Elmo1 Inhibits Ubiquitylation of Dock180

Research Article 923 Elmo1 inhibits ubiquitylation of Dock180

Yoshinori Makino1, Masumi Tsuda1, Shin Ichihara1, Takuya Watanabe1, Mieko Sakai1, Hirofumi Sawa1,*, Kazuo Nagashima1, Shigetsugu Hatakeyama2 and Shinya Tanaka1,‡ 1Laboratory of Molecular and Cellular Pathology, and 2Department of Molecular Biochemistry, Hokkaido University Graduate School of Medicine, N15, W7, Sapporo 060-8638, Japan *Present address: Department of Molecular Pathobiology and 21st Century COE Program for Zoonosis Control, Hokkaido University Research Center for Zoonosis Control, Sapporo 060-8638, Japan ‡Author for correspondence (e-mail: [email protected])

Accepted 15 November 2005 Journal of Cell Science 119, 923-932 Published by The Company of Biologists 2006 doi:10.1242/jcs.02797

Summary Dock180, a member of the CDM family of proteins, plays Dock180 binding, preserved the inhibitory effects on roles in biological processes such as phagocytosis and ubiquitylation of Dock180. Upon EGF stimulation, both motility through its association with the signalling adaptor Dock180 and ubiquitin were demonstrated to translocate protein Crk. Recently, the complex formation between to the cell periphery by immunoﬂuorescence, and we found Dock180 and Elmo1 was reported to function as a bipartite ubiquitylation of Dock180 and its inhibition by Elmo1 to guanine nucleotide exchange factor for Rac. In this study, occur in cellular membrane fractions by in vivo we demonstrated that the amount of Dock180 increased ubiquitylation assay. These data suggest that Dock180 is when Elmo1 was co-expressed. Dock180 was found to be ubiquitylated on the plasma membrane, and also that ubiquitylated and Dock180 protein levels could be Elmo1 functions as an inhibitor of ubiquitylation of augmented by treatment with proteasome inhibitor. The Dock180. Therefore, an ubiquitin-proteasome-dependent ubiquitylation of Dock180 was enhanced by epidermal protein degradation mechanism might contribute to the growth factor (EGF), Crk and adhesion-dependent signals. local activation of Rac on the plasma membrane. Furthermore, Elmo1 inhibited ubiquitylation of Dock180, resulting in the increase in Dock180 levels. The Elmo1 mutant ⌬531, which encompasses amino acids required for Key words: Dock180, Elmo1, Crk, Rac, Ubiquitylation

Introduction an unconventional bipartite guanine nucleotide exchange factor

Journal of Cell Science Dock180 was originally identified as a Src-homology 3 (SH3)- (GEF) for Rac (Brugnera et al., 2002). Recently, it was domain-binding protein of the signalling adaptor protein Crk demonstrated that the small GTPase RhoG interacts directly (Hasegawa et al., 1996; Tanaka et al., 1993). Subsequently, with Elmo1, and that a tri-molecular complex comprised of homologues of Dock180 were identified in Drosophila RhoG, Elmo1 and Dock180 activates Rac1, which then results (Myoblast city) and Caenorhabditis elegans (CED-5), and in integrin-mediated cell spreading, phagocytosis and nerve these were designated as the CDM family of proteins (Cote and growth factor (NGF)-induced neurite outgrowth (deBakker et Vuori, 2002). CDM proteins are evolutionarily conserved and al., 2004; Katoh and Negishi, 2003). have been implicated in various biological responses, including It is well known that ubiquitylation plays a pivotal role in cell migration (Cheresh et al., 1999; Kiyokawa et al., 1998a) physiological cellular responses, including growth-factor- and phagocytosis (Albert et al., 2000), in mammals, mediated signal transduction for cell proliferation and motility. Drosophila (Duchek et al., 2001; Nolan et al., 1998) and C. The epidermal growth factor (EGF) receptor is ubiquitylated elegans (Wang et al., 2003; Wu and Horvitz, 1998). by a RING-finger-type ubiquitin ligase, Cbl (Galcheva- The major SH2 domain targets of Crk are components of Gargova et al., 1995; Joazeiro et al., 1999). The event is focal adhesion p130Cas and paxillin (Feller, 2001), implicating important for regulation of endocytosis of the receptor Crk in cytoskeletal reorganisation. Downstream of Crk, (Mosesson et al., 2003; Soubeyran et al., 2002) and for Dock180 functions as an activator for Rac and regulates cell lysosomal degradation (Longva et al., 2002). Furthermore, motility, filopodia formation and phagocytosis, particularly some of the GEFs, namely Vav and CNrasGEF (for ‘cyclic through stimulation of ␤1 and ␣v␤5 integrins (Albert et al., nucleotide ras GEF’), are ubiquitylated by Cbl and Nedd4, 2000; Gustavsson et al., 2004). respectively (Miura-Shimura et al., 2003; Pham and Rotin, Elmo1 (for ‘engulfment cell motility 1’) was initially 2001). Finally, it has been suggested that ubiquitin-dependent identified as a mammalian homologue of C. elegans Ced-12, protein degradation regulates actin cytoskeletal reorganisation. which is required for cell migration and engulfment of dying In this study, we present the new findings that Dock180 is cells (Gumienny et al., 2001). Elmo1 functionally cooperates ubiquitylated mainly on the plasma membrane; that this is with Crk and Dock180, and promotes phagocytosis and enhanced by EGF, Crk and adhesion-dependent signals; and morphological changes (Grimsley et al., 2004; Gumienny et that its amounts are regulated by an ubiquitin-proteasome- al., 2001; Gustavsson et al., 2004). In addition, Elmo1 binds dependent protein degradation mechanism. Furthermore, we directly to Dock180 (Gumienny et al., 2001) and functions as demonstrated that endogenous Elmo1 could regulate the 924 Journal of Cell Science 119 (5)

