A Novel Interaction between the SH2 Domain of SignalingAdaptor Protein Nck-1 and the Upstream Regulator of theRho Family GTPase Rac1 Engulfment and Cell Motility 1(ELMO1) Promotes Rac1 Activation and Cell Motility*

Received for publication, January 14, 2014, and in revised form, June 8, 2014 Published, JBC Papers in Press, June 13, 2014, DOI 10.1074/jbc.M114.549550

Guo Zhang‡1, Xia Chen‡1, Fanghua Qiu§, Fengxin Zhu‡, Wenjing Lei‡, and Jing Nie‡2

From the ‡State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Division of Nephrology,Nanfang Hospital, Southern Medical University, Guangzhou 510515, China and the §Department of Clinical Laboratory,Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou 510515, China

Background: Nck functions as an adaptor to regulate cytoskeleton organization.Results: The SH2 domain of Nck binds to ELMO1. This interaction promotes ELMO1 translocating to membrane, enhancesRac1 activity and cell migration.Conclusion: Nck-ELMO1 interaction promotes Rac1 activity.Significance: This finding defines a new role for Nck in cell motility.

Nck family proteins function as adaptors to couple tyrosinephosphorylation signals to actin cytoskeleton reorganization.Several lines of evidence indicate that Nck family proteinsinvolve in regulating the activity of Rho family GTPases. In thepresent study, we characterized a novel interaction betweenNck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that theNck-1-ELMO1 interaction is mediated by the SH2 domain ofNck-1 and the phosphotyrosine residues at position 18, 216,395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which theSH2 domain was inactive), or a 4YF mutant of ELMO1 lackingthese four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitortreatment and overexpression of Src family kinase Hck signifi-cantly enhanced Nck-1-ELMO1 interaction. Moreover, wildtype Nck-1, but not R308K mutant, significantly augmented theinteraction between ELMO1 and constitutively active RhoG(RhoGV12A), thus promoted Rac1 activation and cell motility.Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulat-ing Rac1 activity.

Protein-protein interactions play central roles in signaltransduction leading to cell proliferation, differentiation, sur-vival, and migration (1, 2). Many of the protein-protein inter-actions are mediated by adaptor proteins. Nck family proteins

are important in transducing signals from tyrosine phosphor-ylation to actin cytoskeletal reorganization (3, 4). The Nck fam-ily has two members in mammals termed Nck-1 and Nck-2;each of them consists of three N-terminal SH3 domains fol-lowed by a single C-terminal SH2 domain. The SH2 domain ofNck can bind a number of activated receptor tyrosine kinasesand tyrosine-phosphorylated docking proteins. On the otherhand, the Nck SH3 domains engage proline-rich binding siteson a host of effectors involved in the control of cellular actindynamics, such as neural Wiskott Aldrich syndrome protein(N-WASP),3 WASP-interactin protein (WIP), and PAK serine/threonine kinases (5, 6).

Nck adaptor proteins are implicated in cellular signal trans-duction regulating cytoskeleton organization. For example, theDrosophila Nck homolog dreadlocks (Dock) mediates growth-cone guidance and signaling, a process needed for formation offilopodia and lamellipodia protrusions (7, 8). In the actin-basedmotility of Vaccinia virus, Nck coordinates the assembly of anactin nucleation complex at the viral surface by binding to atyrosine- phosphorylated viral protein through its SH2 domainand by recruiting N-WASP through its SH3 domains (9, 10).Given the critical role of Nck in transducing signals from tyro-sine-phosphorylated proteins, we previously performed GSTpull-down assay followed by mass spectrometry to search novelbinding partners of the SH2 domain of Nck-1, and 13 potentialbinding proteins were identified (11). One of them is engulf-ment and cell motility 1 (ELMO1), the subject of the presentstudy.

ELMO1 is the mammalian orthologue of the Caenorhabditiselegans gene ced-12. ELMO1 forms a complex with Dock180and acts as an unconventional two-part guanine nucleotideexchange factor (GEF) specific for Rho family GTPase Rac1 (12,13). Rac1 is a key regulator of actin cytoskeletal dynamics and

* This work was supported by Grant 81288001 from the National ScienceFoundation of China, Grant 2012KJCX0027 from the Guangdong Provin-cial Department of Education, Science, and Technology Innovation Fund(to J. N.), and Grant 81200503 from the National Science Foundation ofChina (to F. Z.).

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: State Key Laboratory of

Organ Failure Research, Guangdong Provincial Institute of Nephrology,Division of Nephrology, Nanfang Hospital, Southern Medical University,1838 North Guangzhou Ave, Guangzhou 510515, China. Tel.: 86-20-62787972; Fax: 86-20-87281713; E-mail: niejing@smu.edu.cn.

3 The abbreviations used are: N-WASP, neural Wiskott Aldrich syndrome pro-tein; WIP, WASP-interactin protein; ELMO, engulfment and cell motility; PV,pervanadate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 33, pp. 23112–23122, August 15, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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This article has been withdrawn at the request of the authors. The authors were recently made aware that the Coomassie Blue image in Fig. 1E was rotated 180°. The Myc immunoblot from lysate in Fig. 2B was reused in Figs. 2C and 6B. A portion of the GAPDH immunoblot in Fig. 2C was reused in Fig. 6B as GAPDH. The last lane of the Myc immunoblot from lysate in Fig. 3A was reused in the first lane of the Myc immunoblot from lysate in Fig. 3C. The first lane of the Myc immunoblot from lysate in Fig. 3E was reused in the last lane of the Myc immunoblot from lysate in Fig. 3G. The last lane of the Myc immunoblot from the IP in Fig. 3E was reused in the first lane of the Myc immunoblot from the IP in Fig. 4C. A portion of the Myc immunoblot from thelysate in Fig. 4A was reused as the HA immunoblot from lysate in Fig. 6C. A portion of the GAPDH immunoblot in Fig. 5A was reused in Fig. 6C as GAPDH. Although the authors believe that the biological conclusions outlined inthe article are valid, the figures presented in the paper do not provide an accurate representation of the original data, and therefore the scientific integrity of the study has been compromised. The authors unanimously agreethat the most appropriate course of action is to withdraw the article. All authors sincerely apologize to the scientific community for not detecting these errors prior to publication and any negative impact this may have caused.

