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Cell Growth & Differentiation Vol. 13, 131-139, March 2002
© 2002 American Association for Cancer Research

Signaling Adaptor Protein v-Crk Activates Rho and Regulates Cell Motility in 3Y1 Rat Fibroblast Cell Line1

Masumi Tsuda, Shinya Tanaka2, Hirofumi Sawa, Hidesaburo Hanafusa and Kazuo Nagashima

Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo 060-8638 [M. T., S. T., H. S., K. N.]; CREST, Japan Science and Technology Corporation [M. T., S. T., H. S., K. N.]; and Osaka Bioscience Institute, Osaka 565-0874 [H. H.], Japan


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The adaptor protein Crk has been reported to associate with focal adhesions and is thought to be involved in integrin-mediated signaling pathway. However, the precise mechanism of Crk-dependent regulation of cytoskeleton still remains under investigation. In this study, we have established a v-Crk-inducible cell line in rat fibroblasts 3Y1 cells and found that v-Crk activated Rho and induced actin stress fiber formation. In addition to the induction of tyrosine-phosphorylation of p130Cas and paxillin, we demonstrated that v-Crk induced threonine-phosphorylated bands sized at 72/78 kDa found specifically in 3Y1 cells. Both of the inhibitors of Rho and Rho-associated kinase, C3 and Y27632, respectively, inhibited these v-Crk-induced biochemical effects. Although v-Crk-induced cells exhibited a decrease of cell motility, integrin stimulation recovered the suppression of motility. Furthermore, v-Crk enhanced motility in chemotactic assay toward fibronectin with additional activation of Rho and the increase of levels of CD44 cleavage. These results suggest that v-Crk activated Rho and induced actin stress fiber formation and CD44 cleavage leading to the regulation of cell motility.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The ECM3 controls various physiological and pathological cellular responses including cell migration, cell growth, tumor cell invasion, and metastasis (1, 2, 3) . Signals from the ECM are mainly transduced via integrins, which are components of multimolecular machinery recognized as focal adhesions in association with talin, vinculin, tensin, paxillin, and p130Cas connect to actin fiber, resulting in the regulation of cytoskeletons. Among these components, p130Cas and paxillin have been reported to be tyrosine-phosphorylated on ECM stimulation and play an important role in the regulation of cell morphology and motility (4 , 5) .

Signaling adaptor protein Crk, mostly composed of src homology (SH)2 and SH3 domains, was originally identified as avian sarcoma virus CT10-encoding oncogene product v-Crk, and subsequently its mammalian homologues, Crk-I, Crk-II, and CrkL were isolated (6, 7, 8) . In v-Crk-transformed cells, v-Crk was found to induce tyrosine phosphorylation of p130Cas and paxillin (9, 10, 11, 12) . Crk associates with p130Cas and paxillin through its SH2 domain and may transmit signals to the multiple downstream effectors by SH3 domain-binding proteins including C3G, Dock180, Sos, and c-Abl (13, 14, 15) . These molecules potentially regulate the activities of small molecular weight GTPases including Rap-1, R-Ras, Rac, or Ras (16, 17, 18) , or of kinases such as c-Jun NH2-terminal kinase, extracellular signal-related kinase, or PI3k (19, 20, 21, 22) . Recently, Crk has been shown to play a role in integrin-mediated signaling pathway controlling cell motility mainly through Dock180-Rac-dependent signaling pathway (23 , 24) .

The Rho family of small GTPases including Rho, Rac, and Cdc42 has been shown to be involved in reorganization of actin cytoskeleton including formation of stress fibers, lamellipodia, and filopodia leading to the regulation of cell motility, cell survival, and cytokinesis (25, 26, 27) . The mechanism of Rho-dependent regulation of actin cytoskeleton has been reported to be complex, and the multiple downstream effectors appear to be implicated. Among them, a family of ROCKs such as p160ROCK/ROCK-I (28) and ROK{alpha}/Rho-kinase/ROCK-II (29 , 30) has been demonstrated to function as one of the effectors of Rho, regulating the acto-myosin cytoskeleton, and accelerated cell motility and tumor cell invasion (31) .

