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Originally published In Press as doi:10.1074/jbc.M401476200 on August 23, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46843-46850, November 5, 2004
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Crk Associates with ERM Proteins and Promotes Cell Motility toward Hyaluronic Acid*

Masumi Tsuda{ddagger}§, Yoshinori Makino{ddagger}§, Toshinori Iwahara¶, Hiroshi Nishihara{ddagger}§, Hirofumi Sawa{ddagger}§, Kazuo Nagashima{ddagger}§, Hidesaburo Hanafusa¶, and Shinya Tanaka{ddagger}§||

From the {ddagger}Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N15, W7, Kita-ku, Sapporo 060-8638, Osaka Bioscience Institute, Suita 565-0874, Osaka, and §CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

Received for publication, February 10, 2004 , and in revised form, July 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins (including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellullar matrix plays an important role in various cellular responses (1–3). Extracellullar matrix drives the spatiotemporal reorganization of the cytoskeleton, which is involved in physiological cell migration, tumor cell invasion, and metastasis. Multiple cell surface molecules have been shown to participate in this extracellullar matrix-dependent signaling mechanism. One of the major molecules is CD44, a transmembrane receptor for hyaluronic acid (4, 5), which associates with the actin cytoskeleton through the ERM family proteins (ERMs),1 including ezrin, radixin, and moesin. The cleavage of CD44 at the extracellular domain by membrane-associated metalloproteinases plays a crucial role in efficient cell detachment during cell migration (6, 7). The binding of CD44 and ERMs is controlled by the threonine phosphorylation of ERMs through Rho-associated kinase (ROCK) and also by N-terminal phospholipid modification of ERMs (8).

The signaling adaptor protein Crk, which is composed of an SH2 domain and two SH3 domains, is considered to be involved in cytoskeletal regulation. Crk has been shown to interact with components of focal adhesion, such as p130Cas and paxillin (9, 10), which were tyrosine-phosphorylated mainly by integrin stimulation. Crk transmits signals to downstream effecters through Crk-SH3 binding proteins C3G and Dock180, which exert a guanine-nucleotide exchange factor activity on Rap-1/R-Ras and Rac, respectively (11–13). Thus, Crk may regulate cytoskeletal movement through these guanine-nucleotide exchange factors and small GTPases. In fact, studies of C3G knockout mice have suggested the regulation of cell adhesion by C3G through Rap-1 (14). The phagocytosis, membrane ruffling, and lamellipodia formation has been shown to be regulated by a Dock180-ELMO-Rac-dependent mechanism (15, 16).

Besides the identification of the activation of Rap-1 or Rac, we and others previously found that Crk could activate another GTPase, RhoA, in rat fibroblast 3Y1 cells or PC-12 neuronal cells (17–19). However, the mechanism of Crk-induced RhoA activation and its role were still under the investigation.

In this study, we analyzed the mechanism and significance of the Crk-induced RhoA activation and found the novel association of Crk and ERMs. Unlike the known Crk-associated molecules, these bindings were not dependent solely on the SH2 or SH3 domain. In agreement with the activation of RhoA by Crk, Rho-GDI was colocalized and coprecipitated with Crk. Thus, the dissociation of Rho-GDI and RhoA by Crk was supposed to be involved in RhoA activation. Furthermore, Crk and the phosphorylated form of ERMs cooperatively induced microvilli formation in fibroblasts. The hyaluronic acid treatment enhanced the activation of RhoA in cell lines stably expressing Crk and ERMs. Finally, Crk and ERMs cooperatively regulated cell motility toward hyaluronic acid. These results suggest that Crk associated with ERMs and activated RhoA, possibly through Rho-GDI. Through this mechanism, Crk plays a role in hyaluronic acid-induced enhancement of cell motility.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Antibodies—Rat fibroblast 3Y1 cells (JCRB0734) and 293T human embryonic kidney cells with simian virus 40 large T antigen were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Establishment of the v-Crk-inducible 3Y1 cell line (3Y1 21-2-1) by the tetracycline-inducible system (Tet-on system; BD Biosciences Clontech) has been described previously (19). The v-Src-transformed 3Y1 cell line (SR-3Y1) was established in the Hanafusa Lab at The Rockefeller University (New York, NY). The mouse monoclonal antibody against viral gag protein (clone 3C2) has been described previously (19) and was used to detect v-Crk. The rat monoclonal antibody for phospho-ERM (297S) was a gift from Dr. Sachiko Tsukita (Kyoto University, Kyoto, Japan). Anti-RhoA (119) and Rho-GDI (A-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Rac1 and anti-Crk antibodies were from BD Transduction Laboratories, anti-Flag M2 antibody was from Sigma, anti-myc antibody (9E10) was a gift from Dr. Hiroshi Ariga (Hokkaido University, Sapporo, Japan), and anti-CD44cyto antibody was a gift from Dr. Hideyuki Saya (Kumamoto University, Kumamoto, Japan).

