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An Evolutionarily Conserved Autoinhibitory Molecular Switch in ELMO Proteins Regulates Rac Signaling

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An Evolutionarily Conserved Autoinhibitory Molecular Switch in ELMO Proteins Regulates Rac Signaling CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Current Biology 20, 2021–2027, November 23, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.10.028 Report An Evolutionarily Conserved Autoinhibitory Molecular Switch in ELMO Proteins Regulates Rac Signaling Manishha Patel,1,2,6 Yoran Margaron,1,6 Nadine Fradet,1 by which ELMO orchestrates Rac signaling in concert with Qi Yang,1,3 Brian Wilkes,1 Michel Bouvier,2,4 Kay Hofmann,5 DOCK180 remain to be established. We used bioinformatics and Jean-Franc¸ois Coˆ te´ 1,2,3,* to search for novel structural elements in ELMO that could 1Institut de Recherches Cliniques de Montre´ al, regulate Rac signaling. Threading analysis performed with 110 Avenue des Pins Ouest, Montre´ al, PQ H2W 1R7, Canada the Phyre algorithm identified Armadillo repeats (ARR) in 2Faculte´ de Me´ decine, Universite´ de Montre´ al, Montre´ al, ELMO1–3 and Drosophila ELMO bearing structural homology PQ H3C 3J7, Canada to ARR found in the formin Dia1 [19] (see Figure S1A available 3Division of Experimental Medicine, McGill University, online). Structural homology between ELMO1 and the formins Montre´ al, PQ H3A 1A3, Canada Dia1 [20, 21] and FHOD1 [22] was also detected with the 3D- 4Institut de Recherches en Immunologie et en Cance´ rologie, Jury structure prediction algorithm (Figure S1B). Finally, 2950 Chemin Polytechnique, Montre´ al, PQ H3T 1J4, Canada BLAST searches uncovered primary amino acid sequence 5Miltenyi Biotec GmbH, D-51428 Bergisch Gladbach, Germany similarity between ELMO1 and FHOD1 (Figure S1C). The region in Dia1 and FHOD1 sharing homology to ELMO is the diaphanous inhibitory domain (DID) and is characterized to Summary engage in intramolecular interactions with a diaphanous autoregulatory domain (DAD) to maintain these proteins in a Dedicator of cytokinesis (DOCK) proteins are guanine nucle- repressed state [23]. A hallmark of this regulatory switch is otide exchange factors (GEFs) controlling the activity of the presence of a GTPase-binding site N-terminal to the DID. Rac1/Cdc42 during migration, phagocytosis, and myoblast Mechanistically, engagement of GTPases to autoinhibited fusion [1–4]. Engulfment and cell motility (ELMO) proteins formins disrupts the inhibitory DID-DAD interactions, thereby bind a subset of DOCK members and are emerging as critical exposing their actin polymerization activity [20, 22]. Because regulators of Rac signaling [5–10]. Although formation of the region in ELMO preceding the ARR interacts with RhoG a DOCK180/ELMO complex is not essential for Rac1 activa- [8], this led us to hypothesize that the N terminus of ELMO tion, ELMO mutants deficient in binding to DOCK180 are may constitute part of a similar autoinhibitory module. unable to promote cytoskeleton remodeling [11]. How We therefore termed the ARR in ELMO as the ELMO inhibitory ELMO regulates signaling through DOCK GEFs is poorly domain (EID) (Figure 1A). Based on sequence alignment with understood. Here, we identify an autoinhibitory switch in FHOD1, the EID is defined by one HEAT domain followed ELMO presenting homology to a regulatory unit described by four ARR (Figure 