The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain Claudia Baumann, Cecilia K. Lindholm*, Donata Rimoldi and Fre ´ de ´ ric Le ´ vy  Ludwig Institute for Cancer Research Ltd, University of Lausanne, Switzerland Introduction Ubiquitin (Ub) ligases play a crucial role in regulating intracellular protein levels in eukaryotes. The reaction catalysed by Ub ligase activity, ubiquitylation, results in a reversible covalent modification of substrate proteins and constitutes a signal for proteasomal and lysosomal degradation. This is an important mecha- Keywords E3 ligase; Itch; protein–protein interaction; sorting nexin 9; ubiquitin Correspondence C. Baumann, Ludwig Institute for Cancer Research Ltd, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland Fax: +41 21 692 59 95 Tel: +41 21 692 59 77 E-mail: Claudia.Baumann@licr.unil.ch Present address *Philip Morris International SA, Neucha ˆ tel, Switzerland  Debiopharm SA, Lausanne, Switzerland (Received 9 January 2010, revised 20 April 2010, accepted 27 April 2010) doi:10.1111/j.1742-4658.2010.07698.x The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting protein 4), possess up to four WW domains for specific association with PY motif-containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using trun- cated proteins, we found that the interaction with SNX9 is mediated by the proline-rich domain (PRD) of Itch, a domain distinct from the conven- tional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD- containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs. Structured digital abstract l MINT-7889719: SNX18 (uniprotkb:Q96RF0) physically interacts (MI:0915) with ITCH (uni- protkb: Q96J02)byanti tag coimmunoprecipitation (MI:0007) l MINT-7889571, MINT-7889619: ITCH (uniprotkb:Q96J02) physically interacts (MI:0915)with SNX9 (uniprotkb: Q9Y5X1)bypull down (MI:0096) l MINT-7889653: Melan-A (uniprotkb:Q16655) physically interacts (MI:0914)withNEDD4 (uniprotkb: P46934) and ITCH (uniprotkb:Q96J02)bypull down (MI:0096) l MINT-7889591, MINT-78896 73, MINT-7890033 : SNX9 (uniprotkb:Q9Y5X1) physically interacts ( MI:0915)withITCH (uniprotkb:Q96J02)byanti tag coimmunoprecipitation (MI: 0007) l MINT-7889689: SNX 9 (uniprot kb:Q9 Y5X1 ) physically i nteracts (MI:0914)withITCH (uniprotkb: Q96J02) and Ubiquitin (uniprotkb:P62988)byanti tag coimmunoprecipitation (MI:0007) l MINT-7889928: Ub (uniprotkb:P62988) physically interacts (MI:0915) with SNX9 (uniprotkb: Q9Y5X1)byanti tag coimmunoprecipitation (MI:0007) l MINT-7889610: SNX9 (uniprotkb:Q9Y5X1) physically interacts (MI:0915) with ITCH (uni- protkb: Q96J02)byanti bait coimmunoprecipitation (MI:0006) Abbreviations GST, glutathione S-transferase; HA, hemagglutinin; PRD, proline-rich domain; SH3, Src homology 3 domain; SNX, sorting nexin; Ub, ubiquitin. FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2803 nism to terminate protein activity and thereby control numerous cellular processes. The specific selection of substrates targeted for ubiquitylation is determined by E3 Ub ligases (EC 6.3.2.19). Itch, or atrophin-1 interacting protein 4 (AIP4, here- after referred to as Itch) (UniProtKB accession num- ber Q96J02), is a member of the NEDD4 family of E3 Ub ligases, and Itch knockout mice display severe immunological disorders. All NEDD4 family members have a C-terminal HECT (homologous to E6-AP C-terminus) domain catalysing the direct transfer of Ub onto substrates. In addition, they have an N-terminal C2 domain for phospholipid ⁄ membrane association and multiple WW domains for protein–protein interac- tions. The latter are members of the group I WW domains with specificity for ligands containing a Pro- Pro-X-Tyr consensus sequence, the so-called PY motif [1]. Many substrate proteins of Itch have been identified (e.g. c-Jun, JunB, ErbB4, CXC-chemokine receptor 4) [2]. We have previously shown that, in melanoma cells, Itch is involved in lysosomal degrada- tion of the melanosomal protein Melan-A (UniProtKB accession number Q16655) [3]. Substrates of Itch generally contain PY motifs that associate with one of the four WW domains of the ligase, and ubiquitylation usually targets them for degradation. Unlike other NEDD4 family members, Itch and NEDD4 (Uni- ProtKB accession number P46934) also possess a short proline-rich domain (PRD). Sorting nexin 9 (SNX9) (UniProtKB accession num- ber Q9Y5X1) belongs to a large family of proteins involved in endocytosis and intracellular trafficking [4,5]. Sorting nexins are characterized by the presence of a phosphoinositide-binding PX domain that mediates interactions with cellular membranes. SNX9, originally identified through its association with metalloproteas- es [6], has since been shown to participate in clathrin- mediated endocytosis of cell-surface receptors such as the transferrin receptor [7]. In addition to the PX mem- brane binding module, SNX9 has a C-terminal Bin ⁄ amphiphysin ⁄ Rvs (BAR) domain and an N-terminal Src homology 3 (SH3) domain. The BAR domain facili- tates homodimerization of SNX9, sensing of membrane curvature and tubulation of membranes [8]. The SH3 domain mediates protein–protein interactions by bind- ing to proline-rich regions present in interacting proteins. Many interaction partners of the SH3 domain of SNX9 have been described [7]. It has also been shown that SNX9 undergoes tyrosine phosphorylation, and that this modification can modulate binding of SNX9 to other proteins [9–11]. Due to its important role in endocytosis, intracellular SNX9 