Intersectin 1 Forms Complexes with SGIP1 and Reps1 in Clathrin-Coated

Biochemical and Biophysical Research Communications 402 (2010) 408–413

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Biochemical and Biophysical Research Communications

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Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits ⇑ Oleksandr Dergai a, ,1, Olga Novokhatska a,1, Mykola Dergai a, Inessa Skrypkina a, Liudmyla Tsyba a, Jacques Moreau b, Alla Rynditch a a Department of Functional Genomics, Institute of Molecular Biology and Genetics, NASU, 150 Zabolotnogo Street, 03680 Kyiv, Ukraine b Molecular Mechanisms of Development, Jacques Monod Institute, Development and Neurobiology Program, UMR7592 CNRS – Paris Diderot University, 15 rue Hélène Brion, 75205 Paris Cedex 13, France article info abstract

Article history: Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endo- Received 7 October 2010 cytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome Available online 12 October 2010 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome pheno- type. Here we report the identification of novel interconnections in the interaction network of this endo- Keywords: cytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth Endocytosis factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP Adaptor proteins associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated Intersectin 1 by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes compris- Protein interactions SGIP1 ing SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluores- cent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction cellular processes from cell survival [3,10], cell polarity and cell cycle progression [11] to dendritic spine development [12–14] Endocytosis is a fundamental process of internalization of and regulation of RTK (receptor tyrosine kinase) trafficking and sig- plasma membrane components, surface receptors, nutrients, naling [15]. Despite the fact that ITSN1 is implicated in a diverse pathogens and extracellular soluble molecules to regulate various set of cellular functions, its major role is thought to be regulation aspects of cell homeostasis and communication with the environ- of endocytic events by orchestrating recruitment of endocytic pro- ment [1]. Except for constituting the main pathway that allows teins such as adaptor Eps15 (epidermal growth factor receptor different substances to enter the cell, endocytosis is a starting point pathway substrate 15), GTPase dynamin, phosphoinositide phos- in a dramatically complicated network of intracellular trafficking. phatase synaptojanin and others [2,16–22]. The fidelity of this process relies on the precision and accuracy ITSN1 functions as a scaffolding protein regulating many of the endocytic components recruited. The endocytic scaffold aspects of membrane traffic. Disruption of ITSN1-containing endo- intersectin 1 (ITSN1) is one of the recruiters. It is an evolutionarily cytic complexes, caused by overexpression of the full-length conserved plasma membrane-associated adaptor protein involved protein, blocks receptor-mediated endocytosis [2]. Depletion of in clathrin-mediated endocytosis, apoptosis, signal transduction, ITSN1 leads to activation of the mitochondrial pathway of apopto- and regulation of cytoskeletal rearrangements [2–5]. ITSN1 is pres- sis in endothelial cells [3], whereas quadruple knockdown of ITSN1 ent in the cell as a set of alternatively spliced isoforms that are and ITSN2 together with Eps15 and Eps15R cause cytosolic locali- differentially expressed in various tissues [6–8]. The short form zation of the adaptor complex AP2 preventing clathrin-coated pit (ITSN1-s) consists of two N-terminal Eps15 homology domains (CCP) formation [23]. Furthermore through interactions with the (EH1 and EH2), a coiled-coil region and five Src homology domains regulators of the cytoskeleton N-WASP and Cdc42, ITSN1 is (SH3A-E) (Fig. 1). The long form (ITSN1-l) contains a C-terminal thought to link clathrin-mediated endocytosis to the actin nucle- extension with DH (a Dbl homology), PH (a pleckstrin homology), ation machinery and participate in actin assembly as a mechanism and a C2 domain [9]. ITSN1 was shown to regulate a plethora of of vesicle protrusion [5,24]. In the current report we attempted to extend our understanding

⇑ Corresponding author. Fax: +380 445260759. of the interaction network of ITSN1-s (further referred to as ITSN1). E-mail address: [email protected] (O. Dergai). An in silico prediction and interaction database search demon- 1 These authors contributed equally to this work. strated the association between the newly identiﬁed endocytic