Plasmids—pGEX-rhotekin-RBD was generated with the use of PCR. The cDNAs of ezrin, radixin, and moesin, a gift from Dr. Sachiko Tsukita (Kyoto University, Kyoto, Japan), were subcloned into the pCXN2-Flag vector. The cDNA for Rho-GDI was a gift from Dr. Yoshimi Takai (Osaka University, Osaka, Japan). The following are Crk mutants: pCAGGS-Myc-CrkII-R38V (an SH2 mutant of c-Crk-II), pCAGGS-Myc-CrkII-W169L (an SH3 mutant of of c-Crk-II), pCAGGS-Myc-vCrk-N273 (an SH2 mutant of v-Crk), and pCAGGS-Myc-vCrk-W405K (an SH3 mutant of v-Crk).

Establishment of Cell Lines—To establish stable cell lines expressing Crk and/or ERMs, mammalian expression plasmids of pCAGGS-Myc-Crk II and/or pCXN2-Flag-ezrin, -radixin, or -moesin were transfected to 3Y1 cells using Fugene 6 transfection reagent (Roche). After the selection with 400 µg/ml of G418 (Calbiochem), the expression levels of Crk and ERMs were examined by immunoblotting using anti-Myc and anti-Flag antibody, respectively.

Immunoprecipitaion and Immunoblotting—Immunoprecipitation and immunoblotting were performed by the method described elsewhere. For detection of CD44 by immunoblotting, cells were pretreated by proteasome inhibitor as described previously (19).

Confocal Laser Scanning—For analysis of the subcellular localization of v-Crk, cells were fixed with 3% paraformaldehyde for 15 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 4 min at RT, and then refixed with 70% methanol for 5 min at –20 °C. Anti-gag monoclonal antibody, 3C2 (1:50 dilution), and Alexa 594-conjugated goat anti-mouse immunoglobulin antibody (1:200 dilution; Molecular Probes) were used as primary and secondary antibodies, respectively. For analysis of the localization of pERMs, cells were fixed with 10% trichloroacetic acid for 15 min on ice. Anti-pERM Ab (297S, culture supernatant without dilution) and Alexa 488-conjugated goat anti-rat immunoglobulin antibody (1:200 dilution; Molecular Probes) were used as primary and secondary antibodies, respectively. The samples were observed using a confocal laser-scanning microscope (FV-300; Olympus, Tokyo, Japan) equipped with a computer.

Pull-down Assay for RhoA and Rac—These methods were described previously (19, 20). In brief, for Rho assay, serum-starved v-Crk-inducible 3Y1 cells with or without Dox were lysed by lysis buffer composed of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5 mM MgCl2, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. Cell lysates were clarified by centrifugation at 5,000 x g, 4 °C, for 5 min, and the supernatants were incubated with 10 µg of GST-rhotekin-RBD fusion protein pre-conjugated with glutathione-beads at 4 °C for 1 h. For Rac assay, cells were lysed with lysis buffer composed of 1% Nonidet P-40, 25 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 10 mM MgCl2, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were clarified by 12,000 rpm at 4 °C for 1 min, and the supernatant was incubated with 10 µg of purified GST-PAK2-RBD and glutathione beads at 4 °C for 1 h. In both Rho and Rac assays, the beads were washed three times with each lysis buffer and subjected to SDS-PAGE with a 12% gel. Precipitated RhoA or Rac1 was detected by immunoblotting using anti-RhoA or Rac1Ab.

Immunoelectron Microscopy—Analysis was performed by the pre-embedding method with double immunostaining. v-Crk-induced 3Y1 cells were fixed with 0.1% glutaraldehyde in 0.1 M cacodylate buffer for 5 min on ice and first incubated with a mixture of primary rat monoclonal antibodies for pERM and a mouse mAb for v-Crk for 3 days at 4 °C. After washing with PBS, the specimens were incubated with 10 nm gold-labeled anti-mouse immunoglobulin Ab for 1 h, followed by incubation for 1 h with biotin-labeled anti-rat Ab, which was further reacted with peroxidase-labeled streptavidin. After re-fixation for 5 min, the enzyme reaction was visualized by using diaminobenzidine as substrate. Cells were re-fixed with 2% OsO4 in 0.1 M phosphate buffer for 50 min and then embedded in Epon. Cells in Epon block were sectioned into 1-µm thicknesses and stained with 1% toluidin blue for confirmation of the status of the cells. Then, ultra-thin sections made with the use of an ultramicrotome (Ultracut, Reihert-Jung, Co, Ansberg, Germany) were stained with 0.2% lead citrate for 5 min. Samples were observed by transmission electron microscopy (Hitachi 7100, Tokyo, Japan).