amount of Dock180 protein through the inhibition of Ubiquitylation of Dock180 in vivo ubiquitylation of Dock180 by Elmo1. We next investigated whether Dock180 is ubiquitylated and degraded by the proteasome, because the ubiquitin-proteasome Results pathway has been recognised as one of the major mechanisms Elmo1 has a stabilising effect on Dock180 protein for the regulation of cellular protein levels. By in vivo To analyse the functions of Elmo1 in the regulation of ubiquitylation assay, an ubiquitylated protein band of over 180 Dock180, we overexpressed Dock180 in the presence or kDa was observed when HA-ubiquitin was co-expressed with absence of Elmo1. In the presence of Elmo1, the amount of Dock180, and this band was significantly enhanced by the Dock180 was increased (Fig. 1A, left panel). In addition, the treatment of proteasome inhibitor MG-132 (Fig. 2A). To amount of the active form of Rac also increased in the presence exclude the possibility that we were detecting only the of Elmo1 (Fig. 1A, left panel). By contrast, Elmo1 did not alter ubiquitylation of a contaminated protein of a size similar to the levels of other co-expressed proteins including Crk (Fig. that of Dock180, we performed a urea-reversal 1A, right panel). immunoprecipitation assay. In support of the idea that the band These data suggested that Elmo1 regulates the stability of is specific to Dock180, we found that the anti-Dock180 Dock180. To test the idea, we performed a pulse-chase antibody could precipitate a protein over 180 kDa that was also analysis. The labelled form of Dock180 decreased by 79% detectable with the anti-HA tag antibody (Fig. 2B). within 24 hours in the absence of Elmo1. However, in the We also examined the effect of MG-132 on endogenous presence of Elmo1, the levels of Dock180 decreased by 68% levels of Dock180 in MCAS cells as compared with those that (Fig. 1B). Three sets of pulse-chase analyses were performed are expressing Dock180 at high levels. We treated cells with and a significant difference between the levels of Dock180 cyclohexamide, an inhibitor of protein synthesis, and then with and without Elmo1 was confirmed as described, and assayed Dock180 levels. The level of Dock180 was markedly shown in a bar graph with the standard error indicated (Fig. decreased after treatment with cyclohexamide, an effect that 1C). To exclude the possibility that Elmo1 activates the can be rescued by treatment with MG-132 (Fig. 2C). In transcription of Dock180, we also performed RT-PCR analysis HEK293T cells, almost the same results were obtained (data and found the unchanged mRNA levels of Dock180 with not shown). These data suggest that the levels of endogenous Elmo1 (Fig. 1D). In this PCR condition, Dock180 bands were Dock180 protein are regulated by an ubiquitin-proteasome- detectable in a dose-dependent manner for templates (Fig. dependent protein degradation mechanism. 1D). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as a control for unchanged levels of Elmo1 inhibits the ubiquitylation of Dock180 mRNA (Fig. 1D). To examine whether the association of Elmo1 regulates Furthermore, we investigated the expression levels of ubiquitylation of Dock180, we examined the effect of wild- Dock180 when protein expression of Elmo1 was suppressed type and mutant forms of Elmo1 on Dock180. Two deletion by short interfering (si)RNA for Elmo1 in the fibrosarcoma cell forms of Elmo1 – T625, which does not bind to Dock180, and line HT1080 to examine whether Elmo1 can inhibit or regulate ⌬531, which contains C-terminus binding sites for Dock180

