Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process Orane Visvikis 1,2 , Patrick Lore ` s 1,2 , Laurent Boyer 3 , Pierre Chardin 4 , Emmanuel Lemichez 3 and Ge ´ rard Gacon 1,2 1 Institut Cochin, Universite ´ Paris Descartes, CNRS (UMR8104), Paris, France 2 INSERM, U567, Paris, France 3 Faculte ´ de Me ´ decine, INSERM U627, Nice, France 4 Institut de Pharmacologie du CNRS, Sophia Antipolis, France In all eukaryotic cells, Rho GTPases (Rho, Rac, Cdc42) control basic cellular functions, including actin cytoskeleton organization, vesicular trafficking, tran- scriptional regulation and cell cycle progression, and are therefore major intracellular regulators of cell growth and division, and cell migration [1,2]. Consis- tent with their key role in cell signaling, dysregulation of Rho-dependent pathways is being found to be caus- ally involved in the pathophysiology of a growing number of human diseases [3]. Hence, mutations in genes encoding Rho regulators are responsible for human developmental defects and, particularly, several Keywords JNK; Rac1b; Rho GTPases; ubiquitination Correspondence G. Gacon, Institut Cochin, De ´ partement Ge ´ ne ´ tique et De ´ veloppement, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France Fax: +33 1 44 41 24 41 Tel: +33 1 44 41 24 70 E-mail: gacon@cochin.inserm.fr (Received 29 August 2007, revised 18 November 2007, accepted 27 November 2007) doi:10.1111/j.1742-4658.2007.06209.x Ubiquitination and proteasomal degradation have recently emerged as an additional level of regulation of activated forms of Rho GTPases. To char- acterize this novel regulatory pathway and to gain insight into its biological significance, we studied the ubiquitination of two constitutively activated forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumori- genic splice variant Rac1b, which is defective for several downstream signaling pathways, including JNK activation. Whereas Rac1L61 under- goes polyubiquitination and subsequent proteasomal degradation in HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more resistant to proteasomal degradation than Rac1L61. Mutational analysis of all lysine residues in Rac1 revealed that the major target site for Rac1 ubiquitination is Lys147, a solvent-accessible residue that has a similar con- formation in Rac1b. Like Rac1L61, Rac1b was found to be largely associ- ated with plasma membrane, a known prerequisite for Rac1 ubiquitination. Interestingly, Rac1b ubiquitination could be stimulated by coexpression of Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1 downstream signaling. Indeed, ubiquitination of Rac1L61 is critically dependent on JNK activation. In conclusion: (a) Rac1b appears to be more stable than Rac1L61 with regard to the ubiquitin–proteasome system, and this may be of importance for the expression and tumorigenic capacity of Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK- activated process, which may explain the defective ubiquitination of Rac1b. The JNK-dependent activation of Rac1 ubiquitination would create a regu- latory loop allowing the cell to counteract excessive activation of Rac1 GTPase. Abbreviations CNF1, cytotoxic necrotizing factor 1; EMT, epithelial–mesenchymal transition; GST, glutathione S-transferase; HA, hemagglutinin; UbR48, ubiquitin mutant lacking Lys48; UbR63, ubiquitin mutant lacking Lys63; UPS, ubiquitin–proteasome system. 386 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS forms of X-linked mental retardation [4]. In addition, a number of bacterial virulence factors and toxins have been found to target Rho GTPases and to induce vari- ous chemical modifications interfering with Rho func- tion and resulting in increased invasive properties of the pathogens [5,6]. Finally, dysregulations of Rho GTPases have been observed in a variety of human tumors [7]; overexpression of Rho GTPases, particu- larly RhoC, frequently contributes to tumor progres- sion and invasiveness [8], and Rac1b, a constitutively active splice variant of Rac1, has been found to be expressed in colorectal and breast tumors [9,10] and to display oncogenic properties in cancer cell lines [11,12]. Rho family GTPases switch between a GDP-bound form localized in the cytosol in association with guan- ine nucleotide dissociation inhibitors, and a GTP- bound form localized on various membranes and mediating interaction with specific effectors to activate downstream signaling pathways [13]. GDP ⁄ GTP cycling is controlled by guanine nucleotide exchange factors and GTPase-activating proteins. An additional level of regulation has emerged from recent studies, including ours, showing that Rho, Rac and