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relays signals from various stimuli such as growth factors, cyto-kines, and adhesion molecules to downstream effectors tomodulate cytoskeleton organization and cell migration (14, 15).Rac1 is preferentially activated at the leading edge of migratingcells where it induces the formation of actin-rich lamellipodiaprotrusions thought to be a key driving force for membraneextension and cell movement (16). One mode of targeting Rac1to the membrane occurs via ELMO binding to the activatedform of another small GTPase, RhoG. The simultaneous inter-action of ELMO with active RhoG and Dock180 serves as anevolutionarily conserved mechanism for Rac1 activation lead-ing to lamellipodia formation and cell migration (17, 18).

Previous studies have shown that ELMO1 binds directly tothe SH3 domain of hematopoietic cell kinase (Hck), a Src familykinase, and is phosphorylated by Hck (19, 20). Tyrosine phos-phorylation of ELMO1 is important for Rac1 activation. How-ever, the mechanism by which tyrosine phosphorylation ofELMO1 influences the GEF activity is not clear. Here wereported that phosphotyrosine residues at position 18, 216, 395,and 511 of ELMO1 mediate the binding to the SH2 domain ofNck-1. The association of Nck-1 with ELMO1 facilitated thebinding of ELMO1 to active RhoG, and thus promoted the GEFactivity of ELMO1/Dock180 complex.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Rabbit polyclonal anti-ELMO1,rabbit polyclonal anti-Nck-1, mouse monoclonal anti-Nckwere purchased from Santa Cruz Biotechnology Inc. (SantaCruz, CA); mouse monoclonal anti-Flag, mouse monoclonalanti-Myc, mouse monoclonal anti-HA were purchased fromSigma; rabbit monoclonal anti-HA, rabbit monoclonal anti-Flag were purchased from Cell Signaling Technology (Beverly,MA); Monoclonal antibodies against phosphotyrosine (4G10antibodies), mouse monoclonal anti-Rac1, Rac1/Cdc42 Activa-tion Magnetic Beads, Ni-NTA His�Bind� Resins, Bug BusterMaster Mix were bought from Millipore (Temecula, CA).

Nck-1 siRNA was designed and synthesized by GenePharma,Shanghai, China. The sense targeting sequence was: GCA-GAAUAAUCCAUUAACUTT. An irrelevant dsRNA with thesense sequence UU-CUCCGAACGUGUCACGUTT was usedas the control.

Cell Culture and Transfection—The HEK293T cells wereobtained from the Cell Bank, Chinese Academy of Medical Sci-ences, Shanghai, China and maintained in high-glucose DMEM(Invitrogen, Gaithersburg, MD) supplemented with 10% FBS(Invitrogen). Pervanadate (100 �M) was added in the culturemedium for 30 min at 37 °C after transfection.

The expression plasmids or siRNA were transfected into cellsusing Lipofectamine 2000 (Invitrogen) according to the man-ufacturer’s instructions. If necessary, carrier DNA or scramblesiRNA was added to keep equal plasmid/siRNA concentrationbetween different groups.

Plasmid Constructs—The full-length ELMO1 cDNA wasamplified and cloned into the pReciever M68 expression vector(FulenGen, Guangzhou, China). Full-length human Nck-1(NCBI accession number BC006403) cDNA was amplified andcloned into the pReciever M11 expression vector by FulenGen,Guangzhou, China. Flag-Dock180 was kindly provided by Dr.

Michiyuki Matsuda (Kyoto University, Kyoto, Japan). The 4YF,3YF, Y511F, Y395F, Y216F, and Y18F mutants of ELMO1 (20)and the R308K, SH3–1�,2�,3�, SH3–1�,2�, SH3–1�,3�,SH3–2�,3� mutants of Nck-1, RNAi-resistant WT Nck-1 andR308K Nck-1 were generated by site-directed mutagenesis kit asdescribed previously (21). GST fusion protein of the SH2, SH31,SH32, and SH33 domain of Nck-1 was generated by PCR usinghuman Nck-1 as the template and ligated into pGEX-4T-3 expres-sion vector. Myc-ELMO11–625, ELMO11–495, ELMO11–315,ELMO1‚531, ARM1, ARM2, and HA-RhoGV12A were gener-ated by FulenGen, Guangzhou, China as described previ-ously (17, 22, 23).

Recombinant Protein Purification—Escherichia coli (BL21)was transformed with pGEX-4T-3 or pGEX-Nck-1-SH2,pGEX- Nck-1-SH31, pGEX-Nck-1-SH32, or pGEX- Nck-1-SH33 and incubated with 0.2 mM isopropyl-�-D-1-thiogalacto-pyranoside (IPTG) for 4 h. The GST fusion proteins were puri-fied from bacterial lysates with GSH-Sepharose 4B beadsaccording to the manufacturer’s instruction (Amersham Bio-sciences). The GST-bound material was then washed with PBSand stored at �80 °C before use.