Wound Healing Assay—The method for the wound healing assay was described previously (19). In brief, after the initial plating of cells for 48 h on uncoated, 10 µg/ml fibronectin- or hyaluronic acid-coated culture dishes, cells were scraped off/wounded by a yellow tip. Subsequently, at 12 and 18 h, the recovery percentage of the wounded portion was measured.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Association of Crk and ERMs—To examine the mechanism of Crk-mediated cytoskeletal movement, the association of Crk and ERMs was examined, because we reported the Crk-dependent activation of RhoA and the cleavage of CD44 (19), and ERMs are known to bind to CD44 regulating actin cytoskeleton. First, we found that anti-Crk antibody coprecipitated transiently expressed ERMs with endogenous Crk in human embryonic kidney 293T cells (Fig. 1A, lanes 1–3). Furthermore, with the overexpression of either v-Crk or human c-Crk-II and ERMs, the association of Crk and ERMs was efficiently demonstrated in 293T cells (Fig. 1A, lanes 4–9). To confirm the association of Crk and ERMs, we established 3Y1 cell lines stably expressing either c-Crk-II or ERMs alone, and c-Crk-II and either of the ERMs. In these cell lines, coprecipitations of EMRs with Crk were clearly demonstrated (Fig. 1B, lanes 6–8).



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  		FIG. 1.
Association of Crk and ERMs. A, 293T cells were transiently transfected by the expression vector of ERMs with Flag tag (lanes 1–3), by both ERMs and v-Crk (lanes 4–6), or by c-Crk-II with myc tag (lanes 7–9). Lane 10, vector control. Top, lysates were immunoprecipitated by anti-Crk Ab, and ERM proteins bound to Crk were detected by immunoblotting (IB) using anti-Flag tag Ab. E, R, and M stand for ezrin, radixin, and moesin, respectively. The expression levels of exogeneous ERMs were examined by anti-Flag Ab (middle) and those of v-Crk and c-Crk-II were by anti-Crk Ab (bottom). B, association of c-Crk-II with ERMs in 3Y1 cell lines. The following were stably expressed in 3Y1 cells: lane 1, C as control vector; lane 2, c-Crk-II; lane 3, ezrin; lane 4, radixin; lane 5, moesin; lane 6, c-Crk-II and ezrin; lane 7, c-Crk-II and radixin; lane 8, c-Crk-II and moesin. c-Crk-II was tagged with myc and ERMs were with Flag. Using cell lysates, immunoprecipitation of anti-Crk Ab was performed, and precipitants were probed with anti-Flag Ab (top). The expression levels of exogenous c-Crk-II and ERMs are shown (middle and bottom). C, association of ERMs and Crk mutants. 293T cells were transiently transfected with the expression vectors as follows: lane 1, vector control; lanes 2–4, either of the ERMs alone; lanes 5–7, either of the ERMs and wild-type c-Crk-II; lanes 8–10, either of the ERMs and SH2-mutant of c-Crk-II, R38V; lanes 11–13, either of the ERMs and SH3-mutant of c-Crk-II, W169L. Cell lysates were immunoprecipitated by anti-Crk Ab and probed with anti-Flag Ab (top). The expression levels of exogenous ERMs and c-Crk-II were examined (middle and bottom).

 
To analyze the binding mechanism of Crk and ERMs, we transiently transfected mutants of c-Crk-II with ERMs in 293T cells. Although the association of c-Crk-II and radixin/moesin seemed to be weakened when we expressed the SH2 or SH3 mutants of c-Crk-II (Fig. 1C, lanes 9, 10, 12, and 13), we could not detect a remarkable suppression of the binding of SH2- or SH3-mutants of c-Crk-II and ERMs (Fig. 1C). Mutational analyses using SH2 or SH3 mutants of v-Crk and CrkL were also performed, and similar results were obtained (data not shown).

Activation of Rho by Crk and ERMs—To examine the effect of the association of Crk and ERMs on Crk-induced activation of RhoA, we performed a pull-down assay using the GST fusion form of Rhotekin-RBD in 293T cells. The activities of RhoA were augmented by coexpression of ERMs by 2.8-fold compared with Crk alone (Fig. 2A, top and graph). In addition, coexpression of Crk and EMRs enhanced Rac activity (Fig. 2B, bottom and graph).