Fig. 1. Schematic representation of the structure of ITSN1 and its binding partners. EH, Eps15 homology; DPF repeat, Asp-Pro-Phe motif of interaction with EH-domains; UIM, ubiquitin-interacting motif; PRD, proline-rich domain; MP, membrane phospholipid-binding; BAR, Bin/amphiphysin/Rvs; FCHo, Fer/Cip4 homology. adaptors Reps1 (RalBP associated Eps15-homology domain protein) Sepharose 4B beads (GE Healthcare) according to the manufac- and SGIP1 (Src homology 3-domain growth factor receptor-bound turer’s instructions. Lysates of transiently transfected HEK293T 2-like (endophilin) interacting protein 1) with ITSN1. cells were prepared in extraction buffer [20 mM Tris–HCl pH In current study we have identified interaction of ITSN1 with 7.4, 150 mM NaCl, 1% Triton X-100, and supplemented with SGIP1 and Reps1. Both Reps1 and SGIP1 colocalize with ITSN1 in complete EDTA-free protease inhibitor cocktail (Roche)] and clathrin-coated pits. Interaction data details evidence for triple centrifuged for 20 min at 12,000g at 4 °C. For pull-down assays complex formation implying scaffolding role for ITSN1. 2–5 lg of purified GST fusion proteins or GST were bound to 30 ll of 50% glutathione-Sepharose 4B beads and incubated with 2. Materials and methods cell lysates for 1 h at 4 °C. The beads were extensively washed and boiled in Laemmli sample buffer (150 mM Tris–HCl pH 6.8, 2.1. Expression constructs 2.5% glycerol, 10% SDS, 3% b-mercaptoethanol and 0.5% bromo- phenol blue). The eluted proteins were resolved by SDS–PAGE The following oligonucleotides were used to amplify the coding and transferred to nitrocellulose membranes. The blots were sequences of SGIP1 and Reps1 respectively: 50-TCTCTGCAGATG blocked for 1 h in 5% non-fat milk, 1 PBS (phosphate-buffered ATGGAAGGATTG-30,50-CCGCGGCCG-CAAACCTTTTCTTGATG-30;50- saline) and 0.1% Tween 20, incubated with the corresponding pri- AGGAATTCATGGAAGGCTTAACGC-30,50-CTCTCGAGTCAAGCGTAAT mary antibodies for 1 h at room temperature and washed. Detec- CTGGAACATCGTATGGGTATTGTAGGTGAGAGAATGG-30 and PCR tion was performed by horseradish peroxidase-labeled secondary products were cloned into the pcDNA4Has/Max vector (Invitro- antibodies (Promega). gen). The glutathione S-transferase (GST) fusion SH3-domains and coiled-coil region of ITSN1 were described previously [25]. 2.4. Cell culture and transfection The region encoding the SH3 domain of amphiphysin 1 (amino acids 413–480) was amplified by PCR and subcloned into the Adherent cell lines HEK293T and MCF-7 cells were maintained pGEX-4T-3 vector (GE Healthcare); the full coding sequence of in Dulbecco’s modified Eagle’s medium (DMEM) supplemented ITSN1 was subcloned into the pEGFP-C1 (Clontech) and pmCher- with 10% fetal calf serum, 50 U/ml penicillin and 100 mkg/ml ry-C1 (Clontech) fluorescent vectors; the ITSN1-HA plasmid was streptomycin. The cells were transiently transfected using polyeth- kindly provided by Dr. Susana de la Luna (Barcelona, Spain); GFP- yleneimine transfection reagent (JetPEI, Polyplus Transfection) and clathrin was a kind gift of Dr. Serhiy Havrylov (Warsaw, Poland). processed 24–36 h after transfection.

2.2. Antibodies 2.5. Immunoprecipitation

Polyclonal antibodies against the EH2 domain of ITSN1 were de- For immunoprecipitation (IP), HEK293 cells were lysed in IP scribed previously [25]. A monoclonal anti-omni (D-8): sc-7270 buffer (20 mM Tris–HCl pH 7.5, 0.5% NP40, 100 mM NaCl, 10% antibody was purchased from Santa Cruz Biotechnology. Anti-HA glycerol, 1 mM PMSF and protease inhibitor cocktail). The cell (hemagglutinin) antibody was purchased from Covance. lysate was mixed with antibodies and protein A/G Agarose beads (Santa Cruz Biotechnology) prewashed in IP buffer. After over- 2.3. Protein expression, pull-down assays and Western blot analysis night incubation at 4 °C the beads were washed three times with IP buffer. Bound proteins were eluted by boiling in 30 llof The recombinant GST fusion proteins were expressed in Laemmli sample buffer and analysed by SDS–PAGE and Western Escherichia coli BL21 (DE3) pLysE and puriﬁed on glutathione- blotting. 410 O. Dergai et al. / Biochemical and Biophysical Research Communications 402 (2010) 408–413