E. coli (BL21) was transformed with His-ELMOl and incu-bated with 0.6 mM IPTG for 4 h at 30 °C. The fusion proteinswere purified by nickel affinity chromatography using Ni-NTAHis�Bind� Resins according to the manufacturer’s recommen-dations. His-ELMO1 fusion Proteins were recovered bysequential elutions with 250 mM imidazole. Eluted proteinswere dialyzed and stored in 10% glycerol at �80 °C, in single-use aliquots.

GST Pull-down and in Vitro Binding Assay—For GST pull-down assay, Cell lysates were prepared and spun at 15,000 � gfor 15 min, and the supernatants were pre-cleared with GST-conjugated Sepharose beads and then incubated with GST orGST-Nck-1-SH2 that conjugated to Sepharose beads for 2 h at4 °C. The proteins bound to Sepharose beads were eluted andthen loaded on SDS-PAGE for Western blot analysis.

For in vitro binding assay, purified His-ELMOl were incu-bated with purified GST or GST-Nck-1-SH2 fusion proteinthat conjugated to Sepharose beads in 500 �l of reaction buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mM

MgCl2, 5 mM NaF, 1% Triton X-100, and protease inhibitormixture) for 12 h at 4 °C. After centrifugation, the proteinsbound to Sepharose beads were washed with ice-cold PBS,mixed with 2� SDS sample buffer. The binding of ELMO1 toNck-1-SH2 was examined by Western blot using anti-ELMO1antibody.

Western Blot and Immunoprecipitation—Cells were lysed inIP buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA,2 mM Na3VO4, 5 mM NaF, 1% Triton X-100, and protease inhib-itor mixture) at 4 °C for 30 min. Protein concentrations weredetermined with a BCA protein assay kit (Thermo Scientific,Rockford, IL). Equal amounts of cell lysates were immunopre-cipitated with indicated antibodies, resolved in SDS samplebuffer and then loaded on SDS-PAGE for Western blot analysis.

Rac1 Activation Assay—The intracellular activity of Rac1 wasexamined using Rac1 activation assay kits according to themanufacturer’s protocols. Briefly, cells were lysed with Mg2�

lysis buffer. After clarifying the cell lysates with glutathione-

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agarose and quantifying the protein concentrations, aliquotswith equal amounts of proteins were incubated with Rac assayreagent (PAK-1 PBD, agarose) at 4 °C for 1 h. The precipitatedGTP-bound Rac1 was then eluted in Laemmli reducing samplebuffer, resolved in a SDS-PAGE, and immunoblotted withmonoclonal anti-Rac1 antibody.

Immunofluorescence Staining—1 � 105 cells were plated onglass coverslips and transfected with various plasmids usingLipofectamine 2000. Cells were fixed with cold methanol(Sigma), permeabilized with 0.3% Triton X-100 min. Cells wereincubated with anti-Myc and anti-Flag antibodies 4 °C over-night, followed by incubating with goat anti-mouse AlexaFluor� 594 and goat anti-rabbit Alexa Fluor� 488 (Invitrogen)for 1 h. After washing, the chambers slides were mounted withSlow Fade�Gold antifade reagent (Invitrogen). All sampleswere observed and analyzed with a Olympus FV1000 confocalmicroscope (Japan).

Migration Assay—Migration assay was performed as de-scribed previously (11). Briefly, cells were seeded onto the filterin the upper compartment of the chamber and incubated for12 h. Cells in the upper surface of the transwell were removedusing cotton swabs. Migrated cells attached on the undersur-

face were fixed with absolute methanol for 15min and stainedwith crystal violet solution (0.5% in PBS) for 30 min. Cells werecounted under microscope at 200�. Microphotographs of 9random fields were taken and the average number of migrat-ing cells was determined for each experimental condition.

Statistical Analysis—Statistical differences between twogroups were determined by the Student’s t test. p � 0.05 wasconsidered statistically significant. The results were expressedas mean � S.D. from at least three experiments.

RESULTS

Characterization of a Direct Interaction between ELMO1 andthe SH2 Domain of Nck-1—We previously identified a novelinteraction between ELMO1 and the SH2 domain of Nck-1 bymass spectrometry (11). To confirm this interaction, Myc-tagged ELMO1 and Flag-tagged Nck-1 were co-transfected intoHEK293T cells and exogenous ELMO1 was immunoprecipi-tated by anti-Myc antibody. As shown in Fig. 1A, exogenousELMO1 was co-immunoprecipitated with exogenous Nck-1.We next analyzed the association of endogenous ELMO1 withendogenous Nck-1. In HEK293T cells, endogenous Nck-1 wasdetected in the immuneprecipitates in the presence of a specific

FIGURE 1. ELMO1 interacts with Nck-1. A, Myc-tagged ELMO1 and Flag-tagged Nck-1 were co-transfected into HEK293T cells. HEK293T cells lysates wereimmunoprecipitated with anti-Myc antibody or IgG followed by anti-Flag immunoblot. Replicate samples of lysates before IP were analyzed by immunoblot toverify expression of exogenous ELMO1 and Nck-1 proteins using anti-Myc and anti-Flag antibodies, respectively. B, endogenous Nck-1 co-precipitates withendogenous ELMO1. Lysates of HEK293T cells were immunoprecipitated using anti-ELMO1 antibody or IgG followed by anti-Nck-1 immunoblot. Replicatesamples of lysates were analyzed by immunoblot to verify equivalent loading. C, schematic representation of Myc-tagged ELMO1 mutants used. D, HEK293Tcells were transiently transfected with ELMO1WT, or indicated ELMO1 mutant. The corresponding cell lysate was incubated with purified GST-Nck-1-SH2. Boundprotein was detected by immunoblot using anti-Myc antibody. Replicate samples of cell lysates were analyzed by immunoblot to verify expression of ELMO1mutant using anti-Myc antibody. E, Nck-1 interacts directly with ELMO1. Nck-1 and ELMO1 were bacterially produced and purified. His-tagged ELMO1 wasincubated with GST or GST-tagged Nck-1-SH2 proteins, and the co-precipitation of ELMO1 was assessed by immunoblotting using anti-ELMO1 antibody.Purified GST-Nck-SH2 or GST added to the reaction was separated by SDS-PAGE and stained with Coomassie Blue (bottom gel).

