923Research Article

IntroductionDock180 was originally identified as a Src-homology 3 (SH3)-domain-binding protein of the signalling adaptor protein Crk(Hasegawa et al., 1996; Tanaka et al., 1993). Subsequently,homologues of Dock180 were identified in Drosophila(Myoblast city) and Caenorhabditis elegans (CED-5), andthese were designated as the CDM family of proteins (Cote andVuori, 2002). CDM proteins are evolutionarily conserved andhave been implicated in various biological responses, includingcell migration (Cheresh et al., 1999; Kiyokawa et al., 1998a)and phagocytosis (Albert et al., 2000), in mammals,Drosophila (Duchek et al., 2001; Nolan et al., 1998) and C.elegans (Wang et al., 2003; Wu and Horvitz, 1998).

The major SH2 domain targets of Crk are components offocal adhesion p130Cas and paxillin (Feller, 2001), implicatingCrk in cytoskeletal reorganisation. Downstream of Crk,Dock180 functions as an activator for Rac and regulates cellmotility, filopodia formation and phagocytosis, particularlythrough stimulation of �1 and �v�5 integrins (Albert et al.,2000; Gustavsson et al., 2004).

Elmo1 (for ‘engulfment cell motility 1’) was initiallyidentified as a mammalian homologue of C. elegans Ced-12,which is required for cell migration and engulfment of dyingcells (Gumienny et al., 2001). Elmo1 functionally cooperateswith Crk and Dock180, and promotes phagocytosis andmorphological changes (Grimsley et al., 2004; Gumienny etal., 2001; Gustavsson et al., 2004). In addition, Elmo1 bindsdirectly to Dock180 (Gumienny et al., 2001) and functions as

an unconventional bipartite guanine nucleotide exchange factor(GEF) for Rac (Brugnera et al., 2002). Recently, it wasdemonstrated that the small GTPase RhoG interacts directlywith Elmo1, and that a tri-molecular complex comprised ofRhoG, Elmo1 and Dock180 activates Rac1, which then resultsin integrin-mediated cell spreading, phagocytosis and nervegrowth factor (NGF)-induced neurite outgrowth (deBakker etal., 2004; Katoh and Negishi, 2003).

It is well known that ubiquitylation plays a pivotal role inphysiological cellular responses, including growth-factor-mediated signal transduction for cell proliferation and motility.The epidermal growth factor (EGF) receptor is ubiquitylatedby a RING-finger-type ubiquitin ligase, Cbl (Galcheva-Gargova et al., 1995; Joazeiro et al., 1999). The event isimportant for regulation of endocytosis of the receptor(Mosesson et al., 2003; Soubeyran et al., 2002) and forlysosomal degradation (Longva et al., 2002). Furthermore,some of the GEFs, namely Vav and CNrasGEF (for ‘cyclicnucleotide ras GEF’), are ubiquitylated by Cbl and Nedd4,respectively (Miura-Shimura et al., 2003; Pham and Rotin,2001). Finally, it has been suggested that ubiquitin-dependentprotein degradation regulates actin cytoskeletal reorganisation.

In this study, we present the new findings that Dock180 isubiquitylated mainly on the plasma membrane; that this isenhanced by EGF, Crk and adhesion-dependent signals; andthat its amounts are regulated by an ubiquitin-proteasome-dependent protein degradation mechanism. Furthermore, wedemonstrated that endogenous Elmo1 could regulate the

Dock180, a member of the CDM family of proteins, playsroles in biological processes such as phagocytosis andmotility through its association with the signalling adaptorprotein Crk. Recently, the complex formation betweenDock180 and Elmo1 was reported to function as a bipartiteguanine nucleotide exchange factor for Rac. In this study,we demonstrated that the amount of Dock180 increasedwhen Elmo1 was co-expressed. Dock180 was found to beubiquitylated and Dock180 protein levels could beaugmented by treatment with proteasome inhibitor. Theubiquitylation of Dock180 was enhanced by epidermalgrowth factor (EGF), Crk and adhesion-dependent signals.Furthermore, Elmo1 inhibited ubiquitylation of Dock180,resulting in the increase in Dock180 levels. The Elmo1mutant ��531, which encompasses amino acids required for

Dock180 binding, preserved the inhibitory effects onubiquitylation of Dock180. Upon EGF stimulation, bothDock180 and ubiquitin were demonstrated to translocateto the cell periphery by immunofluorescence, and we foundubiquitylation of Dock180 and its inhibition by Elmo1 tooccur in cellular membrane fractions by in vivoubiquitylation assay. These data suggest that Dock180 isubiquitylated on the plasma membrane, and also thatElmo1 functions as an inhibitor of ubiquitylation ofDock180. Therefore, an ubiquitin-proteasome-dependentprotein degradation mechanism might contribute to thelocal activation of Rac on the plasma membrane.