2.6. Immunoﬂuorescence and confocal microscopy 3.1. ITSN1 interacts with SGIP1

MCF-7 cells were plated on coverslips and transfected with the SGIP1 is the one of the recently identified endocytic adaptors. appropriate plasmid DNA using the JetPEI reagent. The cells were Initially discovered as a conserved brain-enriched protein regulat- washed 24 h post-transfection in ice-cold 1xPBS, fixed in 4% form- ing energy homeostasis, it was shown to bind the important endo- aldehyde for 15 min, washed three times in 0.2% Triton X-100 for cytic protein, endophilin [27]. The N-terminal MP (membrane 5 min and blocked in 2% BSA, 0.2% and Triton X-100 in 1xPBS for phospholipid)-binding) domain of SGIP1 is responsible for lipid 30 min at room temperature. Then incubations with the appropri- binding and demonstrates membrane tubulating activity similar ate primary antibodies diluted in blocking buffer were performed. to that of the BAR (Bin/amphiphysin/Rvs) and EFC (Extended Fer- For immunofluorescence, Texas Red horse anti-mouse (Vector CIP4 homology) domains. SGIP1 is required for transferrin uptake Laboratories Inc.) antibodies were used. The slides were mounted but it is not needed for EGF (epidermal growth factor) internaliza- using PVA-DABCO (Fluka) and confocal images were taken using tion [28]. a Zeiss LSM510 microscope. All images represent a single confocal To validate the predicted interactions in vivo, immunoprecipita- section, taken from the bottom surface of the expressing cells. tion assays were carried out. HA-tagged ITSN1 readily coprecipitated with omni-SGIP1 using anti-omni mouse monoclonal antibodies 3. Results (Fig. 2A). To explore the binding sites of ITSN1, in vitro binding assays with individual GST-SH3 domains were performed. In the Comprehension of endocytic events in terms of multiprotein range of the five ITSN1 SH3 domains, only the SH3A and SH3E do- complexes rearrangements is incomplete because of insufficient mains mediated interaction with the proline-rich domain (PRD) of information about the number of proteins involved and the con- SGIP1 in vitro (Fig. 2B). To validate the in vivo SGIP1/ITSN1 complex tacts they form. In this study we investigated the interaction net- formation, immunofluorescent analysis was carried out. GFP-ITSN1 work of the endocytic adaptor ITSN1 with proteins that were and omni-SGIP1 significantly colocalized at the plasma membrane shown to participate in endocytosis. We analyzed available inter- (Fig. 2C). ITSN1 is located at the plasma membrane in dense dis- action data and used the on-line service Scansite (http://scan- crete spots that are reported to be CCPs [17]. According to these site.mit.edu) [26] to evaluate the probability that proteins could findings almost all ITSN1 spots of different sizes that we observed, bound to ITSN1; newly identified endocytic proteins were favored. were clathrin-positive [Fig. 1S]. Therefore it can be concluded that Numerous proline-rich stretches and a high score for potential ITSN1 forms a complex with SGIP1 in CCPs. This fact is in line with binding to the SH3A domain of ITSN1 made SGIP1 and Reps1 plau- previously published data showing that SGIP1 is colocalized with sible candidates. Up to twenty motifs for SGIP and nine sites for EGFR (EGF receptor) suggesting its localization with CCPs where Reps1 were predicted. it is codistributed with Eps15 and AP-2 complex [28].

Fig. 2. SGIP1 interacts with adaptor protein ITSN1 in vitro and in vivo. (A) HEK293T cells were cotransfected with plasmids encoding HA-tagged ITSN1 and omni-tagged SGIP1. Extracts of the cotransfected cells were subjected to immunoprecipitation with anti-omni mouse monoclonal antibodies. Protein complexes were analyzed by Western blotting, using anti-omni and anti-HA antibodies. (B) SGIP1 binds to the SH3 domains of ITSN1 in vitro. Immobilized on glutathione beads GST-fused SH3 domains of ITSN1 were incubated with lysates of HEK293T cells overexpressing omni-SGIP1. The amount of GST-fused proteins bound to the beads was checked by Coomassie staining. Omni- SGIP1 was immunoblotted with anti-omni antibodies. (C) ITSN1 and SGIP1 are colocalized at the plasma membrane. MCF-7 cells were plated on coverslips and cotransfected with GFP-ITSN1 and omni-SGIP1. 24 h post-transfection the cells were ﬁxed in 4% formaldehyde. Omni-SGIP was stained with anti-omni antibodies and visualized with Texas Red-conjugated anti-mouse IgG. Confocal images were captured with Zeiss LSM 510 Meta. Scale bar: 5 lm. O. Dergai et al. / Biochemical and Biophysical Research Communications 402 (2010) 408–413 411