Cdc42, particularly when in their GTP-bound form, can be targeted for polyubiquitination and subsequent degra- dation [14,15]. Ubiquitination consists of the covalent attachment to proteins of ubiquitin, an 8 kDa polypep- tide. It is catalyzed by three enzymes: a ubiquitin-acti- vating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a target-specific ubiquitin protein ligase (E3), acting sequentially to form an isopeptide bond between the ubiquitin C-terminus and the e-amino group of lysines of the target protein [16]. Ubiquitin contains seven lysine residues that can be attached to other ubiquitins in a highly processive reaction to form a polyubiquitin chain [17]. Classically, Lys48-linked chains facilitate recognition of and degradation by the 26S proteasome [18], and Lys63-linked chains regulate processes such as signal transduction and DNA repair through proteasome-independent mechanisms, whereas much less is known about the function of chains linked through other lysine residues [19]. In early studies, a bacterial toxin produced by uro- pathogenic Escherichia coli, cytotoxic necrotizing fac- tor 1 (CNF1), which provokes permanent Rho activation by catalyzing deamidation of Gln63 of RhoA (Gln61 in Rac1 ⁄ Cdc42), was found to sensitize Rho proteins to ubiquitination and proteasomal degra- dation [14,15]. Subsequently, Smurf1, a HECT domain E3 ligase, was shown to target RhoA for ubiquitina- tion and degradation [20], thereby regulating epithelial cell polarity and transforming growth factor-b-depen- dent epithelial–mesenchymal transition (EMT) [21], as well as tumor cell motility [22]. Consistent with a major role of Smurf1 as a RhoA E3 ligase, CNF1- induced ubiquitination of RhoA was found to be spe- cifically impaired in Smurf1 ) ⁄ ) cells [23]. In contrast to RhoA, little is known about the regulation of Rac and Cdc42 by the ubiquitin–proteasome system (UPS). In particular, specific E3 ligases for Rac and Cdc42 have not yet been reported. Recently however, UPS-medi- ated degradation of activated Rac1 was shown to be essential during the early stages of hepatocyte growth factor-induced epithelial cell scattering [24]. To gain insight into the mechanism and biological significance of UPS regulation of Rac1, we have stud- ied here the ubiquitination of mutationally activated Rac1 (Rac1L61) and its naturally occurring splice vari- ant Rac1b. We report that, unlike Rac1L61, Rac1b mostly escapes the ubiquitination process and is there- fore resistant to proteasomal degradation. We show by mutational analysis that the main target site for Rac1L61 polyubiquitination is Lys147, a surface resi- due that should not be hampered in Rac1b; we also report that Rac1 ubiquitination is stimulated by JNK, which may explain the defective ubiquitination of Rac1b. Results Rac1b is much less sensitive than Rac1L61 to the UPS As shown in Fig. 1A, the constitutively activated mutant Rac1L61 undergoes a significant degree of polyubiquitination in HEK293 cells. By contrast, both Rac1WT and Rac1N17, which are predominantly GDP-bound, are not detectably ubiquitinated in this system. This is in full agreement with previous obser- vations made in various epithelial and endothelial cell types [14,24]. Using ubiquitin mutants lacking Lys48 (UbR48) or Lys63 (UbR63), we found that ubiquitin chain synthe- sis on Rac1L61 was markedly but not completely inhibited by UbR48 and that, by contrast, UbR63 sup- ported the formation of polyubiquitinated Rac1 to the same extent as wild-type ubiquitin (Fig. 1B). This points to a major role of Lys48 of ubiquitin in Rac1L61 polyubiquitin chain extension. Also in agree- ment with this view, the monoubiquitinated form of Rac1 was found to be clearly accumulated in cells expressing UbR48 (Fig. 1B), indicating that, in the absence of Lys48, the chain cannot be extended. Con- sistently, inhibition of proteasome using MG132 resulted in an increase of polyubiquitinated Rac1 O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 387 species (Fig. 1C) and accumulation of Rac1 (Fig. 1D, right panel). Whereas most of the polyubiquitin chains on Rac1 appear to be extended through Lys48, leading typically to proteasome degradation, the persistence of a significant amount of ubiquitinated Rac1 when UbR48 was used was repeatedly observed. This could be due to mixed chains including endogenous wild-type and mutant K48R ubiquitin. Alternatively, a fraction of Rac1L61 may have undergone homogeneous poly- ubiquitination employing neither Lys48 nor Lys63 chains, or heterogeneous polyubiquitination, as recently reported for other proteins [25]. Unlike activating point mutations of Rac1 (such as