Key words: Dock180, Elmo1, Crk, Rac, Ubiquitylation

Summary

Elmo1 inhibits ubiquitylation of Dock180Yoshinori 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, Sapporo060-8638, Japan‡Author for correspondence (e-mail: tanaka@med.hokudai.ac.jp)

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

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amount of Dock180 protein through the inhibition ofubiquitylation of Dock180 by Elmo1.

ResultsElmo1 has a stabilising effect on Dock180 proteinTo analyse the functions of Elmo1 in the regulation ofDock180, we overexpressed Dock180 in the presence orabsence of Elmo1. In the presence of Elmo1, the amount ofDock180 was increased (Fig. 1A, left panel). In addition, theamount of the active form of Rac also increased in the presenceof Elmo1 (Fig. 1A, left panel). By contrast, Elmo1 did not alterthe levels of other co-expressed proteins including Crk (Fig.1A, right panel).

These data suggested that Elmo1 regulates the stability ofDock180. To test the idea, we performed a pulse-chaseanalysis. The labelled form of Dock180 decreased by 79%within 24 hours in the absence of Elmo1. However, in thepresence of Elmo1, the levels of Dock180 decreased by 68%(Fig. 1B). Three sets of pulse-chase analyses were performedand a significant difference between the levels of Dock180with and without Elmo1 was confirmed as described, andshown in a bar graph with the standard error indicated (Fig.1C). To exclude the possibility that Elmo1 activates thetranscription of Dock180, we also performed RT-PCR analysisand found the unchanged mRNA levels of Dock180 withElmo1 (Fig. 1D). In this PCR condition, Dock180 bands weredetectable in a dose-dependent manner for templates (Fig.1D). Primers for glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were used as a control for unchanged levels ofmRNA (Fig. 1D).

Furthermore, we investigated the expression levels ofDock180 when protein expression of Elmo1 was suppressedby short interfering (si)RNA for Elmo1 in the fibrosarcoma cellline HT1080 to examine whether Elmo1 can inhibit or regulatethe amounts of endogenous Dock180. Among three siRNAsfor Elmo1, designated as Elmo1#1, #2 and #3, Elmo1#3 most-efficiently reduced the expression levels of Elmo1 in HT1080cells; the assay was performed using a negative control and ascramble control against Elmo1#3, and Elmo1#3. Expressionlevels of Dock180 were reduced by Elmo1#3, compared withboth negative control and scramble control (Fig. 1E). Threesets of this siRNA assay were performed, and significantdifferences in the endogenous levels of Dock180 between thecontrols and Elmo1 siRNA were confirmed, as described by abar graph with standard error (Fig. 1F).

To exclude the possibility that Elmo1 or siRNA for Elmo1might affect the amount of mRNA levels of Dock180, weperformed RT-PCR analysis and found that mRNA levels ofendogenous Dock180 were almost equal; by contrast, those ofElmo1 were reduced by siRNA for Elmo1 (Fig. 1G). Moreover,we performed the same assay using siRNA for Elmo1 inHEK293T human embryonic kidney cells. We used Elmo1#2because it showed the best efficiency for reduction of Elmo1in HEK293T cells, and found that protein levels of Dock180decreased in concert with reduction of those of Elmo1 bysiRNA (Fig. 1H).

It is noteworthy that the endogenous levels of Dock180 werenot altered even with the presence of force-expressed Elmo1in HEK293T cells (data not shown). This was probablybecause the physiological levels of Elmo1 were sufficient forstabilisation of endogenous Dock180.

Ubiquitylation of Dock180 in vivoWe next investigated whether Dock180 is ubiquitylated anddegraded by the proteasome, because the ubiquitin-proteasomepathway has been recognised as one of the major mechanismsfor the regulation of cellular protein levels. By in vivoubiquitylation assay, an ubiquitylated protein band of over 180kDa was observed when HA-ubiquitin was co-expressed withDock180, and this band was significantly enhanced by thetreatment of proteasome inhibitor MG-132 (Fig. 2A). Toexclude the possibility that we were detecting only theubiquitylation of a contaminated protein of a size similar tothat of Dock180, we performed a urea-reversalimmunoprecipitation assay. In support of the idea that the bandis specific to Dock180, we found that the anti-Dock180antibody could precipitate a protein over 180 kDa that was alsodetectable with the anti-HA tag antibody (Fig. 2B).

We also examined the effect of MG-132 on endogenouslevels of Dock180 in MCAS cells as compared with those thatare expressing Dock180 at high levels. We treated cells withcyclohexamide, an inhibitor of protein synthesis, and thenassayed Dock180 levels. The level of Dock180 was markedlydecreased after treatment with cyclohexamide, an effect thatcan be rescued by treatment with MG-132 (Fig. 2C). InHEK293T cells, almost the same results were obtained (datanot shown). These data suggest that the levels of endogenousDock180 protein are regulated by an ubiquitin-proteasome-dependent protein degradation mechanism.