3.2. ITSN1 forms a complex with Reps1 was observed. Regarding the localization of ITSN1 in CCPs we also consider Reps1 to be in CCPs. Reps1 belongs to a group of conserved EH domain-containing The immunofluorescence data obtained regarding the localiza- proteins primarily associated with endocytosis and vesicle trans- tion of SGIP1 and Reps1 at the sites of clathrin-mediated endocytosis port. In mammals, EH domain-containing proteins Eps15, ITSN1 raised the question of whether these proteins are components of the and ITSN2 localize in CCPs and are important components of the same or different protein complexes. Antibodies specific of omni-tag endocytic machinery [23]. All the proteins described have a similar efficiently precipitated HA-Reps1 from cell lysates cotransfected structure, and next to the N-terminal EH domains possess a coiled- with omni-SGIP1 and HA-Reps1 constructs (Fig. 3D). Moreover coil region capable of dimerization (Fig. 1). Initially, Reps1 was endogenous ITSN1 was found in this immunoprecipitate suggesting identified as a RalBP1 (Ral binding protein 1)-associated protein its possible role as a scaffold molecule. that undergoes phosphorylation in response to EGF and interacts with the adaptors Grb2 and Crk implicated in signaling [29] as well 3.3. Reps1 and SGIP1 interact with amphiphysin 1 as with the Rab11-FIP2 protein involved in the function of recycling endosomes [30]. Given that SGIP1 and Reps1 associate with endocytic proteins Here we demonstrate the in vivo interaction between Reps1 and and localize to CCPs, we investigated whether they interact with ITSN1. Omni-tagged Reps1 overexpressed in HEK293T cells and amphiphysin 1, an important membrane-deforming SH3 domain- endogenous ITSN1 coprecipitated using polyclonal antibodies raised containing protein that is known to regulate dynamin oligomeriza- against the ITSN1 EH2 domain (Fig. 3A). According to the in vitro tion [31–33]. In vitro binding experiments revealed that Reps1 and binding assays, this interaction is mediated by the ITSN1 SH3C SGIP1 could efficiently be pulled down by the SH3 domain of domain and with low affinity by the SH3A domain (Fig. 3B). The amphiphysin 1 from HEK293T cell extracts (Fig. 4A and B). coiled-coil region of ITSN1 could also provide heterodimerization In contrast to SGIP1, that was previously shown to interact with with the coiled-coil domain of Reps1, but we observed no binding endophilin, Reps1 failed to precipitate with the endophilin SH3 do- of affinity-purified GST-CCR of ITSN1 with full-length Reps1 main (Fig. 4C). Endophilin is another membrane-deforming protein (Fig. 4B). These results indicate that interaction between adaptors implicated in many stages of clathrin-mediated synaptic vesicle is mediated by SH3/PRD interactions. endocytosis. Amphiphysin and endophilin have similar domain Immunofluorescent analysis was used to investigate the intra- organizations (Fig. 1) and similar properties in binding to lipid cellular distribution of the proteins examined. To this end, MCF-7 membranes but differ in their effect on dynamin vesiculation of cells were cotransfected with GFP-Reps1 and mCherry-ITSN1 bilayer tubules – amphiphysin supports dynamin-mediated vesicle constructs; significant codistribution of the expressed proteins formation whereas endophilin inhibits such formation [34].

Fig. 3. ITSN1 forms a complex with Reps1. (A) HEK293T cells were transfected with omni-Reps1. Cell extracts were subjected to immunoprecipitation with anti-EH2 antibodies. Protein complexes were eluted with Laemmli sample buffer and analyzed by Western blotting using anti-omni and anti-ITSN1 antibodies. (B) The SH3C domain of ITSN1 mediates its interaction with Reps1. Immobilized on glutathione beads GST-fused SH3 domains of ITSN1 were incubated with extracts of HEK293T cells overexpressing omni-Reps1. The amount of GST-fused proteins bound to the beads was checked by Coomassie staining. Omni-Reps1 was immunoblotted with anti-omni antibodies. (C) MCF- 7 cells were cotransfected with mCherry-ITSN1 and GFP-Reps1; 24 h post-transfection the cells were ﬁxed in 4% formaldehyde. Scale bar: 5 lm. (D) SGIP1 and Reps1 form a complex in vivo. Omni-SGIP1 and HA-Reps1 were coexpressed in HEK293T cells. The cells were lysed and the extracts subjected to immunoprecipitation with anti-omni antibodies. The precipitated proteins were analyzed by Western blot with anti-ITSN1, anti-HA and anti-omni antibodies. 412 O. Dergai et al. / Biochemical and Biophysical Research Communications 402 (2010) 408–413

