Abstract

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair^{1, 2, 3}. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.

Introduction

The attachment of a single ubiquitin molecule (monoubiquitin) to a variety of cell-surface receptors is sufficient to drive their internalization and degradation^{2, 4, 5, 6}. Several endocytic adaptor proteins that control these processes — such as Eps15, epsins and Hrs — harbour one or more ubiquitin-binding domains (UBDs) that are able to recognize the ubiquitinated receptors and sort them along the endocytic pathway². Interestingly, UBDs often mediate monoubiquitination of the proteins that contain them^{2, 3, 7}. However, it is not yet understood whether and how monoubiquitination of ubiquitin-binding proteins may contribute to the regulation of their functions in vivo.

The suppressors of T-cell receptor signalling (Sts) 1 and 2 are ubiquitin-binding proteins that suppress signalling via T-cell receptors⁸ and regulate endocytic sorting of receptor tyrosine kinases^{9, 10}. Sts1 and Sts2 are recruited to activated epidermal growth factor (EGF) and platelet-derived growth factor receptors via the ubiquitin ligase Cbl and bind to ubiquitinated receptor complexes through their amino-terminal ubiquitin-associated (UBA) domains. Both of these steps are required for the ability of Sts proteins to interfere with EGF receptor (EGFR) endocytosis and degradation, but the molecular mechanisms that underlie regulation of Sts in these processes remain elusive.

Monoubiquitination of Sts1 and Sts2, which is observed as a shift in their mobility on SDS–PAGE gels and which corresponds to the addition of a monoubiquitin to Sts1 and Sts2, is potently enhanced by overexpression of monoubiquitin in the cell (Fig. 1a). As several UBDs promote ubiquitination of their host proteins³, we investigated whether ubiquitination of Sts1 and Sts2 was also dependent on the presence of functional UBA domains. Mutation of glycine (G) and phenylalanine (F) in the highly conserved MGF motif of the UBA of Sts1 (Sts1-GF/AA; Fig. 1b) abolished binding to monoubiquitin (Fig. 1b), as well as its monoubiquitination (Fig. 1a, b, lower panel). The same mutations did not completely block ubiquitin binding of Sts2 (Fig. 1b). However, mutation of Lys 40 in the UBA of Sts2 impaired its interaction with monoubiquitin (Fig. 1b) and monoubiquitination of Sts2 (Fig. 1, b). These results show that the UBA domains of Sts1 and Sts2 are crucial determinants of both their ubiquitin binding and monoubiquitination, and provide the first example of proteins that undergo monoubiquitination that is mediated by UBA domains.

Figure 1: Functional UBA domains are required for monoubiquitination of Sts1 and 2 in cells.

(a) Overexpression of ubiquitin (Ub) leads to UBA-dependent monoubiquitination of Sts1–2. Lysates of HEK293T cells co-expressing the indicated Flag–Sts1–2 constructs and HA–Ub or empty vector were subjected to immunoprecipitation (IP) using an -Flag antibody, followed by western blotting (WB). (b) Alignment of the UBA domains of Sts1 and 2. Black bars indicate residues that have been mutated for Ub-binding and ubiquitination assays. Lysates of HEK293T cells overexpressing Flag–Sts1–2 wild-type (WT) or the indicated UBA mutants were subjected to GST–ubiquitin pull-down assays, followed by western blotting. TCL, total cell lysate. (c) Localization of minor (purple) and major (red, Lys 202) ubiquitination sites of Sts2 (upper panel). Lower panel: Lys 202 was mutated to arginine and the corresponding Flag–Sts2 construct was transfected in HEK293T cells, together with HA–ubiquitin or empty vector. The lysates were subjected to immunoprecipitation and western blotting.