levels are expected to be tightly regulated. Here we identify SNX9 as a new substrate of the E3 Ub ligase Itch. We show that Itch ubiquitylates and regulates the level of SNX9. Unlike most substrates studied previously, we found that binding of SNX9 to Itch occurs through the SH3 domain of SNX9 and the PRD of Itch. We demonstrate that the PRD of Itch is essential for SNX9 binding, ubiquitylation and degra- dation. Taken together, these results extend the pool of substrates of NEDD4 family E3 ligases to proteins containing SH3 domains. Results Itch interacts with sorting nexin 9 We previously showed that, in melanoma cells, the Ub ligase Itch is involved in lysosomal degradation of the melanosomal protein Melan-A [3]. To identify addi- tional substrates of Itch in melanoma cells, we per- formed pulldown experiments by incubating an immobilized GST–Itch fusion protein with cell extracts from pigmented SK-MEL-23 melanoma cells. The recovered associated proteins were identified by mass spectrometry. The Itch sequence used in this experiment (GST–Itch–PWW) contained the proline-rich domain and four WW protein–protein interaction domains, but lacked the phospholipid-interacting C2 domain and the catalytic HECT domain (Fig. 1). SNX9 was identified among the proteins that were found to associate with GST–Itch–PWW but not with GST alone. SNX9 is a Fig. 1. Schematic representations of the constructs used to identify proteins associated with Itch and to analyse interactions between Itch and SNX9. The various domains and their localization within the proteins are indicated. BAR, Bin ⁄ amphiphysin ⁄ Rvs domain; C2, Ca 2+ -dependent phospholipid binding domain; GST, glutathione S-transferase; HECT, homologous to E6-AP C-terminus domain; myc, myc tag; PRD, proline-rich domain; PX, phosphoinositide bind- ing domain; SH3, Src homology 3 domain; WW, WW domain. The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al. 2804 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS cytosolic protein that contains an SH3 domain, a PX domain and a homodimerization BAR domain. In mass spectrometric analysis, seven unique peptides matching the SNX9 protein sequence were detected, covering about 17% of the entire SNX9 sequence. To confirm the interaction between SNX9 and Itch, we repeated the pulldown experiment using GST–Itch–PWW and extracts of HEK293 cells expressing vesicular stomatitis virus-G epitope (VSV)-tagged SNX9. As shown in Fig. 2A, a band of approximately 75 kDa reacting with anti- body against the VSV tag was detectable in the lane containing material immunoprecipitated with GST–Itch– PWW. In parallel, an aliquot of the same cell extract was analysed directly with the antibody against the VSV tag. Having identified SNX9 in GST–Itch–PWW pull- down experiments, we confirmed the interaction in cells. We first analysed this interaction in cells exoge- nously expressing the proteins. HEK293 cells were chosen for this and other over-expression experiments as SK-MEL-23 melanoma cells have a very low trans- fection efficiency. In HEK293 cells co-transfected with VSV-tagged SNX9 and HA-tagged Itch, Itch was found to be associated with SNX9 (Fig. 2B). The inter- action of Itch with SNX9 was further validated in SK-MEL-23 melanoma cells by co-immunoprecipita- tion of endogenous Itch and SNX9 proteins (Fig. 2C). Itch ubiquitylates and regulates the level of SNX9 Next we tested whether SNX9 was ubiquitylated by Itch. Plasmids encoding SNX9 and HA-tagged ubiqu- itin (Ub HA ) were transiently transfected into HEK293 cells. The total pool of ubiquitylated proteins was immunoprecipitated with an antibody against the HA tag and analysed by Western blot with antibody against SNX9. In cells expressing SNX9 and Ub HA , several bands corresponding to ubiquitylated SNX9 were detected (Fig. 3A). We confirmed that Itch directly ubiquitylated SNX9 by performing an in vitro ubiquitylation assay. Immobilized GST–SNX9 fusion protein was incubated with biotinylated Ub and a pro- teasome-depleted lysate of HEK293 cells transfected either with a vector encoding full-length Itch or with an empty vector. GST–SNX9 was eluted and re-immu- noprecipitated with antibody against SNX9. Ubiquity- lated GST–SNX9 was detected by immunoblotting with horseradish peroxidase-conjugated streptavidin. In the presence of over-expressed Itch, a smear of high-molecular-mass proteins was observed, indicating ubiquitylated forms of SNX9 (Fig. 3B). Prolonged exposure of the membrane revealed a similar smear in the sample containing a lysate of control-transfected cells, probably due to endogenous Itch protein (data not shown). No evidence of ubiquitylation was found with GST alone. Note that analysis of SNX9 ubiquity- lation in cells showed a few discrete bands (Fig. 3A), while a smear of ubiquitylated SNX9 was detected in the in vitro assay (Fig. 3B). This may be due to the fact that, in the latter case, a vast excess of ubiquitin was present and proteasomes were absent. The absence of highly ubiquitylated SNX9 in cells may also be due to factors regulating SNX9 ubiquitylation in a cellular A C B Fig. 2. Itch interacts with sorting nexin 9 (SNX9). (A) Extracts from HEK293 cells transfected with VSV-tagged SNX9 were incubated with immobilized GST or GST–Itch–PWW. Bound material was resolved by SDS ⁄ PAGE and analysed by immunoblotting using antibodies against the VSV tag. Cell extracts were analysed in parallel and correspond to 10% of the eluted material. (B) Digitonin-soluble extracts