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  		FIG. 2.
A, analysis of RhoA and Rac activity in Crk and ERMs-expressing cells. 293T cells were transiently transfected with expression vectors of myc-tagged c-Crk-II and Flag-tagged ERMs as follows: lane 1, c-Crk-II; lane 2, ezrin; lane 3, radixin; lane 4, moesin; lane 5, c-Crk-II and ezrin; lane 6, c-Crk-II and radixin; lane 7, c-Crk-II and moesin. GTP-bound forms of endogenous RhoA and Rac were precipitated by GST-Rhotekin-RBD and GST-PAK-RBD, respectively, and detected by immunoblotting by using anti-RhoA and Rac antibodies. Protein expression levels of total RhoA or Rac were also examined, and ratios of the GTP-form of RhoA/Rac and total RhoA/Rac were calculated and described as a bar graph. B, association of Crk with Rho-GDI. In 3Y1 cells stably expressing CrkII and ezrin, the localizations of Rho-GDI and CrkII were analyzed by immunofluorescence microscopy using anti-Rho-GDI Ab (a, green) and anti-Crk Ab (b, red). Co-localization of Rho-GDI and c-Crk-II was demonstrated by merged imaging (c, yellow). C, 293T cells were transiently transfected with the expression vector of c-Crk-II (lane 1), Rho-GDI (lane 2), both c-Crk-II and Rho-GDI (lane 3), c-Crk-II, Rho-GDI, and either of the ERMs (lanes 4–6). c-Crk-II and ERMs were tagged with Flag. The lysates were immunoprecipitated by anti-Rho-GDI Ab and probed with anti-Flag antibody, demonstrating coprecipitated c-Crk-II (top, arrowheads) and ERMs (top, asterisk). The expression levels of exogenous c-Crk-II and ERMs are shown at the bottom.

 
To examine the mechanism of Crk-induced Rho activation, we focused on the association of Crk and Rho-GDI, because no known Rho guanine-nucleotide exchange factor has been reported to bind to Crk. In fact, Rho-GDI contains possible Crk-interacting sequences, such as the YXXP motif for the SH2 domain and proline-rich region for the SH3 domain. The colocalization of these proteins was observed in 3Y1 cells by confocal microscopy (Fig. 2B), and force-expressed Crk was coprecipitated with Rho-GDI by using anti-Rho-GDI antibody in 293T cells (Fig. 2C, arrowhead). The association of ERMs and Rho-GDI was also observed, as reported previously (Fig. 2C, asterisk) (21). It should be noted that we failed to detect the association of endogenous Crk and Rho-GDI (data not shown).

Association of Crk and pERMs and Induction of Microvilli Formation—Because ERM proteins were known to be regulated by phosphorylation, the binding of Crk and the phosphorylated form of ERMs (pERMs) were examined by using a v-Crk-inducible 3Y1 cell line (clone 21-2-1) (19). In the presence of v-Crk, the association of Crk with pERMs was detectable in the cytoplasmic fraction of 3Y1 cells (Fig. 3A).



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  		FIG. 3.
Association of Crk and the phosphorylated form of ERMs. A, analysis of the binding between v-Crk and pERMs in the v-Crk-inducible 3Y1 cell line (clone 21-2-1). After the treatment of doxycycline to induce v-Crk, total cell lysates (lane 1) and each fraction of cell lysates indicated above the (lanes 2–4) were examined by immunoprecipitation using anti-pERM Ab (297S), and probed with anti-gag Ab (3C2) for detection of v-Crk (top). The expression level of v-Crk in each lysate was analyzed by immunoblotting (IB) by using 3C2 Ab (bottom). B, immunofluorescent analysis of pERMs by induction of v-Crk. The v-Crk-induced 3Y1 cells (clone 21-2-1) were treated with doxycycline for 48 h (photographs b–j). Cells with no doxycycline treatment are shown as photo a. Within 48 h, cells were also treated with the ROCK inhibitor Y27632 (e, f, and g) or the phosphatidylinositol 4,5-bisphosphate inhibitor neomycin (h, i, and j). Localizations of pERMs and v-Crk were analyzed by anti-pERM Ab (297S) (green) and anti-gag Ab (red), respectively. Arrowheads indicate the colocalization of v-Crk and pERMs. C, the phosphothreonine levels of ERMs in 3Y1 21-2-1 cells in the absence (lanes 1, 3, and 5) or the presence (lanes 2, 4, and 6) of doxycycline (Dox) were examined by immunoblotting by anti-pERM Ab. The cells were also treated with Y27632 (lanes 3 and 4) or neomycin (lanes 5 and 6).

 
We then analyzed the subcellular localization of pERMs in a v-Crk-inducible 3Y1 cell line. pERMs were observed diffusely in the cytoplasm and partially at the edge of the cytoplasm of 3Y1 cells without v-Crk (Fig. 3B, a). However, with v-Crk induction, pERMs were demonstrated to translocate to cellular microvilli, and co-localization of v-Crk and pERMs was shown by a merged image (Fig. 3B, b–d). In addition, co-localization of v-Crk and pERMs was also detected as dotted patterns in the cytoplasm (Fig. 3B, b–d, arrowheads). To confirm the involvement of ROCK, which was known to phosphorylate ERMs in v-Crk-dependent microvilli formation of pERMs, we used the ROCK inhibitor Y27632 and found that this reagent inhibited the localization of pERMs to microvilli, whereas the remaining co-localization of v-Crk and pERMs was still detectable in the cytoplasm (Fig. 3B, e–g, arrowheads). With Y27632 treatment, phosphothreonine levels of ERMs especially on ezrin and radixin were decreased, but the detectable levels of the phosphorylated form of moesin should be noted (Fig. 3C, lanes 3 and 4).