Journal of Cell Science the amounts of endogenous Dock180. Among three siRNAs (Fig. 3A,B) (Shimazaki et al., 2005) – were used in an in vivo for Elmo1, designated as Elmo1#1, #2 and #3, Elmo1#3 most- ubiquitylation assay. In this experiment, we performed efficiently reduced the expression levels of Elmo1 in HT1080 immunoprecipitation analysis 24 hours after transfection. After cells; the assay was performed using a negative control and a 24 hours, the amounts of Dock180 were still almost equal in scramble control against Elmo1#3, and Elmo1#3. Expression each transfectant. The equal amounts of Dock180 enabled us levels of Dock180 were reduced by Elmo1#3, compared with to compare the ubiquitylation levels of Dock180. Both wild- both negative control and scramble control (Fig. 1E). Three type Elmo1 and the ⌬531 mutant inhibited ubiquitylation of sets of this siRNA assay were performed, and signiﬁcant Dock180, whereas the T625 mutant form of Elmo1 could not differences in the endogenous levels of Dock180 between the (Fig. 3C). It should be noted that levels of the hyper- controls and Elmo1 siRNA were conﬁrmed, as described by a ubiquitylated form of Dock180 (ӷ500 kDa) seemed to bar graph with standard error (Fig. 1F). decrease in the T625 mutant of Elmo1 (Fig. 3C). The To exclude the possibility that Elmo1 or siRNA for Elmo1 pleckstrin-homology (PH) domain of Elmo1 might have some might affect the amount of mRNA levels of Dock180, we effect on the poly-ubiquitylation of Dock180, because Elmo1 performed RT-PCR analysis and found that mRNA levels of was known to bind the Docker domain (DHR-2 domain) of endogenous Dock180 were almost equal; by contrast, those of Dock180 through the PH domain, which was partially Elmo1 were reduced by siRNA for Elmo1 (Fig. 1G). Moreover, contained in the T625 mutant (Lu et al., 2004). we performed the same assay using siRNA for Elmo1 in We also detected ubiquitylated bands around 70 kDa with HEK293T human embryonic kidney cells. We used Elmo1#2 the anti-HA tag antibody when full-length Elmo1 was co- because it showed the best efficiency for reduction of Elmo1 expressed; because the molecular weight of Elmo1 is around in HEK293T cells, and found that protein levels of Dock180 70 kDa, these bands may be ubiquitylation bands of Elmo1 decreased in concert with reduction of those of Elmo1 by (Fig. 3C, upper panel, arrow). In fact, we found that Elmo1 was siRNA (Fig. 1H). also ubiquitylated during an in vivo ubiquitylation assay using It is noteworthy that the endogenous levels of Dock180 were lysates from HEK293T cells expressing both Elmo1 and HA- not altered even with the presence of force-expressed Elmo1 ubiquitin (data not shown). Further study should be carried out in HEK293T cells (data not shown). This was probably to determine the physiological role of ubiquitylation of Elmo1, because the physiological levels of Elmo1 were sufficient for since overexpression of proteins is known to induce their stabilisation of endogenous Dock180. ubiquitylation and thereby ensure their quality control. Inhibition of ubiquitylation of Dock180 by Elmo1 925 Journal of Cell Science

Fig. 1. Elmo1 increases the stability of Dock180 protein. (A) Left panels; HEK293T cells were transiently transfected with mammalian expression plasmids for Flag-Dock180 alone or Flag-Dock180 and myc-Elmo1. After 48 hours, cells were lysed and analysed by immunoblotting (IB) with anti-Flag tag, anti-myc tag and anti-actin antibodies. The active form of Rac was detected in a pull-down assay. Right panels: HEK293T cells were transfected with mammalian expression plasmids for Flag-Crk II alone or Flag-Crk II and myc-Elmo1, and cells were analysed in the same way as for the left panels. (B) For pulse-chase analysis for Dock180, HEK293T cells transiently expressing Dock180 alone (–) or Dock180 and Elmo1 (+) were labelled for 1 hour and then chased for the time indicated at the top of the panel. (C) The signal intensity of Dock180 was measured and shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (D) mRNA levels of exogenous Dock180. RT-PCR analysis was performed using mRNA extracted from HEK293T cells transiently expressing Dock180 alone or Dock180 and Elmo1. GAPDH is a control for the amounts of PCR templates. T/F, transfection; RT, reverse transcriptase. (E) HT1080 cells were transfected with siRNAs for negative control, scramble control and Elmo1#3. After incubation for 96 hours, cell lysates were subjected to immunoblotting for detection of Dock180, Elmo1 and actin. (F) The signal intensity of Dock180, normalised by the intensity of actin, is shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (G) mRNA levels of endogenous Dock180. RT-PCR analysis was performed using the same mRNA extracts as E. GAPDH is a control for the amount of PCR template. (H) HEK293T cells were transfected with negative control and Elmo1#2 and cells were analysed in the same manner as E.