G12V and Q61L), which have not been found to occur in any natural circumstance, Rac1b is a constitutively activated variant expressed in breast and colorectal tumors. Indeed, Rac1b is an alternatively spliced vari- ant of Rac1, containing a 19 amino acid insertion between positions 75 and 76, immediately C-terminal to the switch II region (residues 60–76 in Rac1), which is critically involved in conformational changes during GDP ⁄ GTP cycling [26]. Accordingly, Rac1b exhibits profoundly altered properties, including enhanced nucleotide exchange activity, impaired intrinsic Fig. 1. Ubiquitination and proteasomal degradation of Rac1L61 and Rac1b. (A) Activated Rac1L61 undergoes ubiquitination in HEK293 cells. Ubiquitination of Rac was assessed by transfecting HEK293 cells with a combination of expression plasmids of 6His–myc–Ub, myc– Rac1L61, Rac1L61WT or Rac1L61N17 as indicated. Ubiquitinated proteins were collected on Co beads, and immunoblotted with Rac1-spe- cific antibodies. Expression of transfected and endogenous proteins was monitored in total protein extracts by immunoblotting using the indicated antibodies. (B) In ubiquitinated Rac1L61, ubiquitin moieties are linked through Lys48. HEK293 cells were transfected with a combi- nation of expression plasmids of myc–Rac1L61 and 6His–myc–UbWT or Lys fi Arg mutants at residues 48 (R48) or 63 (R63). Overexposure allows the detection of the monoubiquitinated form of Rac1, in the presence of UbR48. (C) Ubiquitinated forms of Rac1L61 accumulate in the presence of the proteasomal inhibitor MG132. HEK293 cells were transfected with a combination of expression plasmids of myc– Rac1L61 and 6His–myc–Ub as indicated, either treated or not with MG132 (20 l M) 6 h before the ubiquitination assay. (D) Differential ubiqui- tination of Rac1L61 and Rac1b (left panel). HEK293 cells were transfected with a combination of expression plasmids of HA–Rac1L61, HA– Rac1bWT, HA–Rac1bL61 and 6His–myc–Ub as indicated. Proteasomal degradation of Rac1L61 and Rac1b (right panel). Proteasomal degra- dation was assessed by determining Rac1L61, Rac1bWT and Rac1bL61 accumulation in the presence of the proteasome inhibitor MG132 (20 l M), as indicated in Experimental procedures. Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al. 388 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS GTPase activity, and failure to interact with RhoGDI; as a result, Rac1b has been found to exist predomi- nantly in the active GTP-bound state, which can be further stabilized by a GTPase-inactivating mutation (Rac1bL61) [26–28]. Therefore, we examined its ability to undergo ubiquitination and subsequent proteasomal degradation in HEK293 cells. As shown in Fig. 1D (left panel), ubiquitination of Rac1b is much lower than that of constitutively activated Rac1. Introducing the L61 mutation in Rac1b provoked a moderate increase in Rac1b ubiquitination (see white bars in Fig. 3A below for quantitative analysis), but did not sensitize it significantly to proteasome-mediated degra- dation in HEK293 cells (Fig. 1D, right panel). A simi- lar difference between Rac1 and Rac1b was observed in HeLa cells; in these cells, Rac1L61 was found to be weakly ubiquitinated, whereas no ubiquitination of Rac1b or Rac1bL61 could be detected (data not shown). To address the difference(s) between Rac1 and Rac1b that might explain their differential sensitivity to the UPS, we attempted to characterize the require- ments for Rac1 ubiquitination, which may not be fulfilled by Rac1b. The main target site for Rac1 ubiquitination is Lys147, a surface residue not hampered in Rac1b We first reasoned that the site(s) for ubiquitin addition in Rac1 might not be accessible in Rac1b. Therefore, in an attempt to map the ubiquitin acceptor site(s) in IB : Rac IB : actin MG132 (20 µ M)- + Rac1L61 Rac1L61R147 -+ Rac1L61R16 -+ Rac1L61R96 -+ 6His-myc-Ub myc-Rac1L61 + - - + + + Cobalt extraction IB : Rac IB : actin IB : Rac + R147 + R16 + R96 A pull-down GST PAK IB : myc myc-Rac1 GST PA K GST PAK GST PAK WT L61 L61R147L61R16 WT L61 input myc-Rac1 L61R147L61R16 B Lys147 C Fig. 2. Mutation of Lys147 to Arg strongly impairs Rac1L61 ubiq- uitination without altering Rac activation. (A) Mutation of Lys147 or Lys16 impairs Rac1L61 ubiquitination (top panel) and subse- quent proteasomal degradation (bottom panel). HEK293 cells were transfected with a combination of expression plasmids of 6His–myc–Ub, myc–Rac1L61 and myc–Rac1L61 with a Lys fi Arg substitution at position 16 (R16), position 96 (R96), or posi- tion 147 (R147), as indicated. Proteasomal degradation was assessed by determining the accumulation of myc–Rac1L61 and myc–Rac1L61 