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anti-ELMO1 antibody, but not in the presence of an isotypecontrol antibody (Fig. 1B).

Previous study has shown that the C terminus (532–727aa) ofELMO1 mediates the interaction with Dock180 (24), andDock180 interacts with Nck-2 (25). To exclude the possibilitythat the interaction between ELMO1 and Nck-SH2 is mediatedby Dock180, we generated two Myc-tagged ELMO1 mutants,namely ELMO1�531 and ELMO11– 625 (Fig. 1C), as describedpreviously (24). The ELMO1�531 lacks the N-terminal 531amino acid residues, whereas the ELMO11– 625 represent trun-cations at residue 625. GST pull-down assay showed thatELMO11– 625 bound to Nck-1-SH2 as well as that of wild-typeELMO1 (ELMO1WT). Whereas, no obvious interaction wasdetected between Nck-1-SH2 and ELMO1�531 (Fig. 1D), sug-gesting that Dock180 is not involved in the ELMO1-Nck-1interaction. To further map the region of ELMO1 responsiblefor binding to Nck-1-SH2, we generated another two ELMO1deletion mutants, namely ELMO11– 495 and ELMO11–315 (Fig.1C). As shown in Fig. 1D, ELMO11– 495 maintained interactionwith Nck-1-SH2, whereas, ELMO11–315 showed no binding,suggesting that the N-terminal 495 amino acid residues ofELMO1 is required for interacting with Nck-1-SH2.

To examine whether ELMO1 and Nck-1 interacts directly,we analyzed the ability of bacterially produced GST-Nck-1-SH2 and His-ELMO1 to associate in vitro. As shown in Fig. 1E,GST-Nck-1-SH2, but not GST, bound to His-tagged ELMO1,indicating that the Nck-1-SH2 fusion protein is capable ofinteracting with ELMO1 directly in solution.

To further confirm that the SH2 domain of Nck-1 mediatesthe binding with ELMO1, we generated GST fusion protein ofindividual SH2 or each of the three SH3 domains of Nck-1 (Fig.2A). GST pull-down experiments were performed using lysatesfrom Myc-tagged ELMO1-transfected HEK293T cells. Asshown in Fig. 2B, the GST-Nck-1 SH2 domain bound to Myc-tagged ELMO1, whereas, none of the three Nck-SH3 domainscould pull-down Myc-tagged ELMO1.

We next generated series full-length Nck-1 mutants con-taining specific amino acid substitutions W38K, W143K,and W229K in each of the SH3 domains predicted to inhibitinteractions with proline-containing proteins, or R308K inthe SH2 domain predicted to disrupt interaction with p-Yresidues. As described previously (21), Nck-1 mutant pro-teins (SH3–1�,2�, SH3–2�,3�, and SH3–1�,3�) in whichtwo SH3 domains were inactive and therefore had only a

FIGURE 2. The SH2 domain of Nck-1 is sufficient to bind to ELMO1. A, schematic representation of Nck-1 mutants used. B, lysates from Myc-tagged WTELMO1-transfected cells were incubated with purified indicated GST-Nck-1 fusion protein, respectively. Bound protein was detected by immunoblot usinganti-Myc antibody. GAPDH was used to verify equivalent loading. GST or GST-bound fusion proteins added to the reaction were separated by SDS-PAGE andstained with Coomassie Blue (bottom gel). C, Myc-tagged ELMO1 was transfected into HEK293T cells with Flag-tagged WT Nck-1 or indicated Nck-1 mutants.Exogenous ELMO1 was immunoprecipitated (IP) using anti-Myc antibody. The presence of Nck-1 in the immunocomplexes was detected by immunoblot usinganti-Flag antibody. Replicate samples of lysates before IP were analyzed by immunoblot to verify expression of exogenous ELMO1 and Nck-1 mutant proteinsusing anti-Myc and anti-Flag antibodies, respectively.

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single functional SH3 domain. Nck-1 mutant SH3–1�,2�,3�

with inactivating Trp-to-Lys substitutions in all three SH3domains therefore has no functional SH3 domain. Nck-1mutants or WT Nck-1 were co-transfected into HEK293T

cells with Myc-tagged ELMO1, respectively. Co-immuno-precipitation assay showed that R308K interacted veryweakly with Myc-tagged ELMO1 compared with that of WTNck-1. However, SH3–1�,2�,3� bound to ELMO1 as well as