To conﬁrm that the ubiquitylated bands over 180 kDa degree as seen in Fig. 3C by co-expression of full-length observed in Fig. 3C are Dock180, we performed urea-reversal Elmo1 and the ⌬531 mutant (Fig. 3D). In addition, immunoprecipitation assay using the same cell lysates as those ubiquitylated bands of around 70 kDa (Fig. 3C, lane 3) analysed in the experiment shown in Fig. 3C. We found that disappeared (Fig. 3D). Furthermore, to conﬁrm that the the ubiquitylated bands were suppressed to almost the same inhibitory effect of Elmo1 for ubiquitylation of Dock180 is not 926 Journal of Cell Science 119 (5)

Fig. 2. Ubiquitylation of Dock180 in HEK293T cells. (A) Results of an in vivo ubiquitylation assay. HEK293T cells were transfected with the indicated plasmids, and after treatment of 10 ␮M MG-132 for 12 hours, cells were lysed and subjected to immunoprecipitation (IP) and immunoblotting (IB). MIG, normal mouse immunoglobulin; TCL, total cell lysate; Ub, ubiquitin. (B) For the urea-reversal immunoprecipitation, the initial precipitates from the anti- Dock180 mAb were treated with 8 M urea buffer for 1 minute and then immunoprecipitated again using the same antibody. (C) MCAS and HEK293T cells were treated with 20 ␮g/ml cyclohexamide and 10 ␮g/ml MG-132 for 24 hours as indicated at the top of the panel. Cell lysates were immunoblotted with anti-Dock180 antibody to detect endogenous Dock180.

ubiquitylation (Fig. 4A). In contrast to the inhibition of ubiquitylation of Dock180 by Elmo1, protein levels of Dock180 in the membrane fraction were lower when co- expressed with Elmo1 than those without Elmo1 (Fig. 4A). We examined the effect of Elmo1 on the amount of endogenous Dock180 in the membrane fraction in HEK293 cells. The amount of endogenous Dock180 in the membrane fraction was also found to be decreased by Elmo1 (data not shown). For further analysis, we employed immunoﬂuorescent microscopy and observed the subcellular localisation of

Journal of Cell Science ubiquitin and Dock180. Dock180 and HA-ubiquitin were co- expressed in HEK293T cells, and cells were stained with anti- non-specific, we examined an irrelevant protein [glutathione S- Dock180 and anti-HA tag antibody. We found that, in quiescent transferase (GST)] on Dock180 ubiquitylation. Unlike Elmo1, cells, both Dock180 and ubiquitin were partially localised at GST was found to have no effect on the levels of ubiquitylation the cell periphery, although most of them were localised mainly of Dock180 (Fig. 3E). We employed an alternative approach to confirm that Elmo1, when bound to Dock180, suppressed ubiquitylation. Fig. 3. Elmo1 suppresses ubiquitylation of Dock180. (A) A We performed an in vivo ubiquitylation assay using the schematic representation of wild-type and mutant forms of Elmo1 Dock180⌬357 mutant (which has a 357 amino acid deletion used in the study. DUF609, domain of unknown function; FL, full at the N-terminus and does not bind Elmo1) (Fig. 3F) length; LZ, leucine zipper; PH, pleckstrin homology; PxxP, (Brugnera et al., 2002). We found that the Dock180⌬357 polyproline-rich motif. (B) Association between Dock180 and Elmo1 in HEK293T cells. Cells were transiently transfected with the mutant was significantly ubiquitylated, much more so than indicated plasmids, and lysates were immunoprecipitated with anti- wild-type Dock180, both in the absence and presence of myc tag antibody, and then probed with anti-myc tag and anti-Flag Elmo1 (Fig. 3F). These results suggest that Elmo1 functions tag antibodies. TCL, total cell lysate. (C) Ubiquitylation of Dock180 as an inhibitor of the ubiquitylation of Dock180 through a in HEK293T cells in the presence of Elmo1 and its mutants. 24 hours mechanism that is dependent on the interaction between after transfection with the indicated plasmids, cells were lysed and Dock180 and Elmo1. subjected to in vivo ubiquitylation assay. (D) The urea-reversal immunoprecipitation was performed using the same lysates as C. The Ubiquitylation of Dock180 on the plasma membrane IP ratio represents the relative signal intensity of immunoprecipitated Next, we investigated the subcellular localisation for Dock180 in lane 1 as 1.0 (bottom of the panel). (E) The ubiquitylation of Dock180. An in vivo ubiquitylation assay for ubiquitylation of Dock180 in the absence or presence of Elmo1 and GST. The immunoprecipitation was performed using anti-Flag tag Dock180 was performed using either the cytosolic or the antibody. (F) The ubiquitylation of Dock180 and its mutant. Lysates membrane fraction of cell lysates. We found that Dock180 from HEK293T cells expressing the indicated plasmids were treated with the membrane fraction was highly ubiquitylated, immunoprecipitated with anti-Dock180 antibody at in vivo an effect that was inhibited by Elmo1 (Fig. 4A). However, in ubiquitylation assay. The IP ratio represents the relative signal the cytosolic fraction, Elmo1 did not alter the level of Dock180 intensity of immunoprecipitated Dock180 in lane 1 as 1.0. Inhibition of ubiquitylation of Dock180 by Elmo1 927 Journal of Cell Science