Elmo1 inhibits the ubiquitylation of Dock180To examine whether the association of Elmo1 regulatesubiquitylation of Dock180, we examined the effect of wild-type and mutant forms of Elmo1 on Dock180. Two deletionforms of Elmo1 – T625, which does not bind to Dock180, and�531, which contains C-terminus binding sites for Dock180(Fig. 3A,B) (Shimazaki et al., 2005) – were used in an in vivoubiquitylation assay. In this experiment, we performedimmunoprecipitation analysis 24 hours after transfection. After24 hours, the amounts of Dock180 were still almost equal ineach transfectant. The equal amounts of Dock180 enabled usto compare the ubiquitylation levels of Dock180. Both wild-type Elmo1 and the �531 mutant inhibited ubiquitylation ofDock180, whereas the T625 mutant form of Elmo1 could not(Fig. 3C). It should be noted that levels of the hyper-ubiquitylated form of Dock180 (�500 kDa) seemed todecrease in the T625 mutant of Elmo1 (Fig. 3C). Thepleckstrin-homology (PH) domain of Elmo1 might have someeffect on the poly-ubiquitylation of Dock180, because Elmo1was known to bind the Docker domain (DHR-2 domain) ofDock180 through the PH domain, which was partiallycontained in the T625 mutant (Lu et al., 2004).

We also detected ubiquitylated bands around 70 kDa withthe anti-HA tag antibody when full-length Elmo1 was co-expressed; because the molecular weight of Elmo1 is around70 kDa, these bands may be ubiquitylation bands of Elmo1(Fig. 3C, upper panel, arrow). In fact, we found that Elmo1 wasalso ubiquitylated during an in vivo ubiquitylation assay usinglysates from HEK293T cells expressing both Elmo1 and HA-ubiquitin (data not shown). Further study should be carried outto determine the physiological role of ubiquitylation of Elmo1,since overexpression of proteins is known to induce theirubiquitylation and thereby ensure their quality control.

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925Inhibition of ubiquitylation of Dock180 by Elmo1

To confirm that the ubiquitylated bands over 180 kDaobserved in Fig. 3C are Dock180, we performed urea-reversalimmunoprecipitation assay using the same cell lysates as thoseanalysed in the experiment shown in Fig. 3C. We found thatthe ubiquitylated bands were suppressed to almost the same

degree as seen in Fig. 3C by co-expression of full-lengthElmo1 and the �531 mutant (Fig. 3D). In addition,ubiquitylated bands of around 70 kDa (Fig. 3C, lane 3)disappeared (Fig. 3D). Furthermore, to confirm that theinhibitory effect of Elmo1 for ubiquitylation of Dock180 is not

Fig. 1. Elmo1 increases the stability of Dock180 protein. (A) Left panels; HEK293T cells were transiently transfected with mammalianexpression plasmids for Flag-Dock180 alone or Flag-Dock180 and myc-Elmo1. After 48 hours, cells were lysed and analysed byimmunoblotting (IB) with anti-Flag tag, anti-myc tag and anti-actin antibodies. The active form of Rac was detected in a pull-down assay. Rightpanels: HEK293T cells were transfected with mammalian expression plasmids for Flag-Crk II alone or Flag-Crk II and myc-Elmo1, and cellswere analysed in the same way as for the left panels. (B) For pulse-chase analysis for Dock180, HEK293T cells transiently expressing Dock180alone (–) or Dock180 and Elmo1 (+) were labelled for 1 hour and then chased for the time indicated at the top of the panel. (C) The signalintensity 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 transientlyexpressing Dock180 alone or Dock180 and Elmo1. GAPDH is a control for the amounts of PCR templates. T/F, transfection; RT, reversetranscriptase. (E) HT1080 cells were transfected with siRNAs for negative control, scramble control and Elmo1#3. After incubation for 96hours, 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 theamount of PCR template. (H) HEK293T cells were transfected with negative control and Elmo1#2 and cells were analysed in the same manneras E.

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non-specific, we examined an irrelevant protein [glutathione S-transferase (GST)] on Dock180 ubiquitylation. Unlike Elmo1,GST was found to have no effect on the levels of ubiquitylationof Dock180 (Fig. 3E).

We employed an alternative approach to confirm thatElmo1, when bound to Dock180, suppressed ubiquitylation.We performed an in vivo ubiquitylation assay using theDock180�357 mutant (which has a 357 amino acid deletionat the N-terminus and does not bind Elmo1) (Fig. 3F)(Brugnera et al., 2002). We found that the Dock180�357mutant was significantly ubiquitylated, much more so thanwild-type Dock180, both in the absence and presence ofElmo1 (Fig. 3F). These results suggest that Elmo1 functionsas an inhibitor of the ubiquitylation of Dock180 through amechanism that is dependent on the interaction betweenDock180 and Elmo1.

