Volume 21, Issue 5, Pages 959-965 (November 2011) The E3 Ubiquitin-Ligase HACE1 Catalyzes the Ubiquitylation of Active Rac1  Stéphanie Torrino, Orane Visvikis, Anne Doye, Laurent Boyer, Caroline Stefani, Patrick Munro, Jacques Bertoglio, Gérard Gacon, Amel Mettouchi, Emmanuel Lemichez  Developmental Cell  Volume 21, Issue 5, Pages 959-965 (November 2011) DOI: 10.1016/j.devcel.2011.08.015 Copyright © 2011 Elsevier Inc. Terms and Conditions

Developmental Cell 2011 21, 959-965DOI: (10.1016/j.devcel.2011.08.015) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 HACE1 Controls CNF1-Induced Degradation of Rac1 (A) Percentage of siRNA-mediated protection against CNF1-induced Rac1 degradation. Cells were transfected with siRNA control (siCtrl) or targeting each of the 27-known HECT-domain containing E3 ubiquitin-ligases and next intoxicated 24 hr with CNF1 10−8 M. Rac1 levels were assessed by anti-Rac1 Enzyme-linked immunosorbent assay (ELISA). Values corresponding to Rac1 levels in siCtrl+CNF1 and siCtrl nonintoxicated cells were set to 0 and 100%, respectively. Data show mean of triplicates ± SEM (∗p = 0.05; ∗∗p < 0.01). (B) Immunoblots (IB) show the inhibitory effect of HACE1 depletion on CNF1-induced Rac1 degradation. Cells were transfected with control siRNA (siCtrl) or HACE1 targeting siRNAs (siRNA mix: siHm or single sequence from the mix: siHs). Cells were left untreated or treated 8 hr with CNF1 10−9 M. Immunoblots anti-Rac1 and anti-RhoA show cellular levels of these GTPases. Immunoblots anti-RhoGDI or anti-β-actin show equal loading. (C) Percentage of cellular depletion of Rac1 and RhoA in cells transfected with different siRNAs and intoxicated 8 hr with CNF1 10−9 M, as compared to nonintoxicated conditions. Data show mean ± SEM, n = 3 independent experiments (∗∗p < 0.01, ∗∗∗p < 0.001, ns: not significant). Developmental Cell 2011 21, 959-965DOI: (10.1016/j.devcel.2011.08.015) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 HACE1 Ubiquitylates Active Rac1 In Vitro (A and B) In vitro ubiquitylation of Rac1 by HACE1 revealed by anti-ubiquitin immunoblot (IB: Ub). Rac1 immunoblot controls are shown in Figures S2A and S2B. The profile shows ubiquitylated forms of Rac1 (Rac1-Ubn). (A) Ubiquitylation assay performed with recombinant Rac1, HACE1 wild-type, or catalytically inactive mutant C876S (HACE1-CS), together with indicated components. (B) GTP-dependent ubiquitylation of Rac1 by HACE1. Recombinant Rac1 loaded with GTP, GDP, or the Mg2+-free form (EDTA) were used in the ubiquitylation reaction. (C) Direct association of HACE1 and Rac1 in vitro (Pull-down). Beads coupled to glutathione S-transferase (GST) or GST-HACE1 were incubated with recombinant purified Rac1 (Rac1), the Mg2+-free form of Rac1 (EDTA), or Rac1 loaded with GDP or GTPγS, as indicated. HACE1-associated Rac1 (top: Rac1) and input Rac1 (bottom) were determined by Rac1 immunoblotting (IB: Rac1). GST proteins were detected by amido black staining (GST or GST-HACE1). (D) Immunoblotting anti-Rac1 showing, as a control, the specific binding of GTPγS-loaded Rac1 to GST-PAK, (Pull-down). GST-PAK70-106 was incubated with recombinant purified Rac1 (Rac1), the Mg2+-free form of Rac1 (EDTA), or Rac1 loaded with GDP or GTPγS, as indicated. Association of Rac1 with GST-PAK70-106 was revealed by Rac1 immunoblotting (IB: Rac1). GST-PAK70-106 