Fig. 3. See previous page for legend. 928 Journal of Cell Science 119 (5)

in the cytoplasm (Fig. 4B, upper panel, arrowheads). Upon indicate that ubiquitylation of Dock180 occurs mainly on the EGF stimulation, both Dock180 and ubiquitin were plasma membrane and Elmo1 inhibits this ubiquitylation. translocated and colocalised at the edge of the ruffled membrane (Fig. 4B, upper panel, arrows). This EGF-induced Enhancement of the ubiquitylation of Dock180 by Crk colocalisation of Dock180 and ubiquitin were also examined Next we investigated whether a main regulator of Dock180 in Cos-7 cells (Fig. 4B, lower panel). The RFP fusion forms of such as Crk was involved in the ubiquitylation of Dock180 on Dock180 and HA-ubiquitin were co-expressed in Cos-7 cells, the plasma membrane, and found its enhancement by both Crk and HA-ubiquitin was visualised by immunostaining with anti- I and Crk II in the membrane fraction (Fig. 5). By in vivo HA tag antibody. In quiescent cells, most of the detectable ubiquitylation assay using whole cell lysates, no significant Dock180 and ubiquitin were diffusely found in the cytoplasm Crk-dependent enhancement of ubiquitylation of Dock180 was (Fig. 4B, lower panel). However, upon EGF stimulation, both observed (data not shown). In addition, we found that Dock180 Dock180 and ubiquitin were translocated to the cell periphery levels in the membrane fraction were significantly increased by (Fig. 4B, lower panel, arrowheads). co-expression with either Crk I or Crk II (Fig. 5). Furthermore, we confirmed that ubiquitylation of Dock180 in the membrane fraction was enhanced by EGF stimulation, Fibronectin-stimulation-enhanced ubiquitylation of and found that Elmo1 could inhibit this enhancement (Fig. 4C). Dock180 The enhancement of the ubiquitylation of Dock180 in whole As Crk and Dock180 are known to function downstream of cell lysates was not detected (data not shown). These results integrins, especially ␣5␤1 (Iwahara et al., 2004; Kiyokawa et Journal of Cell Science

Fig. 4. Ubiquitylation of Dock180 on the plasma membrane. (A) Ubiquitylation of Dock180 in the membrane fraction. Cell lysates of HEK293T cells expressing the indicated plasmids were separated into cytosolic and membrane fractions. The samples were subjected to an in vivo ubiquitylation assay. The expression levels of transfected proteins were analysed using cell lysates of the cytosol and membrane fractions. Anti-E-cadherin antibody was used as a marker for the membrane fraction. CL, cell lysate. (B) Localisation of Dock180 and ubiquitin in cells. HEK293T cells (upper panels) and Cos-7 cells (lower panels) were transiently transfected with Flag-Dock180 or Dock180-RFP and HA-ubiquitin expression plasmids. After incubation for 36 hours, cells were stained with anti-HA tag antibody and analysed by confocal microscopy. (C) Ubiquitylation of Dock180 in the membrane fraction under EGF stimulation. HEK293T cells expressing the indicated plasmids were stimulated by EGF or not. Cell lysates were subjected to in vivo ubiquitylation assay. Inhibition of ubiquitylation of Dock180 by Elmo1 929

Fig. 5. The enhancement of ubiquitylation of Dock180 by Crk. An in vivo ubiquitylation assay was performed using lysates of the membrane fraction of HEK293T cells expressing the indicated plasmids (left panels). The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0 (bottom of the left panel). The expression levels of Dock180, Crk I and Crk II in the membrane fraction of cell lysates (membrane) and in total cell lysates (total) were determined by immunoblotting (right upper and middle panels). Anti-E-cadherin antibody was used as a marker for the membrane fraction (right lower panel).

Discussion We have investigated the physiological function of Dock180, a member of the CDM family of proteins, and its interacting protein Elmo1, and found that Dock180 could be ubiquitylated and its amounts regulated by an ubiquitin-proteasome- dependent protein degradation mechanism. Initial observations, in which the amount of force-expressed Dock180 al., 1998b; Sakai et al., 1994; Schaller and Parsons, 1995), we increased when Elmo1 was co-expressed in HEK293T cells, carried out a re-plating assay using a ﬁbronectin-coated dish. prompted us to investigate the involvement of ubiquitylation in An increase in ubiquitylation of Dock180 in the membrane regulation of the amounts of Dock180. To exclude the fraction was observable 15 minutes after re-plating and possibility of Elmo1-dependent transcriptional regulation of persisted for at least 6 hours (Fig. 6A). The amount of Dock180 Dock180, we performed RT-PCR and pulse-chase analyses for in the membrane fraction was not altered when cells were re- the amounts of Dock180 with or without Elmo1 in HEK293T plated on a ﬁbronectin-coated dish (Fig. 6A). In these cells, cells, and found that Elmo1 stabilised Dock180. In fact, we Elmo1 suppressed ubiquitylation of Dock180 even in the cells found that Elmo1 inhibited ubiquitylation of Dock180, and that in suspension (Fig. 6B, lanes 1 and 3), and also inhibited the levels of endogenous Dock180 were decreased by siRNA augmentation of the re-plating-induced ubiquitylation of for Elmo1. Since a rapid alteration in the level of Dock180 Dock180 (Fig. 6B, lanes 2 and 4). during the process of maturation of dendritic cells has been