were prepared from HEK293 cells transfected with HA-tagged Itch, VSV-tagged SNX9 or both. Extracts were immunoprecipitated (IP) using anti- bodies against the VSV tag, resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibodies against the HA tag or against the VSV tag as indicated. Cell extracts were analysed in parallel. (C) Digitonin-soluble extracts from SK-MEL-23 melanoma cells were immuno- precipitated using polyclonal antibodies against SNX9 or control polyclonal antibodies against CD2AP (Ctrl Ab). Proteins were analysed by immunoblotting with monoclonal antibody against Itch or polyclonal antibodies against SNX9. Cell extracts were analysed in parallel. C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2805 context, such as co-factors and deubiquitylating enzymes. To investigate whether ubiquitylation of SNX9 led to its degradation, we analysed the impact of modulat- ing Itch levels on intracellular levels of SNX9. We transduced pigmented SK-MEL-23 melanoma cells with a recombinant lentivector delivering a short hair- pin RNA sequence that efficiently silenced Itch expres- sion. Itch silencing caused an increase in the level of endogenous SNX9 protein (Fig. 3C). Quantitative analysis indicated that the level of SNX9 increased approximately twofold (2.1 ± 0.4, mean ± standard deviation from three independent experiments) com- pared to cells transduced with control lentivector. Consistent with this observation, over-expression of Itch in HEK293 cells decreased the level of SNX9 (Fig. 3D). To demonstrate that Itch regulates the degradation of SNX9, we performed pulse–chase experiments using HEK293 cells over-expressing VSV-tagged SNX9 alone or in combination with Itch. We analysed SNX9 degradation for 24 h. In the absence of exogenous Itch, SNX9 appeared to be a very stable protein, with a half-life of approximately 24 h (Fig. 3E). The half- life of SNX9 was reduced in the presence of over- expressed Itch, such that the level of labelled SNX9 protein has decreased to approximately 20% of its initial amount after 24 h (Fig. 3E). Altogether, these results show that the Ub ligase Itch binds to and ubiquitylates SNX9, directly affecting AB CD E Fig. 3. Itch ubiquitylates SNX9 and targets it for degradation. (A) HEK293 cells transfected with plasmids encoding HA-tagged ubiquitin (Ub HA ), SNX9 or both were treated with lactacystin before lysis. Proteins immunoprecipitated (IP) with monoclonal antibody against the HA tag and cell extracts were analysed by immunoblotting using polyclonal antibodies against SNX9. (B) In vitro ubiquitylation assay. GST–SNX9 or GST immobilized on glutathione beads were incubated with cytosolic extracts from HEK293 cells, supplemented with 2 lg biotinylated ubiquitin (Ub-biotin) and ATP. HEK293 cells were transfected with Itch (+) or empty vector ()), as indicated. After the indicated time, the reaction was stopped by boiling the beads for 5 min in 2% SDS. SNX9 was immunoprecipitated and analysed by immunoblotting using horseradish peroxidase-conjugated streptavidin to reveal SNX9-bound Ub-biotin. (C) Extracts from SK-MEL-23 cells transduced with lentivec- tors encoding short hairpin RNA specific for Itch (rec. lv ⁄ siItch) or an irrelevant target gene (rec. lv ⁄ siControl) were resolved by SDS ⁄ PAGE and analysed by immunoblotting with the indicated antibodies. (D) Extracts from HEK293 cells expressing SNX9 alone or together with Itch were analysed by immunoblotting with antibodies against SNX9 and Itch. Note that, in (C) and (D), the level of SNX9 is inversely proportional to the level of Itch. (E) HEK293 cells transfected with VSV-tagged SNX9 or a combination of VSV-tagged SNX9 and myc-tagged Itch were incubated for 30 min in medium containing 35 S-methionine ⁄ cysteine to label newly synthesized proteins (pulse). After labelling, cells were incubated in regular medium for the indicated times (chase). Extracts were prepared and immunoprecipitated using antibody against the VSV tag. Eluted material was resolved by SDS ⁄ PAGE. The left panel shows autoradiography of the gel. Immunoprecipitated radioactivity was quantified using a phosphorimager and the radioactivity remaining was determined relative to time 0 (right panel). The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al. 2806 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS the intracellular level of the latter by promoting its degradation. The SH3 domain of SNX9 binds to the proline-rich domain of Itch Given that substrates of Itch and NEDD4 family E3 ligases generally contain PY motifs, we screened the sequence of SNX9 for such motif but failed to detect one. Previous studies have shown that Itch binds to the SH3 domains of endophilin A1 and beta-p21-activated kinase-interactive exchange factor (bPIX) via its PRD [12,13]. We therefore produced a GST fusion product that contained only the SH3 domain of SNX9 (Fig. 1) and tested whether this truncated version of SNX9 was able to interact with Itch. As shown in Fig. 4A, immo- bilized GST–SNX9(SH3) pulled down Itch from cellu- lar extracts of SK-MEL-23. These results indicate that the SH3 domain of SNX9 mediates the interaction with Itch. It is interesting to note that SNX9(SH3) associated with Itch but not with NEDD4 (Fig. 4A). NEDD4 is a close functional homologue of Itch that shares over 45% amino acid identity but diverges in the sequence of its proline-rich segment. Under the same experimental conditions, the cytoplasmic tail of the melanosomal protein Melan-A associated