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To identify the lysine(s) in Sts proteins that had been monoubiquitinated in vivo, we purified human Sts2 by immunoaffinity columns and subjected the monoubiquitinated form to trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS–MS) analysis (see Supplementary Information, Fig. S1). We identified Lys 202, which accounted for the majority of incorporated ubiquitin in the Sts2 molecule, in addition to three minor sites (Lys 15, Lys 309 and Lys 358) (Fig. 1c; and see Supplementary Information, Fig. S1). Mutation of Lys 202 to arginine (Sts2^K202R) efficiently impaired Sts2 monoubiquitination (Fig. 1c), supporting the notion that Lys 202 is the main monoubiquitination site of Sts2 in vivo.

Monoubiquitination can have several functional consequences for the targeted protein, including changes in binding properties, subcellular localization and activity^{1, 2, 3}. Using in vitro ubiquitin-binding assays, we found that monoubiquitinated Sts1 and Sts2 did not interact with exogenous monoubiquitin (Fig. 2a). We tested whether this phenomenon is also true for other endocytic adaptor proteins that are known to be monoubiquitinated, such as the ubiquitin interacting motif (UIM)-containing Eps15 and Hrs^{7, 11, 12}. Indeed, monoubiquitinated forms of Eps15 (Fig. 2b) and Hrs (Fig. 2c) did not bind to glutathione S-transferase (GST)-fused monoubiquitin, whereas the same unmodified protein efficiently did so. To further validate these findings, we created permanently monoubiquitinated proteins by fusing a ubiquitin moiety to the carboxy-terminal part of Sts1, Sts2, Eps15 and Hrs (Fig. 2f). Sts1–ubiquitin, Sts2–ubiquitin, Eps15–ubiquitin and Hrs–ubiquitin chimerae maintained their ability to interact with non-ubiquitinated targets, including Cbl, epsin and STAM, respectively (see Supplementary Information, Fig. S2). However, when tested for binding to GST-fused ubiquitin, all ubiquitin chimerae were impaired in their ability to interact with exogenous ubiquitin (Fig. 2b–e). To exclude that this effect was due to misfolding imposed by the fusion of ubiquitin to these proteins, we introduced a mutation in the conserved hydrophobic patch of ubiquitin (Ile44) that abolishes its binding to known UBDs³, including the UBA of Sts1 and Sts2 (Fig. 2d, e). Mutation of Ile44 to Ala in the ubiquitin chimerae completely restored the ability of Sts1, Sts2, Eps15 and Hrs to bind to GST–monoubiquitin (Fig. 2b–e). These data support the concept that monoubiquitination of both UIM- and UBA-containing proteins neutralizes their ubiquitin-binding capacities.

Figure 2: Monoubiquitination of Sts1–2, Hrs and Eps15 abolishes their binding to exogenous ubiquitin.

(a) Lysates of HEK293T cells overexpressing either Flag–Sts1 or Flag–Sts2 and HA–ubiquitin were subjected to GST–ubiquitin pull-down assays, followed by western blotting (WB). (b) Neither natively monoubiquitinated Eps15 nor Eps15 with C-terminally fused monoubiquitin (a single ubiquitin molecule) bind to exogenous ubiquitin. Lysates of HEK293T cells overexpressing the indicated constructs were subjected to GST–ubiquitin pull-down assays, followed by western blotting. (c) Monoubiquitinated Hrs does not bind to GST–ubiquitin. Lysates of HEK293T cells overexpressing Myc–Hrs, together with HA–ubiquitin, Myc–Hrs–ubiquitin or Myc–Hrs–ubiquitin^I44A, were subjected to GST–ubiquitin pull-down assays, followed by western blotting. (d) Lysates of HEK293T cells overexpressing Flag–Sts1 wild type or the indicated chimerae were subjected to GST–ubiquitin pull-down experiments and analysed by western blotting. (e) Flag–Sts2 wild type or the indicated chimerae were subjected to GST–ubiquitin pull-down experiments and analysed by western blotting. (f) Schematic representation of ubiquitin chimerae that were used for the described experiments. EH, Eps15-homology domain; FYVE, PtdInsP₃ binding domain; SH3, Src-homology 3 domain; VHS, Vps27/Hrs/STAM domain.