Lys fi Arg mutants in the presence of the protea- some inhibitor MG132 (20 l M), as indicated in Experimental pro- cedures. (B) Unlike the R16 mutation, the R147 mutation does not impair Rac1L61 activation. Myc-tagged wild-type and mutant Rac1 were expressed in HEK293 cells (top panel), pulled down with GST–PAK (bottom panel) and immunoblotted with antibody to myc tag. (C) Superposition of the structures of Rac1 (Protein Data Bank: 1MH1) and Rac1b (Protein Data Bank: 1RYH), both bound to a GTP analog (GppNHp), using PYMOL software. The main differences are highlighted in green (Rac1b). The structure of Rac1b switch I, shown as a green line at the bottom, is clearly further away from the nucleotide (GTP, in red), explaining a decreased affinity for nucleotide in Rac1b. The switch II region of Rac1b (dotted green line on the right), where the 19 amino acid insertion of Rac1b is found, is disordered in the crystal. The mostly disordered structures of the switch II region are compati- ble with important modifications in nucleotide binding and interac- tion with effectors. The positions of the Lys147 residues are almost identical. Lys147 of Rac1 is yellow, and the equivalent residue of Rac1b is shown in orange on top. There is only a minor change in orientation of the lysine side chain. O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 389 activated Rac1, we performed individual substitutions of all lysines present in the Rac1 sequence to arginine and analyzed the mutated proteins for ubiquitination in HEK293 cells. Most of the 18 mutations tested did not notably affect the polyubiquitination pattern of myc–Rac1L61 (supplementary Fig. S1); only two lysine mutants, i.e. Rac1L61R16 and Rac1L61R147, showed a strong decrease in polyubiquitination as compared to control Rac1L61 or to the Rac1L61R96 ‘ubiquitina- tion-irrelevant’ lysine mutant (Fig. 2A, top panel). Consistent with inhibition of their polyubiquitination, both Rac1L61R16 and Rac1L61R147 were found to be resistant to proteasome degradation, as evidenced by their absence of stabilization upon proteasome inhi- bition (Fig. 2A, bottom panel). As the rate of polyubiquitination of Rac GTPase is critically dependent on the active GTP-bound confor- mation, we investigated whether the K16R and K147R substitutions might affect the strength of the L61 acti- vating mutation. As shown in Fig. 2B, Rac1L61R16 was strongly impaired in its ability to bind to glutathi- one S-transferase (GST)–PAK, indicating that the active GTP-bound form of Rac1L61 is altered by the K16R substitution. Indeed, mutations in this region of Ras (residues 15, 16, and 17) result in reduced nucleo- tide binding and impaired activation [29]. Consistently, we observed that Rac1L61R16 was unable to induce membrane ruffling in COS cells. Therefore, it is likely that the poor ubiquitination of Rac1L61R16 is related to a defective activated state. By contrast, GST–PAK pulldown assays revealed that Rac1L61R147 binds the CRIB domain of PAK with the same efficiency as Rac1L61, suggesting a fully activated conformation (Fig. 2B). Also consistent with a bona fide active state, Rac1L61R147 was largely associated with plasma membrane in HEK293 cells (supplementary Fig. S2) and was found to activate JNK to the same extent as Rac1L61 (data not shown); moreover, it was also found to induce ruffles in COS cells (data not shown). Three-dimensional structures of Rac1, either isolated or associated with various molecular partners, indicate that Lys147 is accessible to solvent, is located far from the GTP-binding site (Fig. 2C) and is not involved in contacts with interacting proteins such as effectors [30], GTPase-activating proteins [31,32], guanine nucle- otide dissociation inhibitors [33] or guanine nucleotide exchange factors [34]; these structural data therefore support our results showing that the K147R mutation is conservative with respect to Rac1–GTP conforma- tion and molecular interactions. Regarding Rac1b, structural analysis has revealed that the 19 residue insertion at position 75–76 indu- ces significant changes in the GTP-binding and effector-binding regions, i.e. an open switch I confor- mation and a highly mobile switch II, but does not interfere with the end of the a3-helix, where Lys147 of Rac1 is located (Fig. 2C) [26]. Altogether, the above results point to Lys147 as a major site for ubiquitin addition on activated Rac1 and provide no evidence to suggest that the accessibility of this site might be hampered by Rac1b-specific inser- tion; therefore, we looked for other mechanisms that could explain the defective ubiquitination of Rac1b. Rac1 ubiquitination is