Ubiquitylation of Dock180 on the plasma membraneNext, we investigated the subcellular localisation forubiquitylation of Dock180. An in vivo ubiquitylation assay forDock180 was performed using either the cytosolic or themembrane fraction of cell lysates. We found that Dock180treated with the membrane fraction was highly ubiquitylated,an effect that was inhibited by Elmo1 (Fig. 4A). However, inthe cytosolic fraction, Elmo1 did not alter the level of Dock180

ubiquitylation (Fig. 4A). In contrast to the inhibition ofubiquitylation of Dock180 by Elmo1, protein levels ofDock180 in the membrane fraction were lower when co-expressed with Elmo1 than those without Elmo1 (Fig. 4A). Weexamined the effect of Elmo1 on the amount of endogenousDock180 in the membrane fraction in HEK293 cells. Theamount of endogenous Dock180 in the membrane fraction wasalso found to be decreased by Elmo1 (data not shown).

For further analysis, we employed immunofluorescentmicroscopy and observed the subcellular localisation ofubiquitin and Dock180. Dock180 and HA-ubiquitin were co-expressed in HEK293T cells, and cells were stained with anti-Dock180 and anti-HA tag antibody. We found that, in quiescentcells, both Dock180 and ubiquitin were partially localised atthe cell periphery, although most of them were localised mainly

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Fig. 2. Ubiquitylation of Dock180 inHEK293T cells. (A) Results of an invivo ubiquitylation assay. HEK293Tcells were transfected with the indicatedplasmids, and after treatment of 10 �MMG-132 for 12 hours, cells were lysedand subjected to immunoprecipitation(IP) and immunoblotting (IB). MIG,normal mouse immunoglobulin; TCL,total cell lysate; Ub, ubiquitin. (B) Forthe urea-reversal immunoprecipitation,the initial precipitates from the anti-Dock180 mAb were treated with 8 Murea buffer for 1 minute and thenimmunoprecipitated again using thesame antibody. (C) MCAS andHEK293T cells were treated with 20�g/ml cyclohexamide and 10 �g/mlMG-132 for 24 hours as indicated at thetop of the panel. Cell lysates wereimmunoblotted with anti-Dock180antibody to detect endogenousDock180.

Fig. 3. Elmo1 suppresses ubiquitylation of Dock180. (A) Aschematic representation of wild-type and mutant forms of Elmo1used in the study. DUF609, domain of unknown function; FL, fulllength; LZ, leucine zipper; PH, pleckstrin homology; PxxP,polyproline-rich motif. (B) Association between Dock180 andElmo1 in HEK293T cells. Cells were transiently transfected with theindicated plasmids, and lysates were immunoprecipitated with anti-myc tag antibody, and then probed with anti-myc tag and anti-Flagtag antibodies. TCL, total cell lysate. (C) Ubiquitylation of Dock180in HEK293T cells in the presence of Elmo1 and its mutants. 24 hoursafter transfection with the indicated plasmids, cells were lysed andsubjected to in vivo ubiquitylation assay. (D) The urea-reversalimmunoprecipitation was performed using the same lysates as C. TheIP ratio represents the relative signal intensity of immunoprecipitatedDock180 in lane 1 as 1.0 (bottom of the panel). (E) Theubiquitylation of Dock180 in the absence or presence of Elmo1 andGST. The immunoprecipitation was performed using anti-Flag tagantibody. (F) The ubiquitylation of Dock180 and its mutant. Lysatesfrom HEK293T cells expressing the indicated plasmids wereimmunoprecipitated with anti-Dock180 antibody at in vivoubiquitylation assay. The IP ratio represents the relative signalintensity of immunoprecipitated Dock180 in lane 1 as 1.0.

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927Inhibition of ubiquitylation of Dock180 by Elmo1

Fig. 3. See previous page for legend.

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in the cytoplasm (Fig. 4B, upper panel, arrowheads). UponEGF stimulation, both Dock180 and ubiquitin weretranslocated and colocalised at the edge of the ruffledmembrane (Fig. 4B, upper panel, arrows). This EGF-inducedcolocalisation of Dock180 and ubiquitin were also examinedin Cos-7 cells (Fig. 4B, lower panel). The RFP fusion forms ofDock180 and HA-ubiquitin were co-expressed in Cos-7 cells,and HA-ubiquitin was visualised by immunostaining with anti-HA tag antibody. In quiescent cells, most of the detectableDock180 and ubiquitin were diffusely found in the cytoplasm(Fig. 4B, lower panel). However, upon EGF stimulation, bothDock180 and ubiquitin were translocated to the cell periphery(Fig. 4B, lower panel, arrowheads).