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We hypothesized that the loss of ubiquitin binding of the monoubiquitinated proteins might be due to an intramolecular interaction between the UBD and the monoubiquitin on the same molecule, thereby preventing its binding to neighbouring ubiquitin targets. Given the fact that Eps15, Hrs and Sts proteins are able to dimerize or oligomerize, and are also found in multimeric protein complexes in cells^{2, 9}, it is possible that their monoubiquitinated forms engage in intramolecular (within the single molecule), intermolecular (between different molecules of the homo-oligomeric complex) or transmolecular (between different proteins in heterologous complexes) interactions. We therefore investigated whether the intramolecular binding between monoubiquitin and the UBA of Sts1 and 2 is sufficient for its auto-inhibition. Dimerization-deficient Sts1–PGM (phosphoglycerate mutase domain) and its ubiquitin chimera were expressed in Escherichia coli, which lacks the ubiquitin conjugation system as well as UBD-containing proteins. As shown in Fig. 3a, bacterially expressed Sts1–PGM readily interacted with GST–ubiquitin, whereas Sts1–PGM–ubiquitin was impaired in binding to exogenous ubiquitin. When the same constructs were subjected to chemical cross-linking under conditions in which wild-type Sts1 was completely cross-linked, there was no detectable dimerization of Sts1–PGM–ubiquitin (Fig. 3b). This result confirms that there is no biochemical evidence for intermolecular interactions between the UBA of monomeric Sts1–PGM and the attached ubiquitin to another Sts1–PGM molecule. The same block in ubiquitin binding was also found in the context of the full molecule, and mutation of I44A in Sts1–ubiquitin was able to restore binding to ubiquitin (see Supplementary Information, Fig. S2e). Last, to directly confirm the conformational change resulting from intramolecular UBD–ubiquitin interactions in ubiquitinated Sts1–2–PGM, we took advantage of fluorescence resonance energy transfer (FRET) technology. We attached the FRET donor cyan fluorescence protein (CFP) to the C terminus and the acceptor citrine to the amino terminus of Sts2–PGM (Cit–Sts2PGM–CFP, Fig. 3c). First, we confirmed that this construct retained the features of the untagged variant described above. For this purpose, we checked its ubiquitination as well as its ubiquitin-binding properties, and found that Cit–Sts2PGM–CFP behaved normally in all assays (see Supplementary Information, Fig. S2f). When expressed in HEK293T cells, this construct led to a FRET signal that was significantly higher than when Cit–Sts2–PGM and Sts2–PGM–CFP were co-expressed (Fig. 3d), indicating that citrine and CFP are in close proximity. Importantly, mutation of the major ubiquitination site Lys 202 to arginine resulted in a decrease of the FRET signal to almost background level. The same decrease was observed in a ubiquitin-binding-deficient mutant in which Lys 40 was mutated to arginine (Cit–Sts2PGM–UBA^*–CFP; Fig. 3c, d). This indicates that, in these mutants, citrine and CFP are too distant from each other to enable energy transfer. These data demonstrate that intramolecular interactions between monoubiquitin and the UBA domain in Sts–PGM occur and are sufficient to block the ubiquitin-binding ability of Sts1 and 2.

Figure 3: Attachment of ubiquitin to bacterially expressed Sts1 leads to intramolecular UBD–ubiquitin interactions that impairs binding to exogenous ubiquitin.