with both Itch and NEDD4, as previously shown [3]. Having found that the interaction was mediated by the SH3 domain of SNX9, we tested whether SNX9 binds to the PRD of Itch. We co-transfected myc- tagged full-length Itch protein or Itch lacking the PRD (myc-tagged DPRD-Itch) together with VSV-tagged SNX9 into HEK293 cells (Fig. 4B). Association between SNX9 and full-length Itch was readily observed, but the interaction between SNX9 and DPRD-Itch was drastically reduced, confirming that SNX9 binds to the PRD of Itch. Next, we investigated whether ubiquitylation of SNX9 depends on its interac- tion with the PRD of Itch. We therefore co-transfected either full-length Itch or DPRD-Itch protein together C AB D Fig. 4. SNX9 interacts via its SH3 domain with the proline-rich domain of Itch. (A) Extracts from SK-MEL-23 cells were incubated with immobilized GST, GST–Itch–PWW, GST–SNX9(SH3) containing only the SH3 domain of SNX9 or GST–Melan-A protein. The GST fusion con- structs used are shown in Fig. 1. Proteins that bound to the beads were analysed by immunoblotting with monoclonal antibody against Itch or polyclonal antibodies against NEDD4. Note that SNX9(SH3) does not pulldown NEDD4, a close functional homologue of Itch. The known interaction of Melan-A with Itch and NEDD4 was used as a positive control, and the lack of interaction of GST–Itch–PWW with either protein as a negative control. (B) Extracts were prepared from HEK293 cells co-transfected with VSV-tagged SNX9, myc-tagged Itch and myc-tagged DPRD-Itch (lacking the proline-rich domain) in the indicated combinations. Extracts were immunoprecipitated using antibody against the myc tag, resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibody against the VSV tag or the myc tag as indicated. Cell extracts were analysed in parallel. (C) Extracts were prepared from HEK293 cells co-transfected with VSV-tagged SNX9, myc-tagged Itch and myc- tagged DPRD-Itch (lacking the PRD) in the indicated combinations. Extracts were immunoprecipitated using antibody against the VSV tag, resolved by SDS ⁄ PAGE and subjected to immunoblotting using antibodies against the myc tag, ubiquitin or the VSV tag as indicated. Cell extracts were analysed in parallel. (D) Extracts from HEK293 cells transfected with myc-tagged Itch or myc-tagged DPRD-Itch were analysed by immunoblotting with antibodies against SNX9, actin or the myc tag as indicated. C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2807 with VSV-tagged SNX9 into HEK293 cells. SNX9 was immunoprecipitated and analysed by Western blotting using an antibody to ubiquitin. Ubiquitylated forms of VSV-tagged SNX9 were easily detected upon co-trans- fection of SNX9 with full-length Itch, but not with the deletion variant lacking the PRD (Fig. 4C). In agree- ment with this finding, over-expression of full-length Itch in HEK293 cells decreased the level of endogenous SNX9, but over-expressing Itch protein lacking the PRD had no impact on SNX9 levels (Fig. 4D). The level of endogenous SNX9 in cells over-expressing full-length Itch was about one-third of the amount in non-transfected control cells. Note that, in the analysis of cellular extracts in Fig. 4B,C, co-expression of Itch and SNX9 did not decrease the level of SNX9 compared to expression of SNX9 alone, in contrast to results with endogenous SNX9 shown in Figs 2B and 3D. This apparent discrepancy was due to variability in co-transfection efficiency (not shown). Together, our results show that binding of SNX9 to Itch is mediated by the SH3 domain of SNX9 and the PRD of Itch. Furthermore, interaction with the PRD of Itch is essential for ubiquitylation of SNX9 and its intracellular regulation. Phosphorylated SNX9 binds Itch It has been reported that SNX9 can be phosphorylated at tyrosine residues by ACK2 (activated Cdc42-associ- ated tyrosine kinase 2) during membrane recruitment [9,14]. Furthermore, in Drosophila, ACK was found to phosphorylate Tyr56 within the SH3 domain of SNX9, abrogating as a result the binding to proline-rich sequences [10]. To examine whether tyrosine phosphorylation of the human SNX9 protein had the same impact on interac- tions with PRD-containing proteins as in Drosophila,we investigated whether Itch interacts with tyrosine-phos- phorylated SNX9. HEK293 cells were co-transfected with plasmids encoding Itch and SNX9. Itch was immu- noprecipitated from lysates, and SNX9 protein bound to Itch was eluted and re-immunoprecipitated to analyse its tyrosine phosphorylation status (Fig. 5). Phosphory- lated SNX9 protein was detected, demonstrating that tyrosine phosphorylated SNX9 still bound to Itch. As a control, tyrosine phosphorylation of SNX9 immunopre- cipitated directly from cell extracts was confirmed. Itch interacts with SNX18 SNX18 (UniProtKB accession number Q96RF0) is the closest relative of SNX9, with a very similar domain structure, including an N-terminal SH3 domain. Little is known about the function of SNX18. It has recently been reported to induce membrane tubulation in AP-1- positive endosomal trafficking [15]. In addition, like SNX9, the SH3 domain of SNX18 can bind to dynam- in-2 [15]. Given the similarity between SNX9 and SNX18, we analysed the potential interaction of SNX18 with the Ub ligase Itch by co-transfection of myc-tagged SNX18 and HA-tagged Itch into HEK293 cells (Fig. 6). After immunoprecipitation of myc-tagged SNX18, bound proteins were eluted and subjected to Western blotting. The membrane was probed using an antibody against