protein was detected by amido black staining (GST-PAK). Developmental Cell 2011 21, 959-965DOI: (10.1016/j.devcel.2011.08.015) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 HACE1 Activity Is Essential for Rac1 Ubiquitylation in Cells (A) Immunoprecipitation (IP) anti-Flag from cells cotransfected with expression plasmids for Flag-Rac1L61, Flag-Rac1V12, or Flag-Rac1N17 and HA-HACE1-CS. Immunoblot anti-HA (IB: HA) shows the preferential association of HACE1 with Rac1L61 and Rac1V12 over Rac1N17. Immunoblot anti-Flag shows levels of immunoprecipitated Rac1 mutants. Immunoblots on total lysates show expression of constructs. (B) Immunolocalization of HA-HACE1-CS (HACE1-CS) and either GFP-Rac1L61 (Rac1L61) or YFP-Rac1N17 (Rac1N17). Arrowheads indicate colocalization of HACE1-CS with Rac1L61 at the edge of cell (scale bar represents 10 μm). (C–F) Immunoblots show Rac1 ubiquitylation in cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of Histidine-tagged ubiquitin (His-Ub) wild-type (WT), or K48R mutant (R48) together with HA-Rac1, Flag-Rac1 mutants and myc-tagged Dbl495-826 (referred to as myc-Dbl), or control siRNA (Ctrl), HACE1 siRNA mix (Hm), and single sequence (Hs). His-Ub crosslinked forms of Rac1 were purified (HisP), resolved on 12% (C, E, F) or 10% (D) SDS-PAGE and detected by immunoblot to HA- or Flag-tags (Rac1-Ubn). Immunoblot anti-HA or anti-Flag (lower panel) were performed in parallel to verify the amounts of HA-Rac1 or Flag-Rac1 proteins engaged in the His-Ub purifications. (D, E, F) Immunoblots anti-HA show expression of HA-HACE1 (WT) or (CS). (E) Immunoblot anti-myc shows expression of myc-Dbl. Developmental Cell 2011 21, 959-965DOI: (10.1016/j.devcel.2011.08.015) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 HACE1 Regulates Rac1 Activity (A) Rac1 activation at 0, 3, 6, and 10 hr of cell intoxication by CNF1 10−9 M in cells transfected with control siRNA (siCtrl) or HACE1 targeting siRNA mix (siHm). Immunoblots show levels of GST-PAK associated GTP-bound Rac1 (Rac1-GTP) and total Rac1 in lysates (Rac1). Immunoblots anti-β-actin show equal loading. (B) Quantification of Rac1-GTP normalized to actin in siCtrl, siHACE1 mix (siHm) or single (siHs) transfected cells left untreated or intoxicated by CNF1 10−9 M, 15 hr. Data show mean ± SEM, n = 3 independent experiments. (C) Confocal sections showing actin cytoskeleton and paxillin in cells transfected with siRNAs control, siHACE1 (siHs) and/or siRac1 and intoxicated 18 hr with 10−9 M CNF1 (scale bar represents 10 μm). (D) Quantification of circularity index of cell outline. A circularity value of 1 corresponds to a perfect circle (dotted line). Cells were transfected with siCtrl, siHs, and/or siRac1 and intoxicated by 10−9 M CNF1 for 18 hr. Data show mean ± SEM, n ≥ 72 cells per condition, three independent experiments. (E) Efficiency of UPEC internalization into endothelial cells. HUVEC monolayers were transfected with siCtrl, siHm, or siRac1 and were then left untreated or were intoxicated with 10−9 M CNF1, 18 hr prior to infection with bacteria. Results are expressed as arbitrary units (A.U.). Data show mean ± SEM, n = 3. Equal value of bacterial adhesion to cells was verified in parallel (not shown). Developmental Cell 2011 21, 959-965DOI: (10.1016/j.devcel.2011.08.015) Copyright © 2011 Elsevier Inc. Terms and Conditions