Furthermore, the Rho family of proteins has been also reported to regulate cell motility by mediating the interaction of CD44 and ECM (32) . CD44 is a transmembrane receptor for ECM components including hyaluronic acid, type I collagen, fibronectin, fibrin, laminin, and chondroitin sulfate, and is implicated in a variety of adhesion-dependent cellular function such as lymphocyte homing, wound healing, migration, and tumor invasion/metastasis (33, 34, 35) . Recently, it was reported that Ras, PI3k, Cdc42, and Rac have been reported to mediate CD44 cleavage at the extracellular domain by membrane-associated metalloproteinases, and this cleavage plays a critical role in an efficient cell detachment from ECM during the cell migration (36) . Furthermore, CD44 has been shown to be associated with actin filaments via binding to ERM proteins (ezrin, radixin, and moesin) regulated by Rho (37) .

In this study, to analyze the Crk-dependent signalling pathway, we established a conditional expression system of v-Crk in rat fibroblast 3Y1 cells. We found that v-Crk activated Rho, induced actin stress fiber formation, and enhanced CD44 cleavage resulting in modulating cell motility. Furthermore, v-Crk-induced tyrosine-phosphorylation of p130Cas and paxillin, actin stress fiber formation, and threonine phosphorylation of 72/78 kDa proteins that were also found to be Rho/ROCK-dependent.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Establishment of v-Crk-inducible Rat Fibroblast 3Y1 Cell Line Using the Tet-On System.
To analyze the mechanism of Crk-mediated signaling pathway leading to enhance tyrosine phosphorylation of focal adhesion components and its biological responses, we established a conditional expression system of v-Crk using rat fibroblast 3Y1 cells. In the v-Crk-inducible 3Y1 cell line (clone 21-2-1), an approximately 11–13-fold induction of v-Crk was observed at 48 h after treatment with Dox, which provided maximum levels of induction at a concentration of 2 µg/ml (Fig. 1A)Citation . In association with v-Crk induction, tyrosine-phosphorylated proteins, sized at 130 and 68 kDa, representing p130Cas and paxillin, respectively, were detected as described previously (Fig. 1A)Citation . By chronological analysis, an increase of v-Crk was detectable ~3 h after induction and reached a maximum level at 48 h (Fig. 1B)Citation . Tyrosine phosphorylation of p130Cas and paxillin attained a maximum of 6-fold and 2.1-fold, respectively, at 48 h without the alteration of expression levels of p130Cas and paxillin (Fig. 1B)Citation .



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Fig. 1. Establishment of v-Crk-inducible 3Y1 cell line by Tet-on system. A, conditional expression of v-Crk in 3Y1 cell line (clone 21-2-1) by the treatment of Dox for 48 hours. Top panel, immunoblotting by anti-gag Ab (3C2) to detect v-Crk. Bottom panel, immunoblotting by antiphosphotyrosine Ab. Lane 1, without Dox; Lane 2, with Dox (2 µg/ml); Lane 3, with Dox (10 µg/ml). B, chronological analysis of the induction levels of v-Crk. The levels of v-Crk, p130Cas, paxillin, and phosphotyrosine in total cell lysates at the indicated time were examined by immunoblotting.

 
v-Crk Activated Rho and Induced Actin Stress Fiber Formation.
In v-Crk-induced 3Y1 cells, we found that v-Crk remarkably induced actin stress fiber formation (Fig. 2, A and B)Citation . v-Crk was detected in a dotted pattern localizing at the ends of induced actin stress fibers (Fig. 2B)Citation . The induction of actin stress fiber was time-dependent at least until 48 h without the significant alteration of the levels of actin (Fig. 2A)Citation .



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Fig. 2. Analysis of actin fiber and subcellular localization of v-Crk. A, analysis of actin fibers in v-Crk-inducible 3Y1 cell line (21-2-1) treated with Dox for 0, 12, 24, and 48 h by phalloidin staining (red). Bottom panel, the expression levels of actin at each time point were examined by the immunoblotting using antiactin antibody. B, localization of induced v-Crk in 3Y1 cell line (21-2-1) was analyzed using anti-gag Ab (green) and phalloidin staining (red). C, pull down assay for active form of Rho using v-Crk-inducible cell line (21-2-1) and 3Y1 cells stably expressing v-Crk.