Journal of Cell Science reported (Akakura et al., 2004), we plan to analyse the involvement of ubiquitylation of Dock180 in this process. We also showed that the ⌬531 mutant that binds to Dock180 preserves an inhibitory activity towards ubiquitylation of Dock180. Even so, the precise mechanism of Elmo1-dependent inhibition of ubiquitylation remains obscure. Elmo1 might block the physical

Fig. 6. Enhancement of ubiquitylation of Dock180 in HEK293T cells re-plated on fibronectin-coated dishes. (A) HEK293T cells were transiently transfected with the indicated plasmids for Dock180 and HA-ubiquitin and re-plated on fibronectin-coated dishes for the indicated duration (top of the panel); lysates in the membrane fraction were subjected to in vivo ubiquitylation assay. Sus, suspended cells; FN, fibronectin. Ratio represents the relative signal intensity of Dock180 in the membrane fraction in lane 1 as 1.0 (bottom of the panel). (B) Inhibition of ubiquitylation of Dock180 by Elmo1. HEK293T cells were transiently transfected with the indicated plasmids, and the re-plating assay and in vivo ubiquitylation assay were performed as in A. 930 Journal of Cell Science 119 (5)

association of E3 ubiquitin ligase to Dock180, or it might mask dishes is likely to be dependent on cell attachment rather than the ubiquitylation sites of Dock180. Alternatively, Elmo1 has be specific for integrin stimulation. These data suggest that been reported to induce a conformational change of Dock180, recruitment of Dock180 to the plasma membrane by EGF, Crk which could inhibit its ubiquitylation (Lu et al., 2004; Lu et and adhesion-dependent signals might contribute to the al., 2005). To define the mechanism of Elmo1 action further, it ubiquitylation of Dock180. will be necessary to identify the E3 ubiquitin ligase that is It should be noted that despite the fact that ubiquitylation required for Dock180. It can at least be said that co-expression levels of Dock180 were elevated by stimulation with EGF or of Cbl with Dock180 does not change the levels of fibronection, or forced expression of Crk, the protein levels of ubiquitylation of Dock180 in HEK293T cells (data not shown). Dock180 in the membrane fraction did not decrease. We Recently, ubiquitylation of GEFs as a regulatory mechanism speculate that the reason why Dock180 seemed not to be has been reported for other proteins. In the case of Vav, an removed in the membrane fraction is that Dock180 is activated Vav mutant (Y174F) was shown to be more sensitive translocated from the cytoplasm by Crk. As the supplied to Cbl-dependent ubiquitylation, which suggests the regulation amount of Dock180 may be more than that degraded by the of protein degradation by tyrosine phosphorylation (Miura- proteasome, the apparent amount of Dock180 is not decreased Shimura et al., 2003). Furthermore, binding to Ras has been after being ubiquitylated. As shown in Fig. 7, by integrin shown to be necessary for ubiquitylation of Ras-GRF2 (de stimulation, Crk recruits Dock180 to the focal adhesion Hoog et al., 2001). We demonstrated that Dock180 was mainly complex and Dock180 then activates Rac. In this process, ubiquitylated on the plasma membrane and that this was several modifications of Dock180 including ubiquitylation enhanced by both EGF and Crk. Furthermore, in re-plating might occur and a part of Dock180 is removed from the cells onto fibronectin-coated dishes, ubiquitylation of Dock180 complex comprising Crk and p130Cas; such regulations might on the plasma membrane was also enhanced. It should be noted modulate local Rac activity. Thus, in the local areas of a cell, that various levels of ubiquitylation of Dock180 were observed Dock180 ubiquitylation, which decreases the amount of even when cells were re-plated on dishes coated with poly-L- Dock180, functions as one of the negative-feedback lysine and collagen (data not shown). Thus, the enhancement machineries for the Dock180-dependent activation of Rac, of ubiquitylation of Dock180 observed with fibronectin-coated after integrin-dependent signals are turned on. Journal of Cell Science

Fig. 7. Model of Dock180 regulation. (A,B) Following integrin stimulation by their ligands (including ﬁbronectin), tyrosine kinases are activated and phosphorylate several components of focal adhesion such as p130Cas, and both Crk and Dock180 are recruited from the cytoplasm. (C) Recruited Dock180 is known to activate Rac, and we speculate that recruited Dock180 is ubiquitylated near the plasma membrane by an ubiquitin ligase. (D) Furthermore, ubiquitylated Dock180 might be removed from the complex comprising Crk and p130Cas, and is degraded by the proteasome. Inhibition of ubiquitylation of Dock180 by Elmo1 931