Furthermore, we confirmed that ubiquitylation of Dock180in the membrane fraction was enhanced by EGF stimulation,and found that Elmo1 could inhibit this enhancement (Fig. 4C).The enhancement of the ubiquitylation of Dock180 in wholecell lysates was not detected (data not shown). These results

indicate that ubiquitylation of Dock180 occurs mainly on theplasma membrane and Elmo1 inhibits this ubiquitylation.

Enhancement of the ubiquitylation of Dock180 by CrkNext we investigated whether a main regulator of Dock180such as Crk was involved in the ubiquitylation of Dock180 onthe plasma membrane, and found its enhancement by both CrkI and Crk II in the membrane fraction (Fig. 5). By in vivoubiquitylation assay using whole cell lysates, no significantCrk-dependent enhancement of ubiquitylation of Dock180 wasobserved (data not shown). In addition, we found that Dock180levels in the membrane fraction were significantly increased byco-expression with either Crk I or Crk II (Fig. 5).

Fibronectin-stimulation-enhanced ubiquitylation ofDock180As Crk and Dock180 are known to function downstream ofintegrins, especially �5�1 (Iwahara et al., 2004; Kiyokawa et

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Fig. 4. Ubiquitylation of Dock180 on the plasma membrane. (A) Ubiquitylation of Dock180in the membrane fraction. Cell lysates of HEK293T cells expressing the indicated plasmidswere separated into cytosolic and membrane fractions. The samples were subjected to an invivo ubiquitylation assay. The expression levels of transfected proteins were analysed usingcell 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-7cells (lower panels) were transiently transfected with Flag-Dock180 or Dock180-RFP and HA-ubiquitin expression plasmids. After incubationfor 36 hours, cells were stained with anti-HA tag antibody and analysed by confocal microscopy. (C) Ubiquitylation of Dock180 in themembrane fraction under EGF stimulation. HEK293T cells expressing the indicated plasmids were stimulated by EGF or not. Cell lysates weresubjected to in vivo ubiquitylation assay.

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929Inhibition of ubiquitylation of Dock180 by Elmo1

al., 1998b; Sakai et al., 1994; Schaller and Parsons, 1995), wecarried out a re-plating assay using a fibronectin-coated dish.An increase in ubiquitylation of Dock180 in the membranefraction was observable 15 minutes after re-plating andpersisted for at least 6 hours (Fig. 6A). The amount of Dock180in the membrane fraction was not altered when cells were re-plated on a fibronectin-coated dish (Fig. 6A). In these cells,Elmo1 suppressed ubiquitylation of Dock180 even in the cellsin suspension (Fig. 6B, lanes 1 and 3), and also inhibitedaugmentation of the re-plating-induced ubiquitylation ofDock180 (Fig. 6B, lanes 2 and 4).

DiscussionWe have investigated the physiological function of Dock180,a member of the CDM family of proteins, and its interactingprotein Elmo1, and found that Dock180 could be ubiquitylatedand its amounts regulated by an ubiquitin-proteasome-dependent protein degradation mechanism. Initialobservations, in which the amount of force-expressed Dock180increased when Elmo1 was co-expressed in HEK293T cells,prompted us to investigate the involvement of ubiquitylation inregulation of the amounts of Dock180. To exclude thepossibility of Elmo1-dependent transcriptional regulation ofDock180, we performed RT-PCR and pulse-chase analyses forthe amounts of Dock180 with or without Elmo1 in HEK293Tcells, and found that Elmo1 stabilised Dock180. In fact, wefound that Elmo1 inhibited ubiquitylation of Dock180, and thatthe levels of endogenous Dock180 were decreased by siRNAfor Elmo1. Since a rapid alteration in the level of Dock180during the process of maturation of dendritic cells has been

reported (Akakura et al., 2004), we planto analyse the involvement ofubiquitylation of Dock180 in this process.

We also showed that the �531 mutantthat binds to Dock180 preserves aninhibitory activity towards ubiquitylationof Dock180. Even so, the precisemechanism of Elmo1-dependentinhibition of ubiquitylation remainsobscure. Elmo1 might block the physical

Fig. 5. The enhancement of ubiquitylation ofDock180 by Crk. An in vivo ubiquitylation assaywas performed using lysates of the membranefraction of HEK293T cells expressing theindicated plasmids (left panels). The IP ratiorepresents the relative signal intensity ofimmunoprecipitated Dock180 in lane 1 as 1.0(bottom of the left panel). The expression levelsof Dock180, Crk I and Crk II in the membranefraction of cell lysates (membrane) and in totalcell lysates (total) were determined byimmunoblotting (right upper and middle panels).Anti-E-cadherin antibody was used as a markerfor the membrane fraction (right lower panel).