(a) Bacterial lysates containing dimerization-deficient Sts1–PGM or Sts1–PGM–ubiquitin were subjected to GST pull-downs and then analysed by western blotting (WB). (b) Attachment of ubiquitin does not induce dimerization of Sts–PGM. Bacterial lysates containing Flag–Sts1–PGM or Flag–Sts1–PGM–ubiquitin (left panel) and full-length Sts1 (right panel) were subjected to chemical cross-linking using bis(sulphosuccinimidyl) subarate (BSS) and then analysed by western blotting. K, relative molecular mass in thousands. (c) Schematic representation of the constructs used for fluorescence resonance energy transfer (FRET) experiments. CFP, cyan fluorescence protein. (d) Left panel: Cell lysates expressing the indicated constructs were analysed using a Victor³ multilabel reader (Perkin Elmer). ^*** = P value < 0.001 in a one-tailed Student's t-test with unequal variance. The FRET signal is shown as FRET–CFP in arbitrary units, normalized within each experimental replicate so that the maximum signal equals 1. The error bars represent the mean 1SD. Right panel: The same lysates were subjected to immunoblotting.

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Our experimental results were additionally evaluated by comparing the thermodynamic properties of intramolecular versus transmolecular ubiquitin binding. The presented biophysical estimates and mathematical equations indicate that monoubiquitinated Sts1 and Sts2 in solution will exclusively bind intramolecularly to their own ubiquitin and not to exogenous ubiquitin (see Supplementary Information). However, if these proteins are localized on scaffolds or platforms (for example, on endosomes), they can engage in transmolecular interactions as the equilibrium between intramolecular versus transmolecular binding depends on the geometrical arrangement of the complex and on the number of ubiquitins that are attached to the target protein (see Supplementary Information). Notably, there will be a dynamic exchange between the intramolecular and transmolecular bound state of UBD-containing proteins that allows a flexible adaptation to changes in the local environment and might also explain the observations that Sts–ubiquitin chimerae are monoubiquitinated to a certain extent (data not shown).

Having created the Sts2–ubiquitin chimera, which mimicks permanent monoubiquitination and the Sts2 mutant (Sts2^K20R) that is not monoubiquitinated, we were able to analyse the functional importance of monoubiquitination of Sts proteins in cells. Sts1 and Sts2 have been shown to inhibit EGFR degradation by binding to the ubiquitin ligase Cbl and interacting with ubiquitinated receptor complexes via their UBA domains⁹. To reliably detect differences in the ability of Sts2, Sts2–ubiquitin and Sts2^K202R mutant to interfere with EGFR degradation even in the presence of endogenous Sts1, we overexpressed them along with EGFR. We made use of the green fluorescent protein (GFP)-tagged EGFR, the degradation kinetics of which are the same as those of wild-type and endogenous receptors (data not shown). Expression of Sts2 in HEK293T cells caused stabilization and accumulation of EGFR–GFP following ligand stimulation (Fig. 4a). By contrast, overexpression of Sts2–ubiquitin, but not of the Sts–ubiquitin^I44A, chimerae in cells caused significantly decreased EGFR levels (Fig. 4a). Equivalent data were obtained for Sts1 and Sts1–ubiquitin chimerae on EGFRs at steady state (data not shown) and after EGF stimulation (see Supplementary Information, Fig. S3a). More importantly, Sts2^K202R, which cannot be monoubiquitinated in cells, stabilized EGFRs more significantly than Sts2 wild type following EGF stimulation (Fig. 4a), indicating that monoubiquitination of Sts2 inhibits its capacity to block ligand-induced degradation of EGFRs. Sts2 therefore represents the first UBD-containing protein, which is monoubiquitinated at a defined lysine residue and the mutation of which is functionally significant in vivo.

Figure 4: Monoubiquitination of Sts1–2 and Hrs affects their ability to regulate cargo sorting.

(a) Sts2^K202R shows enhanced stabilization of EGFR compared with wild-type Sts2. HEK293T cells co-expressing EGFR–GFP and either of the indicated Flag–Sts2 constructs were stimulated with EGF and analysed by flow cytometry. The data represent the mean SEM of three experiments. (b) HeLa cells were co-transfected with ubiquitin–TfR, Myc-tagged Hrs, Hrs–ubiquitin or Hrs^S270E and Alexa⁵⁶⁸-transferrin (Tf) was internalized for 15 min. After a 2-h chase period in the presence of cycloheximide and leupeptin, the cells were processed for confocal microscopy. Cell-associated Alexa⁵⁶⁸-transferrin was quantified as described in Methods. Error bars denote SEM. Ub–TfR: n = 10; Ub–TfR + Hrs: n = 40; Ub–TfR + Hrs–Ub: n = 40; Ub–TfR + Hrs^S270E: n = 25.