CAGGAAGAUUU for Elmo1#3 (1561-1585 bp of Elmo1), and CCGGAAG- Such a spatio-temporal alteration of Dock180 levels on the GUAACCGAAGGAAGAUUU for scramble control against Elmo1#3, respectively. plasma membrane could function in cell migration. We hope All siRNA duplex oligoribonucleotides including negative control (Stealth RNAi that future works both on the identification of ubiquitin ligase Negative CTL MED GC) were purchased from Invitrogen. for Dock180 and on the analyses of ubiquitylation of Dock180 Cell fractionation in living cells might further clarify the mechanism for Cell fractions were prepared by the method described previously (Kobayashi et al., ubiquitin-dependent Rac-GEF regulation. 2001) with some modifications. Briefly, cells were scraped and suspended in buffer A containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, Materials and Methods 1 mM PMSF and complete protease inhibitor cocktail. After freeze and thaw, cell Cells suspensions were centrifugated at 1,000 g for 7 minutes and then at 20,000 g for HEK293T (human embryonic kidney 293 cells with SV40 T antigen), Cos-7, 10 minutes. The supernatant was removed (cytosolic fraction). The pellet was MCAS (human ovarian mucinous adenocarcinoma) and HT1080 (human washed once with buffer A, lysed with buffer B containing 20 mM Tris-HCl (pH fibrosarcoma) cells were maintained in Dulbecco’s Modified Eagle’s Medium 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 50 mM NaF, 1 mM Na3VO4, 1 mM (DMEM), supplemented with 100 ␮g/ml penicillin and streptomycin and 10% fetal PMSF and complete protease inhibitor cocktail, and centrifugated at 20,000 g for bovine serum (Sigma), in 5% CO at 37°C. 10 minutes (membrane fraction). One-fifth volume of buffer A was used in the 2 preparation of the membrane fraction. Antibodies The following antibodies were used: anti-influenza hemagglutinin (HA) tag mouse Pull-down assay for Rac activity monoclonal antibody (mAb; clone 12CA5, Roche Diagnostics); anti-Dock180 mAb HEK293T cells were lysed with a lysis buffer composed of 1% NP-40, 25 mM, (clone H4, Santa Cruz Biotechnology); anti-Dock180 polyclonal antibody (clone HEPES (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA, 10 mM MgCl2, 1 mM PMSF and complete protease inhibitor cocktail. Lysates were centrifuged at H70, Santa Cruz Biotechnology); anti-FLAG tag mAb (clone M2, Sigma); anti-myc ␮ tag mAb (clone 9E10, gift from Hiroshi Ariga, Hokkaido University, Sapporo, 14,000 g at 4°C for 1 minute. The supernatants were incubated with 10 g of Japan); anti-Rac mAb (clone 102, BD Transduction Laboratories); anti-actin mAb purified GST-PAK2-RBD and then with glutathione-Sepharose 4B beads (clone C4, Chemicon International); anti-Elmo1 polyclonal antibody (clone ab2239, (Amersham Pharmacia Biotech). The beads were washed three times with lysis Abcam); and anti-E-cadherin mAb (clone 36, BD Transduction Laboratories). The buffer. The precipitants were analysed by immunoblotting with anti-Rac antibody anti-mouse immunoglobulin Ab conjugated with Alexa Fluor 488 and the anti-rabbit to detect GTP-form Rac. immunoglobulin Ab conjugated with Alexa Fluor 594 were purchased from Molecular Probes. Pulse-chase assay HEK293T cells expressing the indicated plasmids were metabolically labelled with ␮ 35 Expression plasmids 100 Ci [ S]methionine for 1 hour at 37°C, washed with phosphate buffered saline The pCXN2-Flag-Dock180, pCAGGS-myc-CrkI and -Crk II vectors were gifts from (PBS), and cultured in DMEM for 0-24 hours. Cell lysates were subjected to M. Matsuda (Osaka University, Osaka, Japan) and the pCGN-HA-Ubiquitin vector immunoprecipitation with anti-Flag tag mAb, the precipitates were separated by SDS- was constructed as described previously (Hatakeyama et al., 2001). The red PAGE, and signals were analysed using the BAS2000 image analyser (Fuji Film). fluorescent protein (RFP) fragment was amplified by PCR and subcloned into XhoI- NotI-digested pCXN2-Flag-Dock180; the resulting plasmid was named pCXN2- In vivo ubiquitylation assay Dock180-RFP. pCXN2-Flag-Crk-II was also constructed (by H. Nishihara, HEK293T cells were transfected with the indicated plasmids. After 12-36 hours, ␮ Hokkaido University, Sapporo, Japan). cells were further cultured in the absence or presence of 10 M proteasome inhibitor The pEBB-Flag-ELMO1 vector was kindly provided by K. Ravichandran MG-132 for 12 hours. The cells were lysed with 1% Tx-100 lysis buffer (see above). (University of Virginia, VA). PCR fragments of full-length Elmo1, T625, and ⌬531 Cell lysates were immunoprecipitated with anti-Dock180 antibody (H4), anti-Flag were subcloned into a pMyc-CMV mammalian expression vector (Clontech tag antibody, or mouse normal IgG (IgG), and immunoblotted with anti-HA tag and Laboratories) that had been digested with XhoI and NotI; the resulting plasmid was anti-Dock180 antibodies. For the denaturing condition, the immunoprecipitates named pCMV-myc-Elmo1 and contains the following changes: T625 [amino acids were incubated with 8 M urea buffer containing 20 mM Tris-HCl (pH 7.4) and 8 (aa.) 1 to 625], and ⌬531 (aa. 532 to 727). pGEX-PAK2-RBD was described M urea for 1 minute, then diluted with 1% Tx-100 lysis buffer and