dependent on JNK activation It has been shown recently in human umbilical endo- thelial cells that preventing the interaction of activated Rac1 with plasma membrane results in abolition of Rac1L61 ubiquitination [35]; using the nonisoprenyla- ble-activated Rac1L61G189 mutant, we confirmed that plasma membrane localization is required for Rac1 ubiquitination to occur in HEK293 cells as well (sup- plementary Fig. S3). We also observed that Rac1b and Rac1bL61 are mainly associated with plasma mem- brane in HEK293 cells (supplementary Fig. S2). Indeed, it has been previously shown that Rac1b fails to interact with RhoGDI, resulting in persistent inter- action with plasma membrane in HT29 and MDCK cells [27,28]. Therefore, the absence of Rac1b ubiquiti- nation is unlikely to be related to a defective mem- brane interaction. While being persistently activated and associated with plasma membrane, Rac1b has an impaired ability to activate several Rac1 downstream signaling path- ways [27,28]. To evaluate the possible effects of acti- vating these pathways on Rac1b ubiquitination, we coexpressed Rac1L61 and Rac1b in HEK293 cells and analyzed the resulting ubiquitination pattern. Whereas Rac1L61 ubiquitination was not modified by Rac1b (data not shown), Rac1b ubiquitination was found to be partially restored in the presence of Rac1L61 (Fig. 3A, left panel); quantitative analysis of the data from three experiments demonstrated a 2.7-fold increase in the relative rate of Rac1b ubiquitination in the presence of Rac1L61 (Fig. 3A, right panel), indicating that the stimulation of Rac1-dependent pathways may, in some way, activate the Rac1 ubiqui- tination machinery. As Rac1b has been shown to display reduced capac- ity to bind POSH [28] and to activate PAK and JNK [27], we addressed the role of these specific pathways in regulating Rac1 ubiquitination. Expression of constitutively activated PAK in HEK293 cells had no effect on Rac1 ubiquitination (not shown); likewise, Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al. 390 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 100% 16% HA-Rac ubiquitination / HA-Rac expression (A.U) 0% 20% 40% 60% 80% 100% HA-Rac1L61 HA-Rac1bWT HA-Rac1bL61 HA-Rac mono-expression HA-Rac coexpressed with myc-Rac1L61 6His-myc-Ub HA-Rac1L61 HA-Rac1b myc-Rac1L61 + – – – + + – – + – WT – + – L61 – + + – + + – WT + + – L61 + Cobalt extraction IB : HA IB : β-tubulin IB : myc IB : HA – IB: Rac IB: P-JNK IB: JNK Cobalt extraction IB: Rac 6His-myc-Ub myc-Rac1 myc-Rac1b + – – + L61 – + WT – + – WT + L61 IB: β-tubulin 6His-myc-Ub HA-Rac1L61 HA-POSH + – – – + – + + – + + + Cobalt extraction IB: Rac IB: Rac IB: HA IB: actin Rac Ubiquitination / Rac Expression (A.U.) 100% 63% 68% 50% 36% 0% 20% 40% 60% 80% 100% – – 2 h 10 µ M 4 h 10 µ M 2 h 20 µ M 4 h 20 µ M SP600125 treatment 6His-myc-Ub myc-Rac1L61 SP600125 (µ M) incubation (h) + – – – + + – – + + 10 2 + + 10 4 + + 20 2 + + 20 4 Cobalt Extraction IB: Rac IB: Rac IB: β–tubulin IB: P–JNK IB: JNK A D B C 37%100% 43% 58% Fig. 3. Rac1 ubiquitination is regulated by JNK. (A) Activation of Rac1 downstream signaling partially restores Rac1b ubiquitination. HEK293 cells were transfected with a combination of expression plasmids of 6His–myc–Ub, HA–Rac1L61, HA–Rac1bWT, HA–Rac1bL61 and myc– Rac1L61 as indicated. Ubiquitinated proteins were immunoblotted with an antibody to HA. The graph represents ubiquitinated HA–Rac1b lev- els, normalized to total HA–Rac1b expression, relative to Rac1L61 controls considered as 100%. The data are representative of three inde- pendent experiments. (B) POSH does not display E3 ligase activity towards Rac1L61. HEK293 cells were transfected with a combination of expression plasmids of 6His–myc–Ub, HA–Rac1L61 and HA–POSH. Ubiquitinated and total proteins were analyzed as described in Experi- mental procedures. (C) Unlike Rac1L61, Rac1b does not activate JNK. HEK293 cells were transfected with a combination of expression plasmids of 6His–myc–Ub, myc–Rac1L61, myc–Rac1WT, myc–Rac1bWT and myc–Rac1bL61 as indicated. Ubiquitinated proteins were immunoblotted with Rac1-specific antibody. JNK activation level was monitored in total protein extracts by anti-phospho-JNK (P-JNK) immunoblotting. (D) JNK inhibition decreases Rac1L61 ubiquitination. HEK293 cells were transfected with a combination of expression plasmids of 6His–myc–Ub and myc–Rac1L61. Twenty hours after transfection, cells were treated with the JNK-specific inhibitor SP600125 at 10 or 20 l M for 2 or 4 h, and then subjected to the ubiquitination assay. Ubiquitinated proteins were immunoblotted with Rac1-specific antibody. JNK activation level was monitored in