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To investigate whether a similar negative regulation by monoubiquitination can be found in other components of the endocytic sorting machinery, we tested the role of monoubiquitination of Hrs in Hela cells. Hrs has been previously implicated in the sorting of ubiquitinated transmembrane receptors into clathrin-coated microdomains of the early endosome^{13, 14, 15}. Because overexpression of Hrs inhibits recycling of an endocytosed ubiquitin-transferrin receptor (TfR) chimera (ubiquitin–TfR) through a mechanism that requires ubiquitin binding⁴, we investigated whether monoubiquitination of Hrs would promote recycling of ubiquitin–TfR. For this purpose, HeLa cells were co-transfected with ubiquitin–TfR and the indicated Hrs constructs, and the intracellular accumulation of endocytosed transferrin following a 2-h chase period was measured. Overexpression of wild-type Hrs or the Hrs–ubiquitin^I44A chimera led to a strong cellular retention of transferrin, whereas Hrs–ubiquitin, which mimics a mutant with a non-functional UIM, Hrs-S270E, was unable to retain transferrin in endosomes (Fig. 4b; see Supplementary Information, Fig. S4c). The ability of Hrs to bind and recruit clathrin is thought to be important for its function as an endosomal sorting receptor¹⁶, and we considered the possibility that ubiquitination could affect clathrin recruitment to endosomes. However, overexpression of both Hrs and the Hrs–ubiquitin chimera equally recruited clathrin to early endosomes, whereas Hrs(1–706), which lacks the clathrin-binding C terminus, did not cause any clathrin recruitment (see Supplementary Information, Fig. S3b). Taken together, monoubiquitination of Hrs does not significantly affect its ability to recruit clathrin to early endosomes, but leads to functional inactivation of the UIM domain, thereby affecting trafficking of ubiquitin–TfR.

Recent studies have shed new light on the possible functions of Eps15, Eps15R and epsin in regulating the endocytic route taken by ubiquitinated cargoes in cells^{17, 18}. It was proposed that the ubiquitin-binding competent Eps15, Eps15R and epsin may engage in trans interactions with ubiquitinated cargoes, thereby promoting clathrin-independent endocytosis following stimulation with high doses of EGF¹⁸. Moreover, it was shown that the UIM of Eps15 is required for its membrane recruitment and co-localization with ligand-bound EGFRs^{19, 20}. To test whether monoubiquitination of Eps15 regulates its co-localization with activated EGFR, we overexpressed Eps15, Eps15–ubiquitin and Eps15–ubiquitin^I44A chimerae in Hela cells that had been treated with high doses of EGF. In these assays, Eps15 and Eps15–ubiquitin^I44A chimerae showed significant co-localization with EGFR-positive vesicles, whereas Eps15–ubiquitin chimerae were diffusely expressed in the cytoplasm and were not associated with endocytosed EGFRs (Fig. 5a; see Supplementary Information, Fig. S4). These data indicate that monoubiquitination of Eps15 inhibits its association and co-localization with EGFR-containing endocytic vesicles.

Figure 5: Monoubiquitination of Eps15 impairs localization of Eps15 to EGFR-positive endosomes.