Fig. 4. Amphiphysin 1 interacts with SGIP1 and Reps 1 in vitro. The bacterially expressed and affinity purified GST-fused SH3 domain of amphiphysin 1 (GST-Amph-SH3), the ITSN1 coiled-coil region (GST-CCR) or GST alone (control) were immobilized on glutathione beads and used as bait to pull down omni-tagged SGIP1 (A) or Reps1 (B) from lysates of HEK293T cells. Omni-SGIP1 and omni-Reps1 were immunoblotted with anti-omni antibodies (upper panel), and GST-fused proteins were visualized with Coomassie staining. (C) Omni-Reps1 was overexpressed in HEK293T cells, and the total cell lysate was incubated with GST-fused SH3 domain of endophilin or GST alone. Reps1 was immunodetected with anti-omni antibodies, GST-fused proteins were stained with Coumassie. (D) Scheme of fragment of endocytic interaction network. Previously established contacts are depicted in solid lines, and newly identified contacts are in dashed lines.

4. Discussion associate in vivo. Analysis of the primary sequences of these proteins revealed no potential binding motifs that could be responsi- ITSN1 is one of the key players in clathrin-mediated endocyto- ble for direct interaction. Moreover, ITSN1 was identified in sis, recently in concert with Eps15 it was shown to regulate the immunoprecipitated material containing SGIP1 and Reps1, indicat- recruitment of the AP2 adaptor and clathrin to sites of CCPs ing that it could serve as platform to bring these proteins together. formation via clustering of membrane-sculpting FCHo (Fer/Cip4 Our immunofluorescence data demonstrate the localization of homology domain-only) proteins considered to be nucleators of these complexes in CCPs. We showed significant codistribution of clathrin-mediated endocytosis [23]. Here we have extended our ITSN1 and SGIP1, however the population of SGIP1 that was understanding of the ITSN1 interaction network by identifying not colocalized with ITSN1 was also detected. Moreover SGIP1- two novel protein partners SGIP1 and Reps1 (Fig. 4D). Reps1 is en- negative ITSN1 foci were also found. These observations could gaged in EGFR signaling and undergoes phosphorylation upon EGF reflect different times of engagement and release of the proteins stimulation [29]. Interestingly, the protein partners identified studied from the CCPs. In contrast to SGIP1 localization, the distri- share similarity with already well established ITSN1 interactors. bution of ITSN1 and Reps1 overlap almost exactly in CCPs implying SGIP1 is a close relative of FCHo whereas Reps1 possesses similar a similar time of recruitment. to Eps15 domain organisation (Fig. 1). ITSN1 is an adaptor protein The human ITSN1 gene is located on chromosome 21 in a Down that could be considered as part of the molecular interface be- syndrome critical region [9]. It is known that individuals with tween the basic machinery of endocytosis, and components of Down syndrome present an increased risk of obesity. Interestingly, the signaling and actin cytoskeleton rearrangements. Current work SGIP1 controls energy balance, and rats with overexpressed SGIP1 provides details on the interactions between ITSN1, SGIP1 and are obese [27]. It is tempting to speculate that in individuals with Reps1. ITSN1 has the highest known number of SH3 domains: five Down syndrome, overexpression of ITSN1 causes disfunction of SH3 domains serve as a platform for interaction with many SGIP1-containing protein complexes with subsequent changes in proteins such as PI3K-C2b, Numb, N-Wasp, c-Cbl, Ruk, Sos1, metabolism leading to obesity. CdGAP, WNK, synaptojanin 1, dynamin 1, SHIP2 and Cbl-b Furthermore we revealed an interaction between SGIP1, Reps1 [10,12,13,15,25,35–40]. Here we have found that two SH3 do- and amphiphysin 1. SGIP1 and amphiphysin 1 can deform mem- mains, namely SH3A and SH3E mediate interaction of ITSN1 with branes. Previously it was shown that SGIP1 interacts with endophi- SGIP1. Reps1 interacts with the SH3C domain and weakly with lin, another membrane-tubulating protein [27]. Thus, it appears the SH3A domain. Interaction data suggest that Reps1 and SGIP1 that the network of interactions between membrane-deforming do not compete for ITSN1 binding as they have different binding proteins and their adaptors is extending. In contrast, Reps1 binds sites. In line with this proposal, we found that SGIP1 and Reps1 only the SH3 domain of amphiphysin 1 but not to endophilin. O. Dergai et al. / Biochemical and Biophysical Research Communications 402 (2010) 408–413 413

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