Fig. 6. Enhancement of ubiquitylation ofDock180 in HEK293T cells re-plated onfibronectin-coated dishes. (A) HEK293T cellswere transiently transfected with the indicatedplasmids for Dock180 and HA-ubiquitin andre-plated on fibronectin-coated dishes for theindicated duration (top of the panel); lysatesin the membrane fraction were subjected to invivo ubiquitylation assay. Sus, suspendedcells; FN, fibronectin. Ratio represents therelative signal intensity of Dock180 in themembrane fraction in lane 1 as 1.0 (bottom ofthe panel). (B) Inhibition of ubiquitylation ofDock180 by Elmo1. HEK293T cells weretransiently transfected with the indicatedplasmids, and the re-plating assay and in vivoubiquitylation assay were performed as in A.

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association of E3 ubiquitin ligase to Dock180, or it might maskthe ubiquitylation sites of Dock180. Alternatively, Elmo1 hasbeen reported to induce a conformational change of Dock180,which could inhibit its ubiquitylation (Lu et al., 2004; Lu etal., 2005). To define the mechanism of Elmo1 action further, itwill be necessary to identify the E3 ubiquitin ligase that isrequired for Dock180. It can at least be said that co-expressionof Cbl with Dock180 does not change the levels ofubiquitylation of Dock180 in HEK293T cells (data not shown).

Recently, ubiquitylation of GEFs as a regulatory mechanismhas been reported for other proteins. In the case of Vav, anactivated Vav mutant (Y174F) was shown to be more sensitiveto Cbl-dependent ubiquitylation, which suggests the regulationof protein degradation by tyrosine phosphorylation (Miura-Shimura et al., 2003). Furthermore, binding to Ras has beenshown to be necessary for ubiquitylation of Ras-GRF2 (deHoog et al., 2001). We demonstrated that Dock180 was mainlyubiquitylated on the plasma membrane and that this wasenhanced by both EGF and Crk. Furthermore, in re-platingcells onto fibronectin-coated dishes, ubiquitylation of Dock180on the plasma membrane was also enhanced. It should be notedthat various levels of ubiquitylation of Dock180 were observedeven when cells were re-plated on dishes coated with poly-L-lysine and collagen (data not shown). Thus, the enhancementof ubiquitylation of Dock180 observed with fibronectin-coated

dishes is likely to be dependent on cell attachment rather thanbe specific for integrin stimulation. These data suggest thatrecruitment of Dock180 to the plasma membrane by EGF, Crkand adhesion-dependent signals might contribute to theubiquitylation of Dock180.

It should be noted that despite the fact that ubiquitylationlevels of Dock180 were elevated by stimulation with EGF orfibronection, or forced expression of Crk, the protein levels ofDock180 in the membrane fraction did not decrease. Wespeculate that the reason why Dock180 seemed not to beremoved in the membrane fraction is that Dock180 istranslocated from the cytoplasm by Crk. As the suppliedamount of Dock180 may be more than that degraded by theproteasome, the apparent amount of Dock180 is not decreasedafter being ubiquitylated. As shown in Fig. 7, by integrinstimulation, Crk recruits Dock180 to the focal adhesioncomplex and Dock180 then activates Rac. In this process,several modifications of Dock180 including ubiquitylationmight occur and a part of Dock180 is removed from thecomplex comprising Crk and p130Cas; such regulations mightmodulate local Rac activity. Thus, in the local areas of a cell,Dock180 ubiquitylation, which decreases the amount ofDock180, functions as one of the negative-feedbackmachineries for the Dock180-dependent activation of Rac,after integrin-dependent signals are turned on.

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Fig. 7. Model of Dock180regulation. (A,B) Followingintegrin stimulation by theirligands (including fibronectin),tyrosine kinases are activated andphosphorylate severalcomponents of focal adhesionsuch as p130Cas, and both Crk andDock180 are recruited from thecytoplasm. (C) RecruitedDock180 is known to activateRac, and we speculate thatrecruited Dock180 isubiquitylated near the plasmamembrane by an ubiquitin ligase.(D) Furthermore, ubiquitylatedDock180 might be removed fromthe complex comprising Crk andp130Cas, and is degraded by theproteasome.

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931Inhibition of ubiquitylation of Dock180 by Elmo1

Such a spatio-temporal alteration of Dock180 levels on theplasma membrane could function in cell migration. We hopethat future works both on the identification of ubiquitin ligasefor Dock180 and on the analyses of ubiquitylation of Dock180in living cells might further clarify the mechanism forubiquitin-dependent Rac-GEF regulation.