 
Because Rho was well known to induce actin stress fiber formation, we examined whether v-Crk activates Rho, and a pull down assay using the Rho-binding region of Rhotekin fused to GST demonstrated that induction of v-Crk induced 2-fold activation of Rho in 3Y1 cells (Fig. 2C)Citation .

v-Crk Induced Rho/ROCK-dependent Actin Stress Fiber Formation.
To confirm the involvement of Rho in v-Crk-dependent actin stress fiber formation, we utilized ADP-ribosylase C3, an inhibitor of Rho (38) , and found that C3 disorganized the v-Crk-induced stress fiber formation (Fig. 3e)Citation .



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Fig. 3. v-Crk induced Rho/ROCK-dependent actin stress fiber formation. Disorganization of v-Crk-induced actin stress fiber formation by Rho inhibitor, C3, and ROCK inhibitor, Y27632. The cells were treated with C3 (b and e), with Y27632 (c and f), or without C3 and Y27632 (a and d). Dox (-), cells without Dox (a–c); Dox (+), cells treated with Dox for 48 h (d–f). Induced v-Crk was stained by anti-gag Ab (green).

 
Furthermore, we examined the effect of the ROCK inhibitor Y27632 (39) , because ROCK, which is one of the downstream effectors of Rho, has been shown to promote actin stress fiber formation, and found that this reagent also suppressed v-Crk-induced stress fiber formation exhibiting the speckled pattern of actin staining diffusely in the cytoplasm of cells (Fig. 3f)Citation . These results suggest that the v-Crk induced actin stress fiber formation by a Rho- and ROCK-dependent mechanism.

v-Crk Induced Threonine Phosphorylation of 72 and 78 kDa Proteins in 3Y1 Cells.
Because ROCK, a serine/threonine kinase, was suggested to be activated by the induction of v-Crk, we investigated the levels of protein phosphorylation after the induction of v-Crk in 3Y1 cells. Using antiphosphothreonine antibody, we found that v-Crk induced positive bands sized at ~72 and 78 kDa in 3Y1 cells (Fig. 4ACitation , upper panel, Lane 12). These bands were also found in 3Y1 cells stably expressing v-Crk (Fig. 4ACitation , upper, Lane 2). To ensure proper preparation of cell lysates, antiphosphotyrosine blotting was also performed, and well-established patterns were confirmed (Fig. 4ACitation , lower panel). Chronological analysis of the induction of phosphothreonine bands in v-Crk-inducible 3Y1 cells showed that the 78 kDa band was detectable at 6 h (Fig. 4B)Citation . In contrast with the tyrosine-phosphorylated bands shown in Fig. 1BCitation , these threonine-phosphorylated bands were diminished at 72 h after the induction of v-Crk. These bands were not detectable in the mouse fibroblast cell line or chicken embryonal fibroblasts expressing v-Crk (Fig. 4ACitation , Lanes 5 and 6).