Given that the accumulation of phosphatidylinositol 4,5-bisphosphate in the plasma membrane through the activation of Rho/ROCK/PI 4-phospho 5-kinase cascade has been shown to be involved in the continuous activation of pERMs (22), we analyzed the effect of neomycin that inhibits the membrane accumulation of phosphatidylinositol 4,5-bisphosphate and found that this reagent produced similar responses to Y27632 (Fig. 3, B, h–j and C, lanes 5 and 6).

To confirm the immunofluorescence study, we performed immunoelectron microscopy using the double staining method. pERMs were visualized with the use of diaminobenzidine, in which they are recognized as electron-dense black substances by transmission electron microscopy, and the presence of anti-v-Crk Ab was demonstrated by secondary antibodies labeled with 10-nm gold particles. pERM labeled with diaminobenzidine was recognized in the cytoplasm of vCrk-expressing 3Y1 cells by light microscopy (Fig. 4a). Immunoelectron microscopy demonstrated that vCrk was colocalized with pERM at the microvilli, cytoplasmic edge, and filamentous structure in the cytoplasm of the cells (Fig. 4, b–g).



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  		FIG. 4.
Immunoelectron microscopic analysis of the association of pERMs and v-Crk. a, immunopositive reaction of pERM in v-Crk-expressing 3Y1 cells observed by light microscopy. pERMs were visualized by enzymatic reaction using diaminobenzidine as a brown color after counterstaining with toluidine blue. b–e, transmission electron microcopy for the colocalization of v-Crk as 10-nm gold particles and pEMR as electron-dense substance at microvilli. The areas in the boxes of b and d are enlarged in c and e, respectively. f and g, colocalization of v-Crk and pERM at the filamentous structure in the cytoplasm (f) and at the cytoplasmic edge (g). Insets of f and g are enlarged images of 10-nm gold particles. Scale bars, b, 300 nm; d, 1 µm; f and g, 100 nm.

 
The Cleavage of CD44 in 3Y1 Cells Expressing Both Crk and ERMs—To analyze the involvement of Crk in the hyaluronic acid-CD44-dependent signaling mechanism, we examined whether Crk increased the cleavage of CD44. Without hyaluronic acid, there was no detectable level of cleavage product of CD44 in Crk or Crk/ERMs-expressing cells (Fig. 5A, top, lanes 3, 7, and 8). Upon hyaluronic acid coating, the cleavage products of CD44 could be observed in Crk-expressing cells and coexpressing cells of c-Crk-II and ezrin or radixin (Fig. 5A, bottom, lanes 3, 7, and 8). Cleaved bands were not observed in Crk/moesin-coexpressing cells (Fig. 5A, bottom, lane 9).



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  		FIG. 5.
Cleavage of CD44 in coexpressing cells of Crk and ERMs. A, the levels of full-length and cleavage products of CD44 were investigated by anti-CD44cyto Ab. The cells were cultured on un-coating (top) or hyaluronic acid-coating (bottom) dishes. Lane 1, parental 3Y1; lane 2, vector control; lane 3, c-Crk-II; lane 4, ezrin; lane 5, radixin; lane 6, moesin; lane 7, c-Crk-II and ezrin; lane 8, c-Crk-II and radixin; lane 9, c-Crk-II and moesin; lane 10, v-Src expressing 3Y1 (SR-3Y1). Arrows indicate the full length of CD44, and arrowheads indicate the cleavage products of CD44. B, analysis of cell motility of Crk- and EMR-expressing 3Y1 cells by wound-healing assay. The cells were plated on uncoated (top graph) or hyaluronic acid-coated (bottom graph) dishes. Light microscopic analyses of the cells were performed at 0, 12, and 18 h after wound formation. The percentage of recovery of wound formation was calculated, and the averages of three independent experiments were plotted as the graph. C, effect of PI-3 kinase inhibitors in 3Y1 cell motility by wound-healing assay. 3Y1 cells stably expressing either of the ERMs or Crk or both of the ERMs or Crk, indicated below the graph, were plated on hemagglutinin-coated culture dishes, and wound-healing assay was performed with or without PI-3 kinase inhibitors, 10 µM LY294002, or 200 nM Wortmannin. Twelve hours after scratching off the plates, the distances of the moved cells were measured. The distance of each cell line without the treatment of inhibitor was designated as 1.0 (bar graph not shown), and the relative distance of the moved cells of each cell line with the inhibitor was described as a bar graph.