Itch. SNX18 was found to interact with over-expressed as well as endogenous Itch pro- tein, suggesting that it may be regulated in a manner similar to SNX9. Discussion In this study, we found that the sorting nexin SNX9 is regulated by the E3 Ub ligase Itch. We showed that the interaction is mediated by the SH3 domain of SNX9 and the PRD of Itch, leading to ubiquitylation Fig. 5. Tyrosine-phosphorylated SNX9 binds to Itch. Extracts were prepared from HEK293 cells transfected with VSV-tagged SNX9, myc-tagged Itch or both, using lysis buffer supplemented with phos- phatase inhibitors. Extracts were immunoprecipitated using antibod- ies against the VSV tag or the myc tag. Proteins eluted from beads coupled to antibody against the myc tag were re-immunoprecipitated using antibody against the VSV tag. Eluted proteins were resolved by SDS ⁄ PAGE and subjected to immunoblotting with antibodies against phospho-tyrosine (4G10), the VSV tag or the myc tag as indicated. A fraction (75%) of eluted material from immunoprecipitated anti- myc + anti-VSV was analysed using 4G10 antibody, and 10% of eluted material was used for detection with antibody against the VSV tag. From material immunoprecipitated using antibody against the VSV tag, a fraction (50%) of eluted material was analysed with 4G10 antibody, and 10% of eluted material was used for detection with antibody against the VSV tag. Cell extracts were analysed in parallel. The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al. 2808 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS and degradation of SNX9. Previous studies revealed that the Itch substrate endophilin A1 also associates via its SH3 domain with the PRD of the ligase [12]. Thus our study represents the second characterization of an interaction between a substrate and a ubiquitin ligase of the NEDD4 family that is mediated by a sequence distinct from the conventional WW domains. Importantly, while the SH3 ⁄ PRD interaction was found to facilitate endophilin ubiquitylation in the case of endophilin A1 [12], the interaction mediated by the PRD of Itch appears to be essential for ubiquitylation and degradation of SNX9. The ability of the PRD to function as a substrate recognition domain in Itch indicates that the pool of Itch substrates is not limited to PY motif-containing proteins. We showed that the interaction of SNX9 with Itch is specific, as SNX9 does not bind to the PRD of the related Ub ligase NEDD4. NEDD4 and Itch can inter- act with the same PY motif-containing substrate pro- tein, such as Melan-A [3], but the two ligases diverge in the sequence of their proline-rich segments. Two types of proline-rich binding motifs for SH3 domains have been described, so-called class I and class II sites, with the consensus sequences Arg-X-X-Pro-X-X-Pro and Pro-X-X-Pro-X-Arg (X being any amino acid), respectively [16,17]. It has been shown that the SH3 domain of SNX9 binds most strongly to the class I ligand motif Arg-X-Ala ⁄ Pro-Pro-X-X-Pro [15,18]. Class I binding motifs are present in the PRDs of both Itch and NEDD4. However, the amino acid context of their binding sites is different, and may affect binding to SH3 domains. Itch has a single discrete class I site with the sequence Arg-Pro-Pro-Pro-Pro-Tyr-Pro, con- forming to the preferred consensus SH3 ligand motif of SNX9 previously reported [18]. In contrast, the PRD of NEDD4 contains two class I sites that overlap and may thus form a structure that cannot interact with the SH3 domain of SNX9. In support of this hypothesis, other SH3 domain-binding partners of SNX9 such as dynamin-2 also present discrete class I sites within their PRDs that mediate binding to SNX9 [15]. In addition to endophilin A1, another SH3 domain-containing protein, bPIX, has been found to interact with the PRD of Itch [12,13]. The complex of bPIX and Itch serves as a scaffold in G protein- coupled receptor signalling [13]. Binding of these two proteins to the PRD of NEDD4 has not been investi- gated, and it is therefore not known whether the inter- action can be mediated by either class I motif present in Itch and NEDD4. It is likely that there are many as yet unidentified proteins regulated by the NEDD4 family ligases Itch or NEDD4 through interactions with their PRDs. We found that SNX18, a close relative of SNX9 with a similar domain structure, also interacts with Itch. Little is known about the function of SNX18 [15,19] and its regulation. SNX9 and SNX18 show more than 40% identity in the amino acid sequence of their SH3 domains, which share a preference for binding to class I ligand motifs and dynamin-2 as a major inter- acting protein [15]. It is therefore conceivable that SNX18 is regulated through ubiquitylation by Itch in a manner similar to SNX9. SNX33 is the third and final member of the SNX family identified to date that possesses an SH3 domain. It is closely related to SNX9 and SNX18, and has also been found to associate with dynamin [20]. It will be interesting to determine whether SNX18 and SNX33 are also substrates of Itch. The SH3 domain of SNX9 is essential for the func- tion of SNX9 in clathrin-mediated endocytosis. This domain was shown to mediate the interaction of SNX9 with dynamin-2 and N-WASP, a homologue of the Wiskott–Aldrich syndrome protein (WASP) [21–24]. Given that the Ub ligase Itch binds to the SH3 domain of SNX9, one could speculate that Itch competes for binding to the SH3 domain with other interaction partners. Dynamin-2 and N-WASP are key components of the