total protein extracts by P-JNK immunoblotting. The graph represents ubiquitinated Rac levels normalized to total Rac expression relative to the control in the untreated condition, considered as 100%. The data are representative of two independent experiments. O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 391 expression of POSH did not appreciably modify Rac1 ubiquitination (Fig. 3B). Therefore, having confirmed that, unlike Rac1L61, Rac1b was unable to activate JNK in HEK293 cells (Fig. 3C), we investigated the possible regulation of Rac1 ubiquitination by a JNK- dependent mechanism. Indeed, we observed that, in response to JNK inhibition by SP600125, ubiquitina- tion of Rac1L61 was impaired in a dose-dependent and time-dependent fashion (Fig. 3D), indicating that JNK activity is a critical regulator of Rac1 ubiquitination. Discussion We have shown that, unlike Rac1L61, the constitu- tively active splice variant Rac1b is poorly ubiquitinat- ed in HEK293 cells, which results in decreased sensitivity to proteasomal degradation. This prompted us to characterize the requirements for Rac1 ubiquiti- nation that are not fulfilled by Rac1b. From our mutational analysis of Rac1 lysine resi- dues, the main ubiquitin addition site in activated Rac1 appears to be Lys147; this result probably explains why Lys147 was found, in a previous study, among the resi- dues required for proteasomal degradation of CNF1- activated Rac1 [36]. Indeed, in Rac2 and Rac3, which are resistant to proteasomal degradation, Lys147 is either absent (Rac3), or is in a different environment as compared to Rac1 (Rac2). Lys147 is not conserved among Rho GTPases, and in RhoA, lysine residues in the N-terminal region (Lys6 and Lys7) have been iden- tified as the acceptors for ubiquitin transferred by Smurf1 [21]. Although a homologous lysine residue, Lys5, is present in Rac1, it is apparently not used for ubiquitin addition. Therefore, the reason why Lys147 rather than Lys5 is the main acceptor site for ubiquitin in Rac1 remains to be elucidated. Of note, however, is that recent studies on the role of the Rab-like GTPase Ypt7 in vacuole fusion in yeast have demonstrated that this small G-protein undergoes ubiquitination and sub- sequent degradation, and that Lys140 and Lys147 con- stitute a major target site for ubiquitin branching in Ypt7 [37]. Interestingly, when the three-dimensional structures of Ypt7 and Rac1 are compared (supplemen- tary Fig. S4), Lys147 has closely similar spatial situations; this observation raises the hypothesis that similar mechanisms and homologous E3 ubiquitin ligases are involved in both cases. On the basis of structural studies, Lys147 is a solvent-accessible residue located far from the GTP-binding site, is not involved in contacts with any of the interacting proteins described so far, and is not affected by the 19-residue insertion in Rac1b. There- fore, the impairment of Rac1b ubiquitination is not likely to be due to inaccessibility or to a local pertur- bation of Lys147 (Lys166 in Rac1b), and nor does it seem to be related to a defective interaction with plasma membrane, as Rac1b was found to be mostly associated with plasma membrane in HEK293 cells, as previously described for other cell types [27,28]. Previous studies have demonstrated that the interac- tion of Rac1 with downstream effectors is required for ubiquitination and degradation [14,35,36]. In agree- ment with this view, we discovered that the rate of Rac1 ubiquitination critically depends on JNK activa- tion. This may indicate that JNK-dependent phosphor- ylation of Rac1 and ⁄ or of its E3 ligase is required to permit efficient Rac1 ubiquitination. Although there is, to our knowledge, no reported evidence for JNK- dependent phosphorylation of Rac1, direct activation of the ligase by JNK-dependent phosphorylation is reminiscent of the recently described activation of the E3 ligase Itch through a phosphorylation-induced con- formational change [38,39]. An attractive hypothesis is that Rac1 ubiquitination depends on a similar regula- tory mechanism, but confirmation of this must await the identification of Rac1 E3 ligase. To date, the only candidate Rac1 E3 ligase has been POSH, a specific partner of activated Rac1 containing a RING finger motif endowed with ubiquitin ligase activity [40,41]; however, our experiments in HEK293 cells did not show any effect of POSH on Rac1L61 ubiquitination and therefore do not support this hypothesis. Except for a recent report on the role of protea- some-mediated degradation of Rac1–GTP in hepato- cyte growth factor-induced epithelial cell scattering, the physiological significance of