 
Analysis of Cell Motility of 3Y1 Cells Expressing Crk and ERMs—To examine whether the association of Crk with ERMs affects cell motility, we performed wound healing assay by using 3Y1 cells stably expressing Crk and ERMs. Without extracellular matrix, the expression of Crk decreased cell motility, as reported previously (19), and expression of either of the ERMs also suppressed motilities (Fig. 5B, top). Coexpression of Crk and either of the ERMs slightly promoted motility, but the levels were still below those observed with wild-type 3Y1 cells. However, upon hyaluronic acid stimulation, double expression of Crk and ERMs enhanced motility more than wild-type level (Fig. 5B, bottom). With integrin stimulation, Crk-expressing cells and the double expression of Crk and ERMs recovered the motility to the levels of wild-type 3Y1 cells but did not provide significant enhancement of motility higher than that of wild-type 3Y1 cells (data not shown).

To confirm the involvement of the CD44 cleavage in v-Crk-regulated cell motility, we examined the effect of PI-3 kinase inhibitors because PI-3 kinase was known to up-regulate CD44 cleavage (23, 24). Wound healing assay demonstrated that PI-3 kinase inhibitors such as LY294002 and Wortmannin tend to suppress the motility of 3Y1 cell lines; however, this suppressive function was most prominently found in 3Y1 cells stably expressing Crk and ezrin (Fig. 5C). PI-3 kinase inhibitors did not affect the motility of cells expressing Crk and moesin (Fig. 5C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Signaling adaptor protein Crk was originally found as an avian sarcoma encoding oncoprotein v-Crk (25). Since human c-Crk-II, the homologue of v-Crk, was isolated, the identification of Crk targets has suggested that Crk links between tyrosine phosphorylated proteins and guanine-nucleotide exchange factors for small GTPases, and regulates cytoskeletal reorganization. In particular, under fibronectin stimulation, the integrin-provoked signal has been shown to be mediated by Crk and transmitted to the downstream effecter Dock180, leading to Rac activation. However, the mechanism of Crk-mediated cell migration or tumor cell invasion has remained under investigation.

In this study, we have found a novel interaction of Crk and the ERM family of proteins that is involved in activation of Rho and hyaluronic acid-CD44 dependent regulation of cell motility (Fig. 6). In contrast to the known Crk binding molecules such as p130Cas, paxillin, C3G, and Dock180, the interaction of Crk and EMRs were not solely SH2- or SH3-dependent. Mutation analysis showed that both SH2 and SH3 mutants of Crk significantly attenuate the association of Crk and ERMs, whereas Crk SH2 mutant binds to ezrin as much as wild-type Crk. Thus, Crk may bind to EMRs by both the SH2 and SH3 domains. On the other hand, the entire conformation of Crk might be required for this association. Determination of the Crk binding site(s) of ERMs may reveal the mechanism.



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  		FIG. 6.
Scheme of the regulation of hyaluronic acid-CD44-dependent cell motility by Crk. Crk bound to ERMs, possibly with Rho-GDI, and promoted hyaluronic acid-CD44-dependent cell motility. The association of Crk and the negative regulator for Rho, such as Rho-GDI, may suppress the function of Rho-GDI, resulting in the activation of Rho. The subsequent activation of ROCK leads to the phosphorylation of ERMs, which link CD44 to actin fibers.

 
According to our previous results, v-Crk activated RhoA in fibroblasts and coexpression of Crk, and ERMs enhanced the activity of RhoA in 293T cells. Given that no known Rho-guanine-nucleotide exchange factor was found to bind to Crk, the mechanism of Crk-dependent activation of Rho was the missing link. Rho-GDI has been shown to bind to the N-terminal FERM domain of ERMs (21), and these data led us to hypothesize that upon extracellullar matrix stimulation, Crk binds to the negative regulator of RhoA (such as Rho-GDI), inactivates Rho-GDI, and leads to the activation of RhoA shown as Fig. 6. Thus, we examined the association of Rho-GDI and Crk. In this study, the association of force-expressed Crk and Rho-GDI was observed in 293T cells, but we failed to show the association of endogenous Crk and Rho-GDI (data not shown). In 293T cells, we did not examine the inhibition of the function of negative regulator Rho-GDI because the simple expression of Rho-GDI did not significantly suppress the activity of RhoA measured by pull-down assay. Furthermore, we also tried to test the effect of Crk on another negative regulator of RhoA, Rho-GAP. However, we did not observe a significant activation of RhoA by the double expression of Crk and Rho-GAP (data not shown). Establishment of a deficient cell line for the negative regulator of RhoA may reveal the Crk-dependent activation mechanism of Rho in future studies.

