (AT3(Q84)) AT3. Left: AT3(Q84) is more heavily ubiquitinated than AT3(Q15). Asterisk: endogenous AT3. AT3(Q15) mice express the protein more highly than the AT3(Q84) mice. Right: stringent immunopurification of 12-month-old AT3(Q84)-expressing brains shows that the HMW bands in left (AT3(Q84)-Ub) are ubiquitinated AT3. Download figure Download PowerPoint Ubiquitination of endogenous AT3 also increased when the unfolded protein response (UPR) was induced by DTT, although DTT did not appreciably alter levels of conjugated Ub (Figure 7B). In contrast, acute or prolonged heat shock did not affect AT3 ubiquitination or Ub levels in Cos7 (Figure 7C), 293, or M17 cells (data not shown). Taken together, these data suggest a physiological function of AT3 ubiquitination during some, but not all, stressors. Lastly, we investigated AT3 ubiquitination in brain lysates from transgenic mice expressing normal (AT3(Q15)) or pathogenic AT3 (AT3(Q84)) driven by the prion promoter (Cemal et al, 2002). Mice expressing AT3(Q84) show motor anomalies as early as 6 weeks of age (Cemal et al, 2002; and our unpublished data). AT3(Q84) was consistently more heavily ubiquitinated than AT3(Q15) in mice aged 4–12 months (Figure 7D). Importantly, this ubiquitinated AT3(Q84) is in the soluble fraction of brain lysates, indicating that it is not sequestered into inclusions. These data suggest that ubiquitination of expanded AT3 may be involved in SCA3/MJD pathogenesis. Discussion We have established that the catalytic activity of a disease-related DUB, AT3, is directly enhanced by ubiquitination. Although ubiquitination increases the enzymatic activity of AT3, it does not alter its preference for cleaving certain Ub linkages in vitro. The N-terminal half of AT3 containing the catalytic Josephin domain is sufficient for ubiquitination-dependent activation, although the C-terminal UIMs that are known to bind and restrict the types of Ub chains cleaved by AT3 are not necessary for this phenomenon (Figure 8). In mammalian cells, the fraction of endogenous AT3 that is ubiquitinated under normal conditions increases when the proteasome is inhibited, when excess conjugated Ub is present, or when the UPR is induced. Our results show that ubiquitination of a DUB can directly regulate its enzymatic properties, and suggest that activation of AT3 by this post-translational modification serves a function in the cellular response to certain stressors. Figure 8.Effects of protein domains on catalytic properties of AT3. Ubiquitination of the Josephin domain is sufficient for activation. Although the UIMs are not necessary for AT3 activation by ubiquitination, they confer Ub linkage preference to the catalytic domain. Download figure Download PowerPoint Ubiquitination as a regulator of enzymatic activity It makes biological sense that DUB activity would be regulated through feedback mechanisms tied to Ub-dependent pathways. Regulation of activity has been reported for some DUBs at various levels, including transcription (upregulation), phosphorylation (downregulation), interaction with single proteins (activation or inhibition), incorporation into complexes (upregulation or downregulation), or proteasomal degradation (inactivation) (reviewed by Nijman et al, 2005; Ventii and Wilkinson, 2008; Yao et al, 2008). In addition, USP1, a DUB that functions in DNA repair, inactivates itself through cleavage upon ultraviolet irradiation (Huang et al, 2006). Cleavage of USP7, which functions in apoptosis, is accomplished by caspase 3 (Vugmeyster et al, 2002). Here, we report for the first time that ubiquitination of a DUB directly regulates its activity. Among the nearly 100 DUBs expressed in humans, at least six are known to be ubiquitinated: USP4, USP6, USP7, UCH-L1, AT3, and Josephin 1 (Shen et al, 2005; Wada and Kamitani, 2006; Fernandez-Montalvan et al, 2007; Meray and Lansbury, 2007; Todi et al, 2007a; and our unpublished data). At least five additional DUBs contain UIMs, a Ub-binding domain that often
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