UPS-mediated regula- tion of Rac GTPase has not been documented. Our results showing that Rac1 ubiquitination critically depends on JNK activation suggest a novel regulatory mechanism allowing the cell to counteract excessive or sustained activation of Rac GTPase. Another significant outcome of our studies regards the tumorigenic capacities of the Rac1b variant. Indeed, Rac1b has been detected in numerous colorec- tal and breast tumors, and although being expressed at a low level in tumor cells, Rac1b has proved to be crit- ical to tumor progression [11,12]. In particular, Rac1b was shown to mediate matrix metalloproteinase-3- induced EMT and genomic instability in mammary epithelial cells; interestingly, in this system, RNAi- mediated downregulation of Rac1b expression resulted in inhibition of EMT and cell migration [11]. There- fore, understanding the molecular basis of the defective ubiquitination ⁄ degradation of Rac1b appears to be an important issue. Indeed, our data suggest that the impairment of Rac1b ubiquitination rests, at least Ubiquitination of Rac1 is regulated by JNK O. Visvikis et al. 392 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS partly, on defective JNK activation, and may provide new ideas for restoring the sensitivity of this tumori- genic variant to UPS. Experimental procedures Cell culture, reagents and transfection HEK293 cells (ATCC reference: CRL-1573) and HeLa cells (ATCC reference: CCL-2) were grown in DMEM (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 100 lg Æ mL )1 strep- tomycin, 100 uÆmL )1 penicillin and 250 ngÆmL )1 fungizone (Gibco, Invitrogen), in humidified atmosphere of 5% CO 2 at 37 °C. Cells were treated as indicated with proteasome inhibitor MG132 (Sigma, St Louis, MO, USA), the protein synthesis inhibitor cycloheximide (Sigma), and the JNK- specific inhibitor SP600125 (Sigma). Cells were transiently transfected using FuGENE 6 Transfection Reagent (Roche, Basel, Switzerland), following the manufacturer’s proce- dure. DNA plasmids and mutagenesis Human cDNA of Rac1 and mutants Rac1N17 and Rac1L61, and mouse cDNA of POSH, cloned in pRK5- myc plasmids, were obtained from A. Hall (University College London, UK). Hemagglutinin (HA)-tagged POSH was obtained by subcloning the cDNA into a pcDNA3– HA plasmid using BamHI and SfiI restriction sites. pXJ– HA–Rac1L61 and pRBG4–6His–myc–Ub plasmids have been used previously [14]. Human cDNAs for Rac1b and Rac1bL61, a gift from P. Jordan (Instituto Nacional de Sau´ de ‘Dr Ricardo Jorge’, Lisbon, Portugal), were sub- cloned into pXJ–HA plasmid using BamH1 and Xho1 restric- tion sites, and into pRK5–myc plasmid using BamH1 and EcoR1 restriction sites. All of the point mutations were generated by using the QuickChange-Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), following the manufacturer’s proce- dure. Each of the 18 lysine mutants of the myc–Rac1L61 sequence and the ubiquitin mutants UbR48 and UbR63 were obtained by replacing the lysine codons AAA and AAG by the arginine codons AGA and AGG respectively. Rac1L61G189 was obtained by mutation of the cysteine codon TGC into the glycine codon GGC. In all cases, the absence of additional mutations was verified by sequencing the entire coding region. Antibodies The primary antibodies used were the 9E10 mouse mono- clonal antibody to myc-tag (Roche), 16B12 mouse mono- clonal antibody to HA-tag (Roche), goat polyclonal antibody to actin (Santa Cruz, San Diego, CA, USA), mouse monoclonal antibody to b-tubulin (Upstate, Milli- pore, Billerica, MA, USA), mouse monoclonal antibody to Rac (USBiological, Swampscott, MA, USA), rabbit poly- clonal antibody to SAPK ⁄ JNK (Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal antibody to phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185; Cell Signaling Technology). For western blotting, secondary peroxidase- conjugated rabbit anti-mouse serum (Dako, Glostrup, Den- mark), secondary peroxidase-conjugated swine anti-rabbit serum (Dako) or secondary peroxidase-conjugated rabbit anti-goat serum (Dako) were used. The secondary fluores- cent antibody used in immunofluorescence studies was Alexa Fluor 488-labeled goat anti-(mouse IgG) (Molecular Probes, Eugene, OR, USA). Western blotting Proteins were resolved in SDS ⁄ PAGE minigels and electro- transferred onto nitrocellulose membrane (Schleicher & Schuell, Millipore). Membranes were probed using the indi- cated primary antibodies and secondary peroxidase-conju- gated antibodies followed by chemiluminescence using the ECL detection system (Amersham, Piscataway, NJ, USA). Quantification of protein expression and ⁄ or ubiquitination was performed