Because it is known that ERMs were phosphorylated in the cytoplasm and translocated to membrane, and that pERMs link between CD44 and actin cytoskeleton, we analyzed the localization of pERMs in v-Crk-inducible fibroblasts. In 3Y1 cells, v-Crk translocated pERMs and induced microvilli formation by a ROCK-dependent signaling mechanism. Although we expected the induction of v-Crk-induced phophorylation of ERMs, we failed to demonstrate such increased phosphorylation of ERMs by Crk in our system (Fig. 3C). We speculate that the relatively high levels of pERMs in the cytoplasm of wild-type 3Y1 cells may mask further phosphorylation of ERMs.

In this study, we showed that the association of Crk and ERMs was involved in the hyaluronic acid-CD44 signaling mechanism to promote cell motility. Considering the mechanism of Crk-dependent enhancement of CD44 cleavage, Crk may also regulate the transcriptional levels of matrix metalloproteinases. As Crk is also known to activate PI-3 kinase (26), which was reported to control the transcriptional levels of matrix metalloproteinases by a Rac-Cdc25-dependent mechanism (23, 24), we examined the effect of PI-3 kinase inhibitors on Crk/ERM-dependent motility of 3Y1 cells on an HA-coated plate. The data shown in Fig. 5C suggest that PI-3 kinase may be involved in the motility of Crk/ezrin- and Crk/radixin-expressing 3Y1 cells. In addition, the Ras/ERK pathway may be involved in Crk-dependent expression of matrix metalloproteinases, because v-Crk has been reported to activate the secretion of matrix metalloproteinase through Ras and ERK (27).

We and others have reported recently that overexpression of Crk, especially Crk-I, was observed in human malignant tumors, such as glioblastoma and lung cancer (28–30). Crk has been shown to be related to the malignant feature of tumor cells. Thus, we should reveal the significance of our present findings, in which Crk regulates cell motility through hyarulonic acid/CD44/ERM, in relation to the invasiveness of human cancers in future studies.


   FOOTNOTES
 
* This work was supported in part by a grant from the Ministry of Education, Science, Culture, and Sports of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 81-11-706-7806; Fax: 81-11-706-7806; E-mail: tanaka{at}med.hokudai.ac.jp.

1 The abbreviations used are: ERMs, ERM family proteins; ROCK, Rho-associated kinase; SH, Src homology; GDI, dissociation inhibitor; RBD, Rac binding domain; Ab, antibody; GST, glutathione S-transferase; pERM, phosphorylated form of ERMs; PI-3, phosphatidylinositol 3. Back


   ACKNOWLEDGMENTS
 
We thank Yasuhisa Fukui (Tokyo Univ., Japan) for useful discussion, Michiyuki Matsuda (Osaka Univ., Japan), Yoshimi Takai (Osaka Univ., Japan) and Sachiko Tsukita (Kyoto Univ., Japan) for plasmids, Hideyuki Saya (Kumamoto Univ., Japan) and Hiroshi Ariga (Hokkaido Univ., Japan) for antibodies, William W. Hall (University College Dublin, Ireland) for critical reading of the manuscript, and Sumie Oikawa, Miho Higuchi, and Masae Maeda for technical assistance. We also thank Mami Sato for immunoelectron microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 REFERENCES
 