actin polymerization machinery [10,25,26]. Interaction of SNX9 with dynamin-2 recruits dynamin-2 to the clathrin-coated pit and stim- ulates the GTPase activity required for vesicle scission [21,23,27,28]. Interaction of SNX9 with N-WASP stim- ulates actin remodelling during endocytosis by enhanc- ing N-WASP activity [22,24,29,30]. It is possible that functionally active SNX9 forming a complex with dynamin-2 or N-WASP at membranes cannot be targeted for degradation as the binding site for Itch is not accessible. It may be speculated that, once the end- ocytic process is completed, Itch competes for binding to the SH3 domain of membrane-bound SNX9, thereby destabilizing the complex of SNX9 with dyn- amin-2 and ⁄ or N-WASP. Itch binding could enable Fig. 6. SNX9 family member SNX18 also interacts with Itch. Extracts were prepared from HEK293 cells transfected with myc-tagged SNX18, HA-tagged Itch or both. Extracts were immunoprecipitated using antibody against the myc tag, resolved by SDS ⁄ PAGE, and sub- jected to immunoblotting with antibodies against Itch or the myc tag as indicated. Cell extracts were analysed in parallel. C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2809 the removal of SNX9 from the membrane, followed by SNX9 ubiquitylation and degradation. Alternatively, Itch may only bind to the SH3 domain of cytosolic SNX9. In the cytosol, SNX9 forms a com- plex with aldolase and dynamin-2 [27]. Thus, in this scenario, Itch would have to compete with dynamin-2 for binding to the SH3 domain of SNX9. Ubiquityla- tion of cytosolic SNX9 by Itch could serve as a mecha- nism to regulate the pool of SNX9 protein in the cell. Our results indicate that SNX9 is a very stable protein with a half-life of approximately 24 h, at least when over-expressed. Although increased Itch levels increased the turnover of SNX9, the half-life of the latter was still relatively long (approximately 15 h). Given that endocytosis is a fast process, the hypothesis of Itch-mediated removal of membrane-bound SNX9 upon endocytic vesicle release seems unlikely. The long half-life of SNX9 instead supports the hypothesis that Itch may regulate the steady-state levels of SNX9. We found that tyrosine-phosphorylated SNX9 can bind to Itch. At odds with our findings, tyrosine phos- phorylation of the SH3 domain of Drosophila SNX9 by ACK was found to reduce interactions with PRD-con- taining proteins [10], hinting at differences between human and Drosophila SNX9. It has also been reported that tyrosine-phosphorylated human SNX9 can bind to the PRD of ACK1 [11], again indicating different roles of tyrosine phosphorylation in the human and Droso- phila SNX9 proteins. Of note, in the human SNX9 protein, the presence of a tyrosine phosphorylation site within the SH3 domain has not yet been formally demon- strated. One could thus speculate that phosphorylation of human SNX9 involves a tyrosine residue that is not located within the SH3 domain, and may therefore not affect the binding of SNX9 to PRD-containing proteins. Future studies should address these issues in detail. Phosphorylation of the ligase Itch might also affect interaction with and ubiquitylation of SNX9. The PRD of Itch was shown to be phosphorylated by the serine ⁄ threonine kinase JNK (c-Jun N-terminal kinase) upon treatment with epidermal growth factor (EGF) [31–33]. This phosphorylation leads to an increase in the ligase activity of Itch, resulting in increased ubiquityla- tion and degradation of substrates such as endophi- lin A1 and Casitas B-lineage lymphoma gene (Cbl) [31]. Both endophilin and Cbl are involved in EGF receptor down-regulation, and their increased degrada- tion after EGF treatment has been proposed to serve as a negative feedback mechanism to terminate internaliza- tion of the EGF receptor [31]. Given that SNX9 is also involved in down-regulation of the EGF receptor after EGF treatment [9], one could speculate that, like endo- philin and Cbl, the level of SNX9 ubiquitylation increases upon EGF-induced phosphorylation of the PRD of Itch. In conclusion, we found that SNX9 is a new sub- strate of the E3 Ub ligase Itch that binds to the PRD, an unconventional substrate recognition domain in Itch. Association of the SH3 domain of SNX9 with the PRD of Itch mediates ubiquitylation and degra- dation of SNX9. Binding of Itch to members of the sorting nexin family with ensuing degradation is an additional level at which E3 Ub ligases can regulate endocytic processes. In the future, more SH3 domain- containing substrates of the Ub ligases Itch and NEDD4 are likely to be identified. Experimental procedures Antibodies The rabbit antiserum against SNX9 was a generous gift from S. Schmid (Scripps Research Institute, La Jolla, CA). Another polyclonal antibody against SNX9 was produced in rabbits by immunization with a full-length GST–SNX9 fusion protein (Eurogentec, Seraing, Belgium). The NEDD4 rabbit antiserum was provided by O. Staub (Department of Pharmacology and Toxicology, University of Lausanne, Switzerland). Monoclonal antibody against the myc tag (clone 9E10) was a gift from R.D. Iggo (Institut Bergonie ´ , Bordeaux, France). Commercial antibodies included: monoclonal antibody against Itch (BD Biosciences ⁄ Pharmingen, San Diego, CA, USA), monoclonal antibody against the HA tag (clone 16B12) (Covance, Princeton, NJ, USA), polyclonal antibody against actin (Sigma-Aldrich, St Louis, MO, USA), monoclonal antibody against the VSV tag (clone P5D4) (Sigma-Aldrich), polyclonal