Materials and MethodsCellsHEK293T (human embryonic kidney 293 cells with SV40 T antigen), Cos-7,MCAS (human ovarian mucinous adenocarcinoma) and HT1080 (humanfibrosarcoma) cells were maintained in Dulbecco’s Modified Eagle’s Medium(DMEM), supplemented with 100 �g/ml penicillin and streptomycin and 10% fetalbovine serum (Sigma), in 5% CO2 at 37°C.

AntibodiesThe following antibodies were used: anti-influenza hemagglutinin (HA) tag mousemonoclonal antibody (mAb; clone 12CA5, Roche Diagnostics); anti-Dock180 mAb(clone H4, Santa Cruz Biotechnology); anti-Dock180 polyclonal antibody (cloneH70, Santa Cruz Biotechnology); anti-FLAG tag mAb (clone M2, Sigma); anti-myctag mAb (clone 9E10, gift from Hiroshi Ariga, Hokkaido University, Sapporo,Japan); anti-Rac mAb (clone 102, BD Transduction Laboratories); anti-actin mAb(clone C4, Chemicon International); anti-Elmo1 polyclonal antibody (clone ab2239,Abcam); and anti-E-cadherin mAb (clone 36, BD Transduction Laboratories). Theanti-mouse immunoglobulin Ab conjugated with Alexa Fluor 488 and the anti-rabbitimmunoglobulin Ab conjugated with Alexa Fluor 594 were purchased fromMolecular Probes.

Expression plasmidsThe pCXN2-Flag-Dock180, pCAGGS-myc-CrkI and -Crk II vectors were gifts fromM. Matsuda (Osaka University, Osaka, Japan) and the pCGN-HA-Ubiquitin vectorwas constructed as described previously (Hatakeyama et al., 2001). The redfluorescent protein (RFP) fragment was amplified by PCR and subcloned into XhoI-NotI-digested pCXN2-Flag-Dock180; the resulting plasmid was named pCXN2-Dock180-RFP. pCXN2-Flag-Crk-II was also constructed (by H. Nishihara,Hokkaido University, Sapporo, Japan).

The pEBB-Flag-ELMO1 vector was kindly provided by K. Ravichandran(University of Virginia, VA). PCR fragments of full-length Elmo1, T625, and �531were subcloned into a pMyc-CMV mammalian expression vector (ClontechLaboratories) that had been digested with XhoI and NotI; the resulting plasmid wasnamed pCMV-myc-Elmo1 and contains the following changes: T625 [amino acids(aa.) 1 to 625], and �531 (aa. 532 to 727). pGEX-PAK2-RBD was describedpreviously (Nishihara et al., 2002). All PCR fragments were verified by sequencing.

RT-PCR analysisRT-PCR analysis was performed by the method described previously (Akakura etal., 2004; Shimazaki et al., 2005). Total RNA was prepared using the RNeasy MiniKit (Qiagen) from HEK293T cells expressing the indicated plasmids and HT1080cells. The forward primer 5�-TGGAGACAAAGTCACGGAGG-3� and the reverseprimer 5�-GATGAGAGGGAAGAGACAGAGG-3� for Dock180 yielded a productof 219 bp. The forward primer 5�-CCGGATTGTGCTTGAGAACA-3� and thereverse primer 5�-CTCACTAGGCAACTCGCCCA-3� for Elmo1 yielded a productof 121 bp. The forward primer 5�-TTCGTCATGGGTGTGAACCA-3� and thereverse primer 5�-GGTCATGAGTCCTTCCACGATAC-3� for GAPDH yielded aproduct of 138 bp.

Transfection, immunoprecipitation and immunoblot analysisHEK293T cells were transfected with plasmids using Lipofectamine 2000(Invitrogen). After incubation for 24-48 hours, the cells were lysed with 1% Tx-100lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA,10% (v/v) glycerol, 1% (v/v) Triton X-100, complete protease inhibitor cocktail(Roche Diagnostics) and 1 mM phenylmethylsulphonylfluoride (PMSF). Lysateswere centrifuged at 20,000 g for 10 minutes at 4°C. The supernatants were incubatedwith the indicated antibodies and then with protein A beads (Protein A Sepharose4 Fast Flow; Amersham Pharmacia Biotech) for 1 hour at 4°C. Precipitates or celllysates were separated by SDS-PAGE, transferred onto PVDF filters (Immobilon),and incubated with primary antibodies. Positive signals were detected by enhancedchemiluminescence (ECL) western blotting reagents (Amersham PharmaciaBiotech) and quantified using a Lumino Image Analyzer (LAS1000; Fuji Film).

siRNA for Elmo1HT1080 and HEK293T cells were transfected with siRNAs indicated below byLipofectamine 2000. After incubation for 96 hours, cells were lysed and subjectedto immunoblotting. Target sequences of siRNA are GGGUGGUCUCUUGCCA-ACCAUGAAU for Elmo1#1 (120-144 bp of Elmo1), GGCACUAUCCUUCGA-UUAACCACAU for Elmo1#2 (216-240 bp of Elmo1), CCGAGAGGAUGAAC-

CAGGAAGAUUU for Elmo1#3 (1561-1585 bp of Elmo1), and CCGGAAG-GUAACCGAAGGAAGAUUU for scramble control against Elmo1#3, respectively.All siRNA duplex oligoribonucleotides including negative control (Stealth RNAiNegative CTL MED GC) were purchased from Invitrogen.