  1. Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84, 359–369[CrossRef][Medline] [Order article via Infotrieve]
  2. Gumbiner, B. M. (1996) Cell 84, 345–357[CrossRef][Medline] [Order article via Infotrieve]
  3. Sheetz, M. P., Felsenfeld, D. P., and Galbraith, C. G. (1998) Trends Cell Biol. 8, 51–54[Medline] [Order article via Infotrieve]
  4. Ponta, H., Sherman, L., and Herrlich, P. A. (2003) Nat. Rev. Mol. Cell. Biol. 4, 33–45[CrossRef][Medline] [Order article via Infotrieve]
  5. O'Neill, G. M., Fashena, S. J., and Golemis, E. A. (2000) Trends Cell Biol. 10, 111–119[CrossRef][Medline] [Order article via Infotrieve]
  6. Tsukita, S., Oishi, K., Sato, N., Sagara, J., and Kawai, A. (1994) J. Cell Biol. 126, 391–401[Abstract/Free Full Text]
  7. Okamoto, I., Kawano, Y., Matsumoto, M., Suga, M., Kaibuchi, K., Ando, M., and Saya, H. (1999) J. Biol. Chem. 274, 25525–25534[Abstract/Free Full Text]
  8. Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., and Tsukita, S. (1998) J. Cell Biol. 140, 647–657[Abstract/Free Full Text]
  9. Brugge, J. S. (1998) Nat. Gen. 19, 309–311[CrossRef][Medline] [Order article via Infotrieve]
  10. Feller, S. M. (2001) Oncogene 20, 6348–6371[CrossRef][Medline] [Order article via Infotrieve]
  11. Tanaka, S., Morishita, T., Hashimoto, Y., Hattori, S., Nakamura, S., Shibuya, M., Matuoka, K., Takenawa, T., Kurata, T., Nagashima, K., and Matsuda, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3443–3447[Abstract/Free Full Text]
  12. Gotoh, T., Hattori, S., Nakamura, S., Kitayama, H., Noda, M., Takai, Y., Kaibuchi, K., Matsui, H., Hatase, O., Takahashi, H., Kurata, T., and Matsuda, M. (1995) Mol. Cell. Biol. 15, 6746–6753[Abstract]
  13. Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T., and Matsuda, M. (1998) Genes Dev. 12, 3331–3336[Abstract/Free Full Text]
  14. Ohba, Y., Ikuta, K., Ogura, A., Matsuda, J., Mochizuki, N., Nagashima, K., Kurokawa, K., Mayer, B. J., Maki, K., Miyazaki, J., and Matsuda, M. (2001) EMBO J. 20, 3333–3341[CrossRef][Medline] [Order article via Infotrieve]
  15. Brugnera, E., Haney, L., Grimsley, C., Lu, M., Walk, S. F., Tosello-Trampont, A. C., Macara, I. G., Madhani, H., Fink, G. R., and Ravichandran, K. S. (2002) Nat. Cell Biol. 4, 574–582[Medline] [Order article via Infotrieve]
  16. Gumienny, T. L., Brugnera, E., Tosello-Trampont, A. C., Kinchen, J. M., Haney, L. B., Nishiwaki, K., Walk, S. F., Nemergut, M. E., Macara, I. G., Francis, R., Schedl, T., Qin, Y., Van Aelst, L., Hengartner, M. O., and Ravichandran, K. S. (2001) Cell 107, 27–41[CrossRef][Medline] [Order article via Infotrieve]
  17. Iwahara, T., Akagi, T., Shishido, T., and Hanafusa, H. (2003) Oncogene 22, 5946–5957[CrossRef][Medline] [Order article via Infotrieve]
  18. Altun-Gultekin, Z. F., Chandriani, S., Bougeret, C., Ishizaki, T., Narumiya, S., de Graaf, P., Van Bergen en Henegouwen, P., Hanafusa, H., Wagner, J. A., and Birge, R. B. (1998) Mol. Cell. Biol. 18, 3044–3058[Abstract/Free Full Text]
  19. Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., and Nagashima, K. (2002) Cell Growth Differ. 13, 131–139[Abstract/Free Full Text]
  20. Nishihara, H., Maeda, M., Tsuda, M., Makino, Y., Sawa, H., Nagashima, K., and Tanaka, S. (2002) Biochem. Biophys. Res. Commun. 296, 716–720[CrossRef][Medline] [Order article via Infotrieve]
  21. Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita, S., and Takai, Y. (1997) J. Biol. Chem. 272, 23371–23375[Abstract/Free Full Text]
  22. Matsui, T., Yonemura, S., and Tsukita, S. (1999) Curr. Biol. 9, 1259–1262[CrossRef][Medline] [Order article via Infotrieve]
  23. Kawano, Y., Okamoto, I., Murakami, D., Itoh, H., Yoshida, M., Ueda, S., and Saya, H. (2000) J. Biol. Chem. 275, 29628–29635[Abstract/Free Full Text]
  24. Kamikura, D. M., Khoury, H., Maroun, C., Naujokas, M. A., and Park, M. (2000) Mol. Cell. Biol. 20, 3482–3496[Abstract/Free Full Text]
  25. Mayer, B. J., Hamaguchi, M., and Hanafusa, H. (1988) Nature 332, 272–275[CrossRef][Medline] [Order article via Infotrieve]
  26. Akagi, T., Shishido, T., Murata, K., and Hanafusa, H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7290–7295[Abstract/Free Full Text]
  27. Liu, E., Thant, A. A., Kikkawa, F., Kurata, H., Tanaka, S., Nawa, A., Mizutani, S., Matsuda, S., Hanafusa, H., and Hamaguchi, M. (2000) Cancer Res. 60, 2361–2364[Abstract/Free Full Text]
  28. Miller, C. T., Chen, G., Gharib, T. G., Wang, H., Thomas, D. G., Misek, D. E., Giordano, T. J., Yee, J., Orringer, M. B., Hanash, S. M., and Beer, D. G. (2003) Oncogene 22, 7950–7957[CrossRef][Medline] [Order article via Infotrieve]
  29. Nishihara, H., Tanaka, S., Tsuda, M., Oikawa, S., Maeda, M., Shimizu, M., Shinomiya, H., Tanigami, A., Sawa, H., and Nagashima, K. (2002) Cancer Lett. 180, 55–61[CrossRef][Medline] [Order article via Infotrieve]
  30. Takino, T., Nakada, M., Miyamori, H., Yamashita, J., Yamada, K. M., and Sato, H. (2003) Cancer Res. 63, 2335–2337[Abstract/Free Full Text]

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