antibody against CD2AP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal antibody against ubiquitin (clone FK2) (Biomol, Plymouth Meeting, PA, USA) and monoclonal anti- body against phospho-tyrosine (clone 4G10) (Upstate Biotechnology, Charlottesville, VA, USA). Secondary anti- bodies were horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (Amersham Biosciences, Pisca- taway, NJ, USA) and horseradish peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark). Plasmids and lentivirus production The plasmids pGEX ⁄ Melan-A and pEGFP ⁄ Itch-HA have been described previously [3]. The plasmid encoding Ub HA was a gift from S. Rothenberger (Institute of Microbiology, University Hospital Lausanne, Switzerland). The pGEX ⁄ Itch-PWW vector was constructed by inserting a blunted XbaI–BglII fragment from the pEGFP ⁄ Itch-HA vector [3] into the SmaI site of the pGEX-4T-3 vector (Amersham Biosciences). The full-length SNX9 coding sequence was The E3 ligase Itch regulates sorting nexin 9 C. Baumann et al. 2810 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS cloned by RT-PCR from mRNA extracted from SK-MEL- 23 melanoma cells. The amplified fragment was digested with EcoRI and XhoI and inserted into the corresponding sites of the pCR-3 ⁄ Met-VSV vector (gift from P. Schneider, Department of Biochemistry, University of Lausanne, Swit- zerland) or the pGEX-4T-1 vector (Amersham Biosciences) to generate the pCR3 ⁄ VSV-SNX9 and the pGEX ⁄ SNX9 plasmids, encoding SNX9 with an N-terminal VSV tag or fused to GST, respectively. pGEX ⁄ SNX9(SH3) was con- structed by PCR amplification of the SH3 domain of SNX9 from the same cDNA as above. The PCR product was inserted into pGEX-4T-1 vector. The plasmids encoding myc-tagged Itch and myc-tagged DPRD-Itch were a gener- ous gift from T.P. Sakmar (Laboratory of Molecular Bio- logy and Biochemistry, Rockefeller University, New York, NY, USA). The plasmid encoding myc-tagged SNX18 was a kind gift from S.R. Carlsson (Department of Medical Bio- chemistry and Biophysics, Umea ˚ University, Sweden). The plasmid pSuper-Itch, encoding Itch siRNA, was produced by annealing two complementary DNA oligonucleotides tar- geting nucleotides 88-108 of the ORF encoding human Itch, as described previously [34], and ligating the fragment into the pSuper vector (a gift from R. Agami, Nederlands Kan- ker Instituut, Amsterdam, The Netherlands). Recombinant lv ⁄ siItch was constructed by excising this siItch sequence together with the H1-RNA promoter from the pSuper vec- tor, and inserting the fragment into the pAB286 lentivector (gift from R.D. Iggo, Institut Bergonie ´ , Bordeaux, France). The same strategy was used to produce the control lentivec- tor recombinant lv ⁄ siLamin to silence lamins A and C. The plasmid pSuper ⁄ siLamin was a gift from R.D. Iggo (Institut Bergonie ´ , Bordeaux, France) and has been described previ- ously [35]. All constructs were verified by sequencing. Lenti- virus production was performed as described elsewhere [3]. Cells and transfections Human embryonic kidney HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics. Pigmented Melan-A + SK- MEL-23 human melanoma cells were maintained in RPMI- 1640 medium, supplemented with 10% fetal bovine serum and antibiotics. Transfections were performed using Fugene (Roche Diagnostics, Indianapolis, IN, USA) or Lipofecta- mine (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ instructions. Transfected cells were main- tained for 48 h at 37 °C. Silencing of Itch Expression of Itch and lamins A and C (control) was silenced in SK-MEL-23 melanoma cells by recombinant lentivector transduction. Transduced cells were selected using puromycin (2.5 lgÆmL )1 ) on day 2 and analysed within 1–4 weeks of culture. Ubiquitin assays HEK293 cells were transfected with indicated plasmids. Twenty-four hours after transfection, the cells were treated for 2 h with 10 lm lactacystin (Biomol), a proteasome inhibitor. Ubiquitylated proteins were immunoprecipitated from cell lysates using monoclonal antibody against the HA tag coupled to protein G–Sepharose. Proteins were resolved by SDS ⁄ PAGE and immunoblotted using anti- body against SNX9. For the in vitro ubiquitylation assay, bacterially produced GST–SNX9 or GST was adsorbed onto glutathione beads and incubated for 0 or 24 h with cytosolic extracts from 3 · 10 6 HEK293 cells per reaction, supplemented with 2 lg biotinylated ubiquitin (Biomol), 10 mm ATP, 5 mm Mg acetate and 0.2 mm DTT. To avoid degradation, protea- somes were removed from cytosol prior to the reaction with GST–SNX9, as described previously [3]. Where indicated, HEK293 cells were transfected with the plasmid encoding Itch 48 h prior to lysis. After the ubiquitylation reaction, 10 mm EDTA was added and the immobilized material was washed and eluted as described previously [3]. The superna- tant was then diluted tenfold in cold lysis buffer and re-im- munoprecipitated using antibody against SNX9. The immunoprecipitated material was resuspended in SDS sample buffer, resolved by SDS ⁄ PAGE and transferred onto nitrocellulose. The ubiquitylated material was revealed using horseradish peroxidase-conjugated streptavidin (1 : 5000, Dako). Preparation of GST fusion proteins and pulldown assays Bacteria cultures were grown to an attenuance at 600 nm of 0.6–0.7. Protein expression was induced with 0.5 mm iso- propyl thio-b-d-galactoside for 2 h at 37 °C, except for GST–SNX9, which was induced for 4 h at 25 °C. Fusion proteins were purified using