FIGURE 3. ELMO1 interacts with Nck-1 in a tyrosine phosphorylation-dependent manner. A, PV treatment enhances the tyrosine phosphorylation ofoverexpressed ELMO1. HEK293T cells were transfected with Myc-tagged ELMO1. 24 h after transfection, cells were treated with 100 �M PV for 20 min, and thencell lysates were harvested. Cell lysates were immunoprecipitated by anti-Myc antibody or IgG followed by anti-4G10 immunoblot. C, pervanadate treatmentpromotes ELMO1-Nck-1 interaction. Cell lysates used in A were incubated with purified GST-Nck-1-SH2. Bound protein was detected by immunoblot usinganti-Myc antibody. E, Hck enhances the tyrosine phosphorylation of ELMO1. HEK293T cells were transfected with Myc-tagged ELMO1 in the presence orabsence of Hck. Cell lysates were immunoprecipitated by anti-Myc antibody followed by anti-4G10 immunoblot. G, Hck promotes ELMO1-Nck-1 interaction.Cell lysates used in E were incubated with purified GST-Nck-1-SH2. Bound protein was detected by immunoblot using anti-Myc antibody. B, D, F, H, graphicrepresentation of the ratio of phosphorylated ELMO1 (B, F) or Nck-1-bound ELMO1 (D, H) to total ELMO1. Data are expressed as mean � S.D. of threeindependent experiments. *, p � 0.05 versus untreated cells.

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that of WT Nck-1 (Fig. 2C). These data indicate that the SH2domain of Nck-1 is sufficient to mediate the interaction withELMO1.

Interaction of ELMO1 and Nck-1 Is Tyrosine PhosphorylationDependent—SH2 domains are known to bind to phosphoty-rosine residues in proteins (26). We thus examined whether theNck-1-ELMO1 interaction is dependent on tyrosine phosphor-ylation of ELMO1. To address this issue, we transfected Myc-tagged ELMO1 into HEK293T cells and then treated cells withtyrosine phosphatase inhibitor pervanadate (PV). The tyrosinephosphorylation (p-Y) of ELMO1 was detected by immunopre-cipitation using anti-Myc antibody followed by immunoblotusing anti-4G10 antibody. As shown in Fig. 3, A and B, the p-Yof ELMO1 was significantly enhanced by PV treatment. GSTpull-down assay revealed that the interaction between GST-Nck-1-SH2 and Myc-tagged ELMO1 was enhanced by PVtreatment, and the increase was statistically significant whencompared with that of untreated control cells (Fig. 3, C and D).Previous study reported that ELMO1 is a substrate of Hck andoverexpression of Hck leads to tyrosine-phosphorylation ofELMO1 (19, 20). Consistent with previous reports, the p-Y ofELMO1 was enhanced in Hck overexpressing cells (Fig. 3, E andF). GST pull-down assay demonstrated that the Nck-1-SH2-ELMO1 interaction was also significantly enhanced (Fig. 3, Gand H). These data provide evidence that the p-Y of ELMO1 isimportant in binding to Nck-1.

There are 5 tyrosine residues (Y18, Y216, Y395 or Y511, andY720) on ELMO1 have been identified to be phosphorylated byHck (20) and 4 of them are located in the N-terminal 531 amino

acids of ELMO1. To determine the contribution of these tyro-sine residues to Nck-1 binding, we generated Myc-taggedELMO1 constructs with Y18, Y216, Y395, or Y511 substitu-tions, and then transiently transfected them into HEK293Tcells, respectively. 24 h after transfection, the interactionbetween Myc-tagged ELMO1 and Nck-1-SH2 was analyzed byGST pull-down assay. As shown in Fig. 4, A and B, mutation ofindividual Y18, Y216, Y395, or Y511 residue of ELMO1 atten-uated the interaction with Nck-1-SH2 when compared withthat of WT ELMO1. We next mutated three (18, 216, and 395),or four tyrosine residues (18, 216, 395, and 511) together; thesemutants were designated 3YF and 4YF, respectively. GST pull-down assay revealed that binding of Nck-1-SH2 to 4YF mutantwas markedly decreased (Fig. 4B). To further confirm this find-ing, WT ELMO1 or 4YF mutant was co-transfected intoHEK293T cells with HA-tagged Hck and Flag-tagged Nck-1. Asshown in Fig. 4, C and D, p-Y of 4YF was dramatically lowerthan that of WT ELMO1. In line with the weak p-Y, the bindingof 4YF to Nck-1 was substantially reduced compared with WTELMO1. However, 4YF did not eliminate the binding to Nck-1.Collectively, these data indicate that p-Y of Y18, Y216, Y395,and Y511 is necessary, but not sufficient, for binding to Nck-1.

ELMO1-Nck-1 Interaction Promotes Rac1 Activation—ELMO1 and Dock180 act as a bipartite GEF to activate Rac1(24). To explore the functional importance of the ELMO1-Nck-1 interaction, Myc-tagged ELMO1, and Flag-taggedDock180 were co-transfected into HEK293T cells eitherwith Nck-1 WT or Nck-1 R308K mutant. Consistent withprevious report, co- expression of ELMO1 and Dock180 sig-

FIGURE 4. Phosphorylation of Y18, Y216, Y395, and Y511 residues of ELMO1 is important for ELMO1-Nck-1 interaction. A, HEK293T cells were trans-fected with WT ELMO1, or indicated ELMO1 mutant. 24 h after transfection, cell lysates were harvested and the corresponding cell lysate was incubated withpurified GST-Nck-1-SH2. ELMO1 bound to GST-Nck-1-SH2 were detected by immunoblot using anti-Myc antibody. Expression of Myc-tagged WT ELMO1 orELMO1 mutant proteins was assessed by immunoblot using anti-Myc antibody. C, HEK293T cells were transfected with WT ELMO1 or 4YF mutant ELMO1together with Flag-tagged Nck-1 and HA-tagged Hck. Cell lysates were immunoprecipitated by anti-Myc antibody followed by anti-Flag or anti-4G10 immu-noblot. Replicate samples of lysates before IP were analyzed by immunoblot to verify expression of exogenous ELMO1, Nck-1, and Hck proteins using anti-Myc,anti-Flag antibodies, and anti-HA, respectively. B and D, graphic representation of the ratio of Nck-1-bound ELMO1 to total ELMO1. Data are expressed asmean � S.D. of three independent experiments. *, p � 0.05 versus WT ELMO1 transfected cells.