using imagej software. Immunofluorescence HEK293 cells were plated at low confluence on glass 18-mm-diameter poly(l-lysine)-coated (0.1 mgÆmL )1 ; Sigma) coverslips in 12-well plates, and transfected the next day. Twenty hours after transfection, cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with NaCl ⁄ P i and 0.2% Triton X-100 for 5 min, and blocked with NaCl ⁄ P i and 1% BSA for 1 h. Cells were then incubated in the same solution with primary antibody for 1 h, and this was followed by 1 h of incubation with a fluorescent secondary antibody. For DNA staining, diaminidophenyl- indol (0.5 lgÆmL )1 ) was added in the last NaCl ⁄ P i wash. Coverslips were mounted using Vectashield Hardset (Vector Laboratories, Burlingame, CA, USA). Cell preparations were observed under a Zeiss (Go ¨ ttingen, Germany) Axiophot epifluorescence microscope; images were digitally acquired and processed using Adobe (San Jose, CA, USA) photoshop 7.0. Activated Rac1 GTPase pulldown assay HEK293 cells seeded in 100 mm Petri dishes were transfect- ed with 5 lg of pRK5–myc–Rac1WT and mutants. The next day, cells were lysed in 500 lL of lysis buffer [50 mm HEPES, pH 7.5, 10 mm MgCl 2 , 150 mm NaCl, 1% Tri- ton X-100, 0.5% NP40, and protease inhibitor cocktail O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 393 (Amersham)]. Proteins (500 lg in 150 lL) were incubated for 2 h at 4 °C with 15 lg of GST or GST–PAK coupled to 20 lL of glutathione–sepharose beads (Amersham). Pel- leted beads were washed twice with washing buffer (50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl 2 ,1mm dithiothreitol, 0.1% Triton X-100, 0.2 mgÆmL )1 BSA, and protease inhibitor cocktail). Bound proteins were recovered by boiling beads in Laemmli sample buffer 2X (SB2X, Sigma) and analysed by western blotting. Ubiquitination assay HEK293 or HeLa cells seeded in 100 mm Petri dishes were transfected with 1–1.5 lg of each plasmid encoding 6His– myc–Ub and various GTPases. Twenty hours after transfec- tion, cells were washed in NaCl ⁄ P i and lysed in 1 mL of denaturing buffer (8 m urea, 20 mm Tris ⁄ HCl, pH 7.5, 200 mm NaCl, 10 mm imidazole, 0.1% Triton X-100, 5 mm N-ethylmaleimide, 10 mm iodoacetic acid). Fifty microliters of lysate were resuspended in SB2X to evaluate the total quantity of proteins. The 6His–myc-ubiquitinated fraction of proteins was recovered by incubating the remaining lysate for 1 h 30 min with 30 lL of cobalt-chelated resin (BD Talon Metal Affinity resin; BD Bioscience, Lexington, KY, USA), previously incubated in denaturing buffer with 0.2 mgÆmL )1 BSA (RIA grade; Sigma). Beads were then washed four times in 1 mL of denaturing buffer, and resus- pended in 25 lL of SB2X. Total lysates and ubiquitinated protein fractions were resolved by SDS ⁄ PAGE and ana- lyzed by western blotting. Assay for proteasomal degradation HEK293 cells seeded in 100 mm Petri dishes were transfect- ed with 5 lg of the indicated plasmids. In order to obtain an identical transfection rate, each pool of transfected cells was trypsinized 8 h after transfection and seeded again into two poly(l-lysine)-coated wells of 12-well Petri dishes. Twenty-four hours post-transfection, protein synthesis and proteasome activity were blocked by adding cycloheximide (1 lgÆmL )1 ) and MG132 (20 lm). Six hours after treatment, cells were lysed with SB2X, and the lysates were resolved by SDS ⁄ PAGE and analyzed by western blotting. Acknowledgements We thank Dr Peter Jordan and Dr Maria-Carla Parrini for plasmids. We also thank Dr Romain Gautier for the pictures of GTPase structures, and Anne Doye for tech- nical assistance. 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Ubiquitination of Rac1L61 lysine mutants. Fig. S2. Subcellular localization of Rac1L61 and Rac1b in HEK293 cells. Fig. S3. Mutation of the prenyl acceptor C189 abol- ishes Rac1 ubiquitination. O. Visvikis et al. Ubiquitination of Rac1 is regulated by JNK FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS 395 [...]...Ubiquitination of Rac1 is regulated by JNK O Visvikis et al Fig S4 Equivalent position of Lys1 47 in the structures of Rac1 (upper panel) and YPT7 (lower panel), both bound to a GTP analog (GppNHp) This material is available as part of the online article from http://www.blackwell-synergy.com 396 Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials... Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS . Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process Orane Visvikis 1,2 , Patrick. proteasomal degradation of CNF1- activated Rac1 [36]. Indeed, in Rac2 and Rac3, which are resistant to proteasomal degradation, Lys1 47 is either absent (Rac3),