(a) Eps15 and Eps15–ubiquitin^I44A strongly colocalized with endoctyosed endogenous EGFR in Hela cells that have been stimulated with 100 ng ml^-1 EGF for 5 min, whereas Eps15-ubiquitin was mostly cytoplasmic. Colcalization was quantified by counting endosomal vesicles that were double-positive vesicles for endogenous EGFR and the indicated constructs. Statistical analysis was performed using the two-sided Wilcoxon test: Eps15 WT > Eps15–ubiquitin (^***, P < 0.0001); Eps15–ubiquitin < Eps15–ubiquitin^I44A (^***, P < 0.0001); Eps15 WT = Eps15–ubiquitin^I44A (P = 0.34). Eps15: n = 21; Eps15–ubiquitin: n = 21; Eps15–ubiquitin^I44A: n = 21. (b) Proposed mechanism of monoubiquitin-mediated regulation of endocytic adaptor proteins: In solution, monoubiquitinated UBD-containing proteins adopt a closed, auto-inhibited conformation due to intramolecular UBD–ubiquitin interactions. This pool of proteins will be inactive with respect to transmolecular binding to ubiquitinated targets; for example, cargo sorting. At the same time, a significant pool of the adaptor protein is captured on scaffolds or platforms (for example, on endosomes or complexes on EGFRs). Depending on the protein, different scenarios will take place: Monoubiquitination precludes localization of the adaptor on the scaffold (as is the case for Eps15). Therefore, the adaptor must be de-ubiquitinated to actively participate in cargo sorting. Alternatively, the monoubiquitinated adaptor can be recruited to the scaffold but transmolecular UBD interactions are dependent on the geometrical arrangement of the domains. Multiple monoubiquitination of cargo can, additionally, shift the equilibrium from intra- to transmolecular ubiquitin binding.

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Our results demonstrate that monoubiquitination of UBD-containing proteins triggers intramolecular interactions with the UBDs, thereby preventing them from binding in trans to ubiquitinated targets. This is a common phenomenon for several UBDs, including UBA (Fig. 2) and UIM (Fig. 2), as well as the novel UBM and UBZ domains²¹. Changes in their ubiquitination status seem to induce a conformational switch from a ubiquitin-binding state of these proteins to an intramolecular monoubiquitin-inhibited state (Fig. 5b). This could explain how UBD proteins that constitute the endocytic sorting machinery can dynamically exchange their ubiquitinated cargoes along the endosomal compartments. Although the main outcome of monoubiquitination of UBD proteins is inhibition of their ubiquitin-binding capacity, broader functional consequences can also be conceived, including changes in enzymatic activity^{1, 3}, binding properties^{17, 22} or intracellular localization (Fig. 5a). Biophysical calculations reveal an important difference in the behaviour of proteins in solution and of proteins that are anchored on scaffolds. Freely diffusible monoubiquitinated UBD-containing proteins will invariably engage in intramolecular UBD–ubiquitin interactions due to the high local concentration of ubiquitin being attached to the same molecule. However, a significant pool of proteins is embedded into multimeric complexes in vivo, which constrains the mobility of the protein components. In such conditions, the reaction equilibrium shifts towards transmolecular interactions (Fig. 5b). At the same time, the attached monoubiquitin becomes accessible and is either cleaved off or, alternatively, can be available as an additional binding surface, thereby positively promoting the assembly of ubiquitin-linked protein networks. Taken together, ubiquitin plays a dual role in endocytic pathways: it acts as a sorting tag on trafficking cargoes and as a regulatory signal on UBD-containing proteins.

Acknowledgements

We thank S. Urbe, S. Polo and P.P. DiFiore for discussions and help with these studies, as well as W. Mueller-Esterl and members of the Dikic laboratory for constructive comments and critical reading of the manuscript. We are very thankful to M. Offterdinger and P. Bastiaens for help with the FRET experiments and to I. Konrad for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DI 931/1-1) and Boehringer Ingelheim Fonds (to I.D.) and the Danish National Research Foundation (to M.M.). C.R. receives a postdoctoral fellowship from the Norwegian Cancer Society.

Competing interests statement

The authors declare no competing financial interests.

Received 25 October 2005; Accepted 19 December 2005; Published online 22 January 2006.

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