Cell fractionationCell fractions were prepared by the method described previously (Kobayashi et al.,2001) with some modifications. Briefly, cells were scraped and suspended in bufferA containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4,1 mM PMSF and complete protease inhibitor cocktail. After freeze and thaw, cellsuspensions were centrifugated at 1,000 g for 7 minutes and then at 20,000 g for10 minutes. The supernatant was removed (cytosolic fraction). The pellet waswashed once with buffer A, lysed with buffer B containing 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 50 mM NaF, 1 mM Na3VO4, 1 mMPMSF and complete protease inhibitor cocktail, and centrifugated at 20,000 g for10 minutes (membrane fraction). One-fifth volume of buffer A was used in thepreparation of the membrane fraction.

Pull-down assay for Rac activityHEK293T cells were lysed with a lysis buffer composed of 1% NP-40, 25 mM,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 at14,000 g at 4°C for 1 minute. The supernatants were incubated with 10 �g ofpurified GST-PAK2-RBD and then with glutathione-Sepharose 4B beads(Amersham Pharmacia Biotech). The beads were washed three times with lysisbuffer. The precipitants were analysed by immunoblotting with anti-Rac antibodyto detect GTP-form Rac.

Pulse-chase assayHEK293T cells expressing the indicated plasmids were metabolically labelled with100 �Ci [35S]methionine for 1 hour at 37°C, washed with phosphate buffered saline(PBS), and cultured in DMEM for 0-24 hours. Cell lysates were subjected toimmunoprecipitation with anti-Flag tag mAb, the precipitates were separated by SDS-PAGE, and signals were analysed using the BAS2000 image analyser (Fuji Film).

In vivo ubiquitylation assayHEK293T cells were transfected with the indicated plasmids. After 12-36 hours,cells were further cultured in the absence or presence of 10 �M proteasome inhibitorMG-132 for 12 hours. The cells were lysed with 1% Tx-100 lysis buffer (see above).Cell lysates were immunoprecipitated with anti-Dock180 antibody (H4), anti-Flagtag antibody, or mouse normal IgG (IgG), and immunoblotted with anti-HA tag andanti-Dock180 antibodies. For the denaturing condition, the immunoprecipitateswere incubated with 8 M urea buffer containing 20 mM Tris-HCl (pH 7.4) and 8M urea for 1 minute, then diluted with 1% Tx-100 lysis buffer andimmunoprecipitated again with the anti-Dock180 antibody (urea-reversalimmunoprecipitation).

Confocal laser scanning microscopical studyCos-7 and HEK293T cells expressing the indicated plasmids were fixed with 3%paraformaldehyde for 15 minutes and permeabilised with 0.1% Triton X-100containing PBS for 4 minutes at room temperature (RT). The cells were washed andincubated with 1% BSA containing PBS, and then with only anti-HA tag antibody,or both anti-HA tag and anti-Dock180 (H70) antibodies at 4°C overnight. The cellswere next incubated with Alexa Fluor 488-conjugated anti-mouse immunoglobulinand/or Alexa Fluor 594-conjugated anti-rabbit immunoglobulin antibodies for 1hour at RT with a light shield protecting the samples from photobleaching. For thenegative control, cells were processed the same way but without primary antibody.The cells were observed using a confocal laser-scanning microscope equipped witha computer (MRC-1024; Bio-Rad Microscience Division).

Re-plating assayHEK293T cells transfected with the indicated plasmids were incubated for 36-48 hours and harvested in 0.25% trypsin-EDTA solution (Sigma). Cells were thenput into suspension in Opti-MEM (Invitrogen) containing 1 mg of soybeantrypsin inhibitor (suitable for neutralisation of 2.5 mg of trypsin). The cells wereheld in suspension for 3 hours at 37°C. Suspended cells were then distributedonto cell culture dishes pre-coated with fibronectin (Iwaki) and the cellsincubated at 37°C for the indicated times. Cells were rinsed in cold PBS priorto protein extraction.

We thank M. Matsuda (Osaka University, Japan) and Kodi S.Ravichandran (University of Virginia, VA) for plasmids and TadakiSuzuki (Hokkaido University, Japan) for useful discussion. This studywas supported in part by Grants-in-Aid from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan, and fromthe Ministry of Health, Labor and Welfare, Japan, and by the YasudaMedical Research Foundation.

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