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nificantly increased Rac1 GTP loading. Overexpressing WTNck-1 further enhanced Rac1 GTP loading, whereas R308Kmutant did not (Fig. 5, A and B). To further confirm thisresult, endogenous Nck-1 was silenced by siRNA (Fig. 5C).Compared with scramble siRNA, transfecting Nck-1 siRNAled to a significant decrease of Rac1 activity. Furthermore,transfecting siRNA-resistant-WT Nck-1, but not siRNA-resistant-R308K, rescued the reduced Rac1 GTP loading(Fig. 5D). These data indicate that Nck-1-ELMO1 interac-tion could enhance the ability of the Dock180/ELMO1 com-plex to promote Rac1 activity.

Nck-1 Enhances ELMO1-RhoG Interaction—We next ex-plored the underlying mechanism of Nck-1-promoted Rac1activity. Previous study demonstrated that the GTP-boundform of RhoG binds to the N-terminal 115 residues ofELMO1 comprising the Armadillo repeats 1 (9 – 49 aminoacid residues) and 2 (65–110 amino acid residues) and thuspromotes Rac1 activity (23). Since Nck-1 also binds to the Nterminus of ELMO1, we thus examined whether ELMO1binds to active RhoG and Nck-1 simultaneously. To this end,we generated ARM1 and ARM2 ELMO1 mutants by mutat-ing 2 residues in each repeat based upon their high degree ofconservation among CED12/ELMO proteins as well as otherARM repeat-containing proteins (Fig. 6A) (23). As shown in

Fig. 6B, Nck-1-SH2 bound to ARM1 and ARM2 ELMO1mutants as well as WT-ELMO1, indicating that ELMO1associates with Nck-1 through a region distinct from that ofactive RhoG.

We next evaluated the impact of Nck-1 on the interactionbetween ELMO1 and active RhoG. Flag-tagged WT Nck-1,R308K, or SH3–1�,2�,3� (in which all three SH3 domains ofNck-1 are inactivated) was co-transfected with Myc-taggedELMO1 and HA-tagged RhoGV12A (constitutively activeRhoG) into HEK293T cells. As shown in Fig. 6C, the interactionbetween ELMO1 and RhoGV12A was dramatically enhanced byoverexpressing WT Nck-1 when compared with empty vectortransfected cells. However, R308K mutant of Nck-1 had no sig-nificant effect on ELMO1-RhoGV12A interaction. On the otherhand, SH3–1�,2�,3� (in which all three SH3 domains are inac-tivated) showed comparable promoting effect as that of WTNck-1, indicating that the SH2 domain of Nck-1 is sufficient topromote ELMO1-RhoG interaction. Since 4YF mutant ofELMO1 did not interact with Nck-1-SH2, we assume that itsbinding to active RhoG might be reduced. To test this issue,WT ELMO1 or 4YF was transfected into HEK293T cellstogether with RhoGV12A. Co-immunoprecipitation assay re-vealed that the intensity of 4YF mutant co-precipitated withRhoGV12A was dramatically decreased compared with that of

FIGURE 5. Nck-1 promotes ELMO1/Dock180-induced Rac1 activity. A, Myc-ELMO1 and Flag-tagged Dock180 were transfected into HEK293T cells with WTNck-1 or R308K mutant of Nck-1, respectively. 24 h after transfection, the amount of GTP-bound Rac1 was determined as described under “ExperimentalProcedures.” C, HEK293T cells were transfected with Nck-1 siRNA alone, or together with siRNA-resistant WT Nck-1, or R308K plasmid, respectively. 24 h aftertransfection, the amount of GTP-bound Rac1 was determined as described in the method. If necessary, empty vector was added to ensure that equal plasmidamounts were transfected in each condition. B and D, graphic representation of the ratio of GTP-Rac1 to total Rac1. Data are expressed as mean � S.D. of threeindependent experiments. B, *, p � 0.05 versus untransfected cells. #, p � 0.05 versus ELMO1 and Dock180 co-transfected cells. D, *, p � 0.05 versus untrans-fected cells. #, p � 0.05 versus Nck siRNA transfected cells.

ELMO1-Nck-1 Interaction Promotes Rac1 Activation

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WT ELMO1 (Fig. 6D), providing further evidence that Nck-1promotes ELMO1-RhoG interaction.

ELMO1-Nck-1 Interaction Promotes Relocation of ELMO1and Cell Migration—To assess the biological relevance of Nck-1-ELMO1 interaction, we first investigated whether Nck-1affects the localization of ELMO1 by immunofluorescencestaining. As previously found (24), Myc-tagged ELMO1 wasobserved in the cytoplasm when expressed alone. When co-ex-pressing Myc-tagged ELMO1 with WT Nck-1, ELMO1 wasobserved in plasma membrane and co-stained with Nck-1. Incontrast, Myc-tagged ELMO1 remained cytosolic whenco-transfected with R308K Nck (Fig. 7A). These data indicate arole of Nck in recruiting ELMO1 to plasma membrane.