the ProFound pulldown GST protein:protein interaction kit (Pierce, Rockford, IL, USA), according to the manufacturer’s instructions. Glutathione beads (30 lL) were used for each purification and subse- quent pulldown experiments. Pulldown experiments were performed as described previously [3]. Immunoprecipitations and Western blots Mammalian cells were lysed with Triton X-100 as described previously [3], except for SNX9 immunoprecipitations, where cells were lysed in 0.5% digitonin. For analysis of tyrosine phosphorylated proteins, lysis buffer was supple- mented with 50 mm sodium fluoride and 10 mm sodium ortho-vanadate to inhibit phosphatases. The protein con- tent was determined using a BCA protein assay kit (Pierce). For immunoprecipitations, cleared cell extracts were incu- bated either with agarose-conjugated antibodies against the C. Baumann et al. The E3 ligase Itch regulates sorting nexin 9 FEBS Journal 277 (2010) 2803–2814 ª 2010 The Authors Journal compilation ª 2010 FEBS 2811 HA tag (clone 3F10) (Roche) or antibodies against the VSV tag (clone P5D4) (Sigma-Aldrich), Sepharose-conju- gated antibodies against the myc tag (clone 9E10), or pro- tein G beads (Pierce) plus polyclonal antibodies against SNX9 or CD2AP (Santa Cruz Biotechnology). After 1–2 h of incubation at 4 °C, immunoprecipitated material was extensively washed, eluted in SDS sample buffer and boiled at 95 °C for 5 min. Proteins were reduced, resolved on SDS ⁄ PAGE and transferred onto nitrocellulose. Mem- branes were subsequently incubated with blocking solution (5% milk in NaCl ⁄ P i , except for 4G10 monoclonal anti- body against where 1% gelatine in NaCl ⁄ P i ⁄ Tween was used) and primary antibody as indicated. Immunoreactivity was detected using horseradish peroxidase-conjugated sec- ondary antibodies and ECL (Amersham Biosciences) according to the manufacturer’s instructions. Quantitative analysis was performed using ImageJ quantification soft- ware (National Institutes of Health, Bethesda, MD, USA). Pulse–chase experiments HEK293 cells were plated on 60 mm plates and transfected with VSV-tagged SNX9 or a combination of VSV-tagged SNX9 and myc-tagged Itch. Twenty-four hours after transfection, cells were starved for 30 min in Dulbecco’s modified Eagle’s medium lacking cysteine and methionine (MP Biomedicals, Solon, OH, USA) supplemented with 10% dialysed fetal bovine serum, then incubated with Easy- Tag Express 35 S Protein Labeling Mix (Perkin Elmer, Waltham, MA), using 110 lCi per 2 mL per plate. After 30 min (pulse), radioactive medium was removed and cells were incubated in Dulbecco’s modified Eagle’s medium sup- plemented with 10% fetal bovine serum, 10 mm HEPES and antibiotics for 6 or 24 h (chase). Cells were lysed for 20 min on ice in a buffer containing 80 mm Tris pH 7.6, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 1% sodium deoxycholate and 0.05% SDS, supplemented with a cock- tail of protease inhibitors. Aliquots of lysates were used to measure the incorporated radioactivity using a liquid scintillation counter. Lysates were subjected to immunopre- cipitation as described above. Eluted material was resolved by 10% SDS ⁄ PAGE. The gel was then fixed in 40% metha- nol ⁄ 10% acetic acid for 15 min, dried, and exposed to an imaging plate (Fuji Medical Systems, Stamford, CT, USA) overnight. The plate was scanned using an FLA-3000 phos- phorimager (Fuji Medical Systems) and BASReader 3.14 software (Raytest, Straubenhardt, Germany). aida image analyzer software (Raytest) was used for quantitative analy- sis. Autoradiography was performed by exposing the dried gel to X-ray film (Kodak, Rochester, NY, USA). Mass spectrometry Gel bands were excised from SDS ⁄ PAGE and subjected to in-gel proteolytic cleavage using sequencing-grade trypsin (Promega, Madison, WI, USA) [36]. Proteolytic peptides were analysed by LC-MS ⁄ MS on a SCIEX QSTAR Pulsar hybrid quadrupole-time of flight instrument (Concord, Ontario, Canada) equipped with a nanoelectrospray source and interfaced to an Ultimate HPLC system (LC Packings, Amsterdam, Netherlands). Peptides were separated on a PepMap reverse-phase capillary C18 column (internal diam- eter 75 lm, length 6.15 cm) at a flow rate of 200 nLÆmin )1 using a 52 min gradient of acetonitrile (0–40%). The Ana- lyst software (Applied Biosystems, Concord, Ontario, Canada) was used for peak detection and to automatically select peptides for collision-induced fragmentation. Non- interpreted peptide tandem mass spectra were used for direct interrogation of the uniprot (Swissprot + TrEMBL) database using mascot 2.1 (http://www.matrixscience. com), limited to the subset of sequences from Homo sapiens. The database used contained 64 386 sequences after taxonomy filter. Generally, only proteins matched by at least two peptides were accepted. Acknowledgements We thank Manfredo Quadroni (Department of Bio- chemistry, University of Lausanne, Switzerland) and the Protein Analysis Facility (Center for Integrative Genomics, Faculty of Biology and Medicine, Univer- sity of Lausanne, Switzerland) for mass spectrometry analysis. We gratefully acknowledge the excellent tech- nical assistance of Anne-Lise Peitrequin and Nicole Le ´ vy. 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The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain Claudia Baumann, Cecilia K SNX9 is a new sub- strate of the E3 Ub ligase Itch that binds to the PRD, an unconventional substrate recognition domain in Itch. Association of the SH3 domain