Journal of Cell Science previously (Nishihara et al., 2002). All PCR fragments were verified by sequencing. immunoprecipitated again with the anti-Dock180 antibody (urea-reversal immunoprecipitation). RT-PCR analysis RT-PCR analysis was performed by the method described previously (Akakura et Confocal laser scanning microscopical study al., 2004; Shimazaki et al., 2005). Total RNA was prepared using the RNeasy Mini Cos-7 and HEK293T cells expressing the indicated plasmids were fixed with 3% Kit (Qiagen) from HEK293T cells expressing the indicated plasmids and HT1080 paraformaldehyde for 15 minutes and permeabilised with 0.1% Triton X-100 cells. The forward primer 5Ј-TGGAGACAAAGTCACGGAGG-3Ј and the reverse containing PBS for 4 minutes at room temperature (RT). The cells were washed and primer 5Ј-GATGAGAGGGAAGAGACAGAGG-3Ј for Dock180 yielded a product incubated with 1% BSA containing PBS, and then with only anti-HA tag antibody, of 219 bp. The forward primer 5Ј-CCGGATTGTGCTTGAGAACA-3Ј and the or both anti-HA tag and anti-Dock180 (H70) antibodies at 4°C overnight. The cells reverse primer 5Ј-CTCACTAGGCAACTCGCCCA-3Ј for Elmo1 yielded a product were next incubated with Alexa Fluor 488-conjugated anti-mouse immunoglobulin of 121 bp. The forward primer 5Ј-TTCGTCATGGGTGTGAACCA-3Ј and the and/or Alexa Fluor 594-conjugated anti-rabbit immunoglobulin antibodies for 1 reverse primer 5Ј-GGTCATGAGTCCTTCCACGATAC-3Ј for GAPDH yielded a hour at RT with a light shield protecting the samples from photobleaching. For the product of 138 bp. negative control, cells were processed the same way but without primary antibody. The cells were observed using a confocal laser-scanning microscope equipped with Transfection, immunoprecipitation and immunoblot analysis a computer (MRC-1024; Bio-Rad Microscience Division). HEK293T cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen). After incubation for 24-48 hours, the cells were lysed with 1% Tx-100 Re-plating assay lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, HEK293T cells transfected with the indicated plasmids were incubated for 36- 10% (v/v) glycerol, 1% (v/v) Triton X-100, complete protease inhibitor cocktail 48 hours and harvested in 0.25% trypsin-EDTA solution (Sigma). Cells were then (Roche Diagnostics) and 1 mM phenylmethylsulphonylfluoride (PMSF). Lysates put into suspension in Opti-MEM (Invitrogen) containing 1 mg of soybean were centrifuged at 20,000 g for 10 minutes at 4°C. The supernatants were incubated trypsin inhibitor (suitable for neutralisation of 2.5 mg of trypsin). The cells were with the indicated antibodies and then with protein A beads (Protein A Sepharose held in suspension for 3 hours at 37°C. Suspended cells were then distributed 4 Fast Flow; Amersham Pharmacia Biotech) for 1 hour at 4°C. Precipitates or cell onto cell culture dishes pre-coated with fibronectin (Iwaki) and the cells lysates were separated by SDS-PAGE, transferred onto PVDF filters (Immobilon), incubated at 37°C for the indicated times. Cells were rinsed in cold PBS prior and incubated with primary antibodies. Positive signals were detected by enhanced to protein extraction. chemiluminescence (ECL) western blotting reagents (Amersham Pharmacia Biotech) and quantified using a Lumino Image Analyzer (LAS1000; Fuji Film). We thank M. Matsuda (Osaka University, Japan) and Kodi S. Ravichandran (University of Virginia, VA) for plasmids and Tadaki siRNA for Elmo1 Suzuki (Hokkaido University, Japan) for useful discussion. This study HT1080 and HEK293T cells were transfected with siRNAs indicated below by was supported in part by Grants-in-Aid from the Ministry of Lipofectamine 2000. After incubation for 96 hours, cells were lysed and subjected to immunoblotting. Target sequences of siRNA are GGGUGGUCUCUUGCCA- Education, Culture, Sports, Science and Technology, Japan, and from ACCAUGAAU for Elmo1#1 (120-144 bp of Elmo1), GGCACUAUCCUUCGA- the Ministry of Health, Labor and Welfare, Japan, and by the Yasuda UUAACCACAU for Elmo1#2 (216-240 bp of Elmo1), CCGAGAGGAUGAAC- Medical Research Foundation. 932 Journal of Cell Science 119 (5)

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