Because Rac1 lies downstream of the ELMO1/Dock180 com-plex in signaling pathways leading to cell migration, we nextcarried out cell migration assays with wild-type and R308Kmutant forms of Nck-1. As reported previously (24), co-expres-sion of Dock180 and ELMO1 enhanced cell migration. Co-ex-pressing WT Nck-1 with Dock180 and ELMO1 furtherenhanced ELMO1/Dock180-promoted cell migration. How-ever, R308K mutant forms of Nck-1 failed to augment ELMO1/Dock180-promoted cell migration (Fig. 7B).

In reciprocal experiments, we examined whether knock-down endogenous Nck-1 would inhibit cell migration. Asexpected, silencing endogenous Nck-1 by siRNA resulted in a�60% reduction on cell migration compared with scramblesiRNA transfected cells. Re-expression of siRNA-resistant WTNck-1, but not R308K mutant, rescued the cell mobility (Fig.7C). Together, these data indicate that Nck-1 functions syner-

gistically with ELMO1/Dock180 complex to promote cellmotility.

DISCUSSION

In the present study, we characterized a novel interactionbetween ELMO1 and the SH2 domain of Nck-1. SH2 domainsare known to bind to phosphotyrosine residues in proteins (26).In the present study, we demonstrated that four Hck-depen-dent pTyr sites (Y18, Y216, Y395, and Y511) on ELMO1 arenecessary for binding to Nck-SH2. Mutation of all four tyrosineresidues, but not single residue, substantially reduced the bind-ing to Nck-SH2. Moreover, we noted that mutation of all 4tyrosines does not completely eliminate the binding. It is pos-sible that other non-Hck-dependent pTyr sites on ELMO1 alsobind to Nck-SH2. This type of multisite binding has been doc-umented for many protein-protein interactions. For example,multisite phosphorylation of cyclin-dependent kinase inhibitorSic1 is required for its degradation by SCF ubiquitin ligase path-way (27). In addition, three pYDxV binding sites on Nephrinhave been found and mutation of all three tyrosine residues inYDxV motifs abolished the binding to the SH2 domain of Nck(28). These data suggest that multisite phosphorylation may bea more general mechanism to set thresholds in regulated pro-tein-protein interactions.

Conventional SH2 domains normally bind to their cognateligands in a two-pronged mode mediated primarily by the pTyrresidue. However, it has been reported that the SH2 domain ofSAP interacts with its ligand at residues positions �2 and �3relative to pTyr and the pTyr residue itself. The SAP/SH2D1A

FIGURE 6. Nck-1 promotes the interaction between ELMO1 and active RhoG. A, schematic representation of WT ELMO1, ARM1, or ARM2 mutant of ELMO1used. B, Myc-tagged WT ELMO1, ELMO1 ARM1, or ARM2 mutant was transfected into HEK293T cells, respectively. Cell lysates were incubated with purifiedGST-Nck-1-SH2. Bound protein was detected by immunoblot using anti-Myc antibody. Expression of Myc-WT ELMO1 or ELMO1 mutant proteins was assessedby immunoblot using anti-Myc antibody. C, Myc-tagged ELMO1 and HA-tagged RhoGV12A were co-transfected with Flag-tagged WT Nck-1, R308K, or SH3–1�,2�,3� into HEK293T cells. Cell lysates were immunoprecipitated by anti-Myc antibody followed by anti-HA, anti-Flag immunoblot. Expression of ELMO1,Nck-1, or RhoGV12A proteins was assessed by immunoblot using anti-Myc, anti-Flag, or anti-HA antibody. D, Myc-tagged WT ELMO1 or Y4F mutant wasco-transfected into HEK293T cells with HA-tagged RhoGV12A. Cell lysates were immunoprecipitated by anti-HA or anti-IgG antibody followed by anti-Mycimmunoblot. Expression of exogenous ELMO1 or RhoGV12A proteins was assessed by immunoblot using anti-Myc or anti-HA antibody.

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SH2 domain can recognize a peptide ligand using either allthree residues for maximal affinity or a combination of any two(29, 30). Whether the low level binding observed between 4YFmutant and GST-Nck-1-SH2 is caused by the binding ofNck-1-SH2 to non pTyr residue of ELMO1 needs furtherinvestigation.

Previous study reported that tyrosine phosphorylation ofELMO1 is important for the GEF activity of ELMO1/Dock180complex (18, 20). However, the underlying mechanism remainsunknown. The present study revealed that the interaction

between ELMO1 and active RhoG was substantially reducedwhen all four tyrosine residues are mutated. In addition, 4YFmutant could not recruit ELMO1 to the plasma membrane. Inline with our finding, previous studies have shown that phos-phorylation of a tyrosine residue in the conserved acidic regionof Vav1, Vav2, or Vav3 is required for their GEF activity (31).The present study adds ELMO1/Dock180 complex to the list ofSrc-activated GEFs targeting Rac1 and/or Cdc42, whichincludes Vav proteins, �-PIX, RGRF1 (also known as CDC25 orGRF1) and FRG (also known as FARP2) (32–35). It will be inter-