1B). Searching for the equivalent of the for Dia formins. One part of the switch, composed of formins’ DAD in ELMO is not straightforward because this a Ras-binding domain (RBD) and Armadillo repeats, is functional region is not a domain but rather a short amphi- positioned N-terminally while the other is housed in the pathic helix. We nevertheless identified a C-terminal region C terminus. We demonstrate interaction between these frag- in ELMO that resembles the formins’ DAD [19, 24], and we ments, suggesting autoinhibition of ELMO. Using a biolumi- named it the ELMO autoregulatory domain (EAD) (Figures 1A nescence resonance energy transfer biosensor, we estab- and 1C). lish that ELMO undergoes conformational changes upon If the EID and EAD of ELMO behave like the analogous disruption of autoinhibition. We found that engagement of domains in formins, they should interact directly. We tested ELMO to RhoG, or with DOCK180, promotes the relief of whether ELMO11–315 can interact with ELMO1315–727 and found autoinhibition in ELMO. Functionally, we found that ELMO that these two ELMO1 fragments specifically coprecipitated mutants with impaired autoregulatory activity promote with DOCK180 (Figure 1D). The critical residues of Dia1 and cell elongation. These results demonstrate an unsuspected FHOD1 DIDs involved in binding the DAD, alanine 256 and level of regulation for Rac1 signaling via autoinhibition of valine 228, respectively, are located in a hydrophobic region ELMO. of the last helix of the third ARR [19, 22]. Structure-based align- ment of the ELMO EID with the DIDs of Dia1 and FHOD1 sug- Results and Discussion gested L202, I204, and L205 as candidate residues potentially important for the function of the ELMO EID. By analyzing the The guanine nucleotide exchange factor (GEF) activity of dedi- Phyre -generated 3D model of the ELMO1 EID and comparing cator of cytokinesis (DOCK) proteins is mediated by the DOCK it to the structures of Dia1 and FHOD1, we found I204 to be homology region 2, a module exclusive to this family of GEFs surface exposed and thus likely to contribute to EAD binding [5, 12–14]. The identification of upstream regulators of the (Figure S1D). We found that two mutants in this hydrophobic DOCK180-Rac pathway revealed a role for this GEF in devel- patch, ELMO11–315(I204D) and ELMO11–315(L202E/I204D/L205E), lost opmental and pathological processes [2, 15–18]. Previous the ability to interact with ELMO1315–727 in both coimmunopre- studies demonstrated a total requirement for engulfment and cipitation and yeast two-hybrid assays (Figures 1D and 1E). cell motility (ELMO) proteins in biological processes controlled Mutation of another nearby residue in the ELMO1 EID, by DOCK180 [1, 11]. Nevertheless, the molecular mechanisms Y216F, did not affect the EID/EAD interaction (Figures 1D and 1E). Next, we investigated which residues in the EAD are critical in EID binding. To provide evidence that the EAD is *Correspondence: [email protected] included in the predicted a helix located between amino 6These authors contributed equally to this work acids 681 and 701 of ELMO1 (Figure 1C), we used the yeast Current Biology Vol 20 No 22 2022 A C Intramolecular inhibitory interaction hELMO1 689 LLSMEIKLRLLDLENIQ hELMO2 682 LLSMEMKLRLLDLENIQ Dia Family RBD DID DD CC FH1 FH2 DAD (EAD) ELMO hELMO3 731 LLTMETKLRLLELENVP Family dELMO 687 LLSMEIKLRLLDTEGVD ELMO Family RBD EID ELM aPH EAD PxxP Ced-12 694 MLKMELRVRLLNVK.LT (DAD) FHOD1 1050 HASMKSLLTSRPEDTTH Dia Dia1 1196 TGVMDSLLEALQSGAAF Bai1 DOCKs ERMs Family Daam1 1030 SGEFDDLVSALRSGEVF RhoG B Helix-1 Helix-2 Helix-3 HEAT-1 80 ...............SPAQNAQQLHERIQSS................SMDAKLEALKDLASLSRD...... ARR-2 114 VTFAQEFINLD........GISLLTQMVESGTE..RYQKLQKIMKPCFGDMLSFTLTAFVELM........ ARR-3 167 ...DHGIVSWD......TFSVAFIKKIASF..............VNKSAIDISILQRSLAILESMVLNSHD ARR-4 214 ..LYQKVAQEIT........IGQLIPHLQGS................DQEIQTYTIAVINALFLKAPD... ARR-5 255 ERRQEMANILAQK....QLRSIILTHVIRAQ..............RAINNEMAHQLYVLQVLTFNLLE... D +DOCK180 +DOCK180 E 1-315 315-727 LexA B42 -Leu (ELMO1 ) (ELMO1 ) +Leu 315-727 WT WT 315-727 + ELMO1 315-727 I204D WT 315-727 (R697A/L698A/L699A) 315-727 315-727 315-727 315-727 L202E/I204D/L205E WT + ELMO1 + ELMO1 ELMO1 ELMO1 ELMO1 ELMO1 WT M692A/E693A + + + + + ELMO1 Y216F WT 1-315 1-315 1-315 (I204D) 1-315 (L202E/I204D/L205E) 1-315 1-315 1-315 1-315 (Y216F) IP: anti-FLAG or H-70 WT R697A/L698A/L699A kDa ELMO1 ELMO1 ELMO1 ELMO1 ELMO1 ELMO1 ELMO1 ELMO1 (DOCK180) 37 Blot: anti-Myc (ELMO11-315) F 50 315-727 HC Blot: anti-Myc (ELMO1 ) 1-315 LexA B42 -Leu (ELMO1 ) (ELMO1) +Leu Blot: anti-DOCK180 (H-70) 150 WT 532-727 37 TCL 10% input 1-315 Blot: anti-Myc (ELMO1 ) WT 532-707 50 Blot: anti-Myc (ELMO1315-727) WT 532-675 150 Blot: anti-DOCK180 (H-70) Figure 1. Intramolecular Interactions in ELMO1 through Novel Domains (A) Schematic representation of the structural homology between ELMO and Dia-family formins. (B) The ELMO1 EID domain is composed of HEAT and Armadillo repeats (ARR). Predicted a helices are shown in gray, hydrophobic residues of the ARR consensus sequence in yellow, and polar residues in blue and red. I204 in ARR-3 (green) is a conserved residue of ELMO proteins. (C) Sequence alignment of the autoregulatory domains of ELMO (EAD) and Dia-related formins (DAD). Red arrows indicate highly conserved residues form- ing the core motif. (D and E) Mutation of critical EID or EAD residues disrupts EID/EAD interaction in coimmunoprecipitation (D) and the yeast two-hybrid system (E). (D) Lysates of HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation with an anti-FLAG (lanes 1–4) or anti-DOCK180 H-70 (lanes 5–8) antibody. Immunoblots were analyzed with anti-Myc (ELMO1) and anti-DOCK180 (H-70) antibodies. ‘‘HC’’ indicates IgG heavy chain. (E) Yeasts cotransformed with LexA fusion construct of ELMO11–315 and B42 fusion constructs of ELMO1315–727 were grown on nonselective and selective (2Leu) media for a nutrient-selective growth assay. (F) Mapping of critical EAD region boundaries. Yeasts cotransformed with the indicated plasmids were assayed as in (E). See also Figure S1. two-hybrid system. We found that nested C-terminal trunca- DID [19, 24](Figure 1C). Therefore, the equivalent methionine tions (ELMO1532–727 and ELMO1532–707) maintained interaction 692 and the highly conserved glutamate 693 of ELMO1 were with ELMO11–315, whereas further deletion of the region con- both mutated to alanine. We found that ELMO11–315 was inca- taining the predicted EAD (ELMO1532–675) diminished the bind- pable of binding ELMO1315–727(M692A/E693A) in a yeast two- ing (Figure 1F). In both FHOD1 and Dia1, the conserved methi- hybrid interaction assay, yet this mutant retained the ability onine of the DAD is responsible for extensive contacts with the to bind DOCK180 (Figure 1E; Figure S1E).
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