FIGURE 7. A, Nck-1 modulates ELMO1 subcellular localization. HEK293T cells were transfected with Myc-tagged ELMO1 alone, or together with WT Nck-1 orR308K mutant of Nck-1. The localization of ELMO1 and Nck-1 was analyzed by immunofluorescence staining with confocal microscopy. Alexa594-conjugatedsecondary antibody was used to visualize ELMO1 (red), Alexa 488-conjugated secondary antibody was used to visualize Nck-1 (green). B, Nck-1 promotesELMO1/Dock180-induced cell migration. Myc-tagged ELMO1 and Flag-tagged Dock180 were transfected into HEK293T cells with WT Nck-1 or R308K mutantof Nck-1, respectively. Boyden chamber motility assay was performed as described under “Experimental Procedures.” Data are expressed as mean � S.D. ofthree independent experiments. *, p � 0.05 versus vector transfected cells. #, p � 0.05 versus ELMO1 and Dock180 co-transfected cells. C, HEK293T cells weretransfected with Nck-1 siRNA alone, or together with siRNA-resistant WT Nck-1, R308K plasmid, respectively. If necessary, empty vector was added to ensurethat equal plasmid amounts were transfected in each condition. Boyden chamber motility assay was performed as described under “Experimental Procedures.”Data are expressed as mean � S.D. of three independent experiments. *, p � 0.05 versus scramble siRNA-transfected cells. #, p � 0.05 versus NcksiRNA-transfected cells. Cells from each transfection condition were saved separately, lysed, and immunoblotted to confirm expression of Dock180, ELMO1, WTNck-1, and R308K mutant of Nck-1.

ELMO1-Nck-1 Interaction Promotes Rac1 Activation

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esting to examine whether phosphorylation of tyrosine residueswould cause conformational change of these Src-activatedGEFs.

A number of studies have shown a role of Nck in Rac1 acti-vation. For example, Pak serves as a common effecter protein ofRac/cdc42 (36). Nck recruits Pak1 to the plasma membrane tobind and to be activated by Rac/cdc42 (37). On the other hand,Yoshii et al. showed that PDGF stimulation causes associationof the �PIX, a GEF for Rac, with the p85 subunit of PI-3K andNck. This association results in activation of the �PIX (38). Inthis case, Nck appears to acts upstream of Rac by relocating thePak1-bound �PIX to the membrane. The present study showeda novel function of Nck-1 acting upstream of Rac1: Nck-1 pro-motes ELMO1 binding to active RhoG and thus recruitsELMO1/Dock180 complex to plasma membrane. Previousstudy demonstrated that engagement of RhoG to ELMO causesconformational changes of ELMO and disrupts autoinhibitionof ELMO (39, 40). It is possible that binding to Nck-1 promotesconformational changes of ELMO1, or stabilizes the open con-formation of ELMO induced by active RhoG.

It has been shown that GTP-loaded Rac1 activates theArp2/3 complex via WAVE proteins to induce actin-basedplasma membrane projection (41, 42). Our study showed thatELMO1 directly interacts with the SH2 domain of Nck-1. SinceNck binds with N-WASP through its SH3 domains to stimulateArp2/3 complex and induces actin nucleation (43), the presentdata suggested that Nck-1-ELMO1-Dock180 complex mightfunction as another link between GTP-Rac1 and Arp2/3 com-plex. Future detail experiments may help to determine whetherNck-ELMO1-Dock180 complex could mediate Rac1-inducedactivation of Arp2/3 complex and actin reorganization.

Acknowledgment—We thank Dr. Michiyuki Matsuda (Kyoto Univer-sity, Kyoto, Japan) for kindly providing the Flag-Dock180 construct.

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ELMO1-Nck-1 Interaction Promotes Rac1 Activation

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Guo Zhang, Xia Chen, Fanghua Qiu, Fengxin Zhu, Wenjing Lei and Jing NieMotility 1 (ELMO1) Promotes Rac1 Activation and Cell Motility

Celland the Upstream Regulator of the Rho Family GTPase Rac1 Engulfment and A Novel Interaction between the SH2 Domain of Signaling Adaptor Protein Nck-1

doi: 10.1074/jbc.M114.549550 originally published online June 13, 20142014, 289:23112-23122.J. Biol. Chem. 

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VOLUME 289 (2014) PAGES 23112–23122DOI 10.1074/jbc.W119.012036

Withdrawal: A novel interaction between the SH2 domainof signaling adaptor protein Nck-1 and the upstreamregulator of the Rho family GTPase Rac1 engulfment andcell motility 1 (ELMO1) promotes Rac1 activation and cellmotility.Guo Zhang, Xia Chen, Fanghua Qiu, Fengxin Zhu, Wenjing Lei, and Jing Nie

This article has been withdrawn at the request of the authors. Theauthors were recently made aware that the Coomassie Blue image in Fig.1E was rotated 180°. The Myc immunoblot from lysate in Fig. 2B wasreused in Figs. 2C and 6B. A portion of the GAPDH immunoblot in Fig.2C was reused in Fig. 6B as GAPDH. The last lane of the Myc immuno-blot from lysate in Fig. 3A was reused in the first lane of the Myc immu-noblot from lysate in Fig. 3C. The first lane of the Myc immunoblot fromlysate in Fig. 3E was reused in the last lane of the Myc immunoblot fromlysate in Fig. 3G. The last lane of the Myc immunoblot from the IP in Fig.3E was reused in the first lane of the Myc immunoblot from the IP in Fig.4C. A portion of the Myc immunoblot from the lysate in Fig. 4A wasreused as the HA immunoblot from lysate in Fig. 6C. A portion of theGAPDH immunoblot in Fig. 5A was reused in Fig. 6C as GAPDH.Although the authors believe that the biological conclusions outlined inthe article are valid, the figures presented in the paper do not provide anaccurate representation of the original data, and therefore the scientificintegrity of the study has been compromised. The authors unanimouslyagree that the most appropriate course of action is to withdraw thearticle. All authors sincerely apologize to the scientific community fornot detecting these errors prior to publication and any negative impactthis may have caused.

WITHDRAWALS/RETRACTIONS

20262 J. Biol. Chem. (2019) 294(52) 20262–20262

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