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Fig. 4. Analysis of threonine and tyrosine phosphorylation levels in v-Crk-inducible 3Y1 cell line. A, the levels of threonine phosphorylation and tyrosine phosphorylation of various cells were investigated by antiphosphothreonine (p-Thr) Ab (top panel) and anti-phosphotyrosine (PY) Ab, respectively (bottom panel). Lane 1, 3Y1; Lane 2, v-CRK-3Y1; Lane 3, SR-3Y1; Lane 4, NIH3T3; Lane 5, v-CRK-NIH3T3; Lane 6, CEF infected with v-Crk-encoding retrovirus CT10; Lane 7, CEF infected with v-Src-encoding retrovirus XD2; Lane 8, 293T; Lane 9, 293T transiently transfected with v-Crk; Lane 10, 293T transiently transfected with v-Src; Lane 11, v-Crk-inducible 3Y1 (clone 21-2-1) without Dox; Lane 12, v-Crk-inducible 3Y1 with Dox. Arrowheads indicate 72 and 78 kDa bands. B, chronological analysis of threonine phosphorylation levels of 72/78 kDa proteins in v-Crk-inducible 3Y1 cell line. The cells treated with Dox for various duration indicated at the top were analyzed by immunoblotting using anti-pThr Ab. C, effect of Y27632 on threonine phosphorylation levels of 72/78 kDa proteins. The cells were treated with Dox for 48 h (Lanes 2–5) or without Dox (Lane 1). Within 48 h after the induction of v-Crk, cells were also treated with 10 µM of Y27632 for 30 min (Lane 3), 12 h (Lane 4), and 18 h (Lane 5). Cell lysates were immunoblotted by anti-pThr Ab. D, suppression of v-Crk-induced tyrosine phosphorylation of p130Cas by ROCK inhibitor Y27632. v-Crk-inducible 3Y1 (21-2-1) were treated with Dox for 48 h (Lanes 2–5) or without Dox (Lane 1). Within 48 h, cells were also treated with 10 µM of Y27632 for 30 min (Lane 3), 12 h (Lane 4), and 18 h (Lane 5), and cell lysates were immunoblotted by anti-PY Ab.

 
We also examined whether the induction of the 72/78 kDa phosphothreonine bands was dependent on the activation of Rho/ROCK. After treatment of cells by Y27632 for 12 h, we found that these bands were not detected by antiphosphothreonine antibody at 48 h after the induction of v-Crk (Fig. 4C)Citation .

Because there are several reports indicating that p130Cas and paxillin can be tyrosine phosphorylated by Rho-dependent mechanism (40 , 41) , we analyzed the involvement of the Rho pathway in v-Crk-induced phosphorylation of p130Cas and paxillin. After treatment of the v-Crk-inducible 3Y1 cell line with Y27632 for 18 h, we found that Y27632 did not completely suppress, but significantly attenuated v-Crk-induced tyrosine phosphorylation of p130Cas (Fig. 4D)Citation . The 68-kDa band corresponding to paxillin was also under the level of detection after treatment with Y27632 for 18 h.

v-Crk Regulates Cell Motility of 3Y1 Cells.
Because the activation of Rho has been closely related to the regulation of cell motility, we examined the effect of v-Crk on cell motility by three independent assays. In the phagokinetic track assay, we observed that v-Crk significantly decreased cell motility at 48 h after induction, and this was recovered by the treatment of C3 or Y27632 (Fig. 5A)Citation . The suppression of cell motility was also found in the wound-healing assay, and this was also released by the treatment of C3 or Y27632. Furthermore, the integrin stimulation by fibronectin coating also disclosed the suppression of motility (Fig. 5B)Citation . In chemotactic assay, the induction of v-Crk enhanced cell motility with fibronectin stimulation (Fig. 5C)Citation .



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Fig. 5. Analysis of cell motility of v-Crk inducible 3Y1 cell line. A, analysis of cell motility by phagokinetic track assay. 3Y1 cells or v-Crk-inducible 3Y1 cells were plated on gold particles in the presence or the absence of Dox. Photographs showed the moved cells and their tracks at 48 h. Bottom, the graph indicates the averages of the area, which several cells moved on gold particles; bars ± SE. *, value was significantly different from Dox-untreated 3Y1 cells (clone 21-2-1) at P < 0.01, by Student’s t test. B, analysis of cell motility by wound healing assay. The v-Crk-inducible 3Y1 cells (clone 21-2-1) were plated on fibronectin uncoating (left) or coating (right) dish, and cultured without Dox (panels a and b) or with 10 µg/ml of Dox (c and d). Light microscopical analysis of the cells immediately after wound formation at 0 h (a and c) and at 48 h (left, b and d), 36 h (right, b and d) after wound formation. Bottom, the graph indicates the averages of the number of moved cells obtained in three independent experiments; bars, ± SD. Statistical analyses were performed by Student’s t test. **, value was significantly different from Dox-untreated cells at 48 h after wound formation, at P < 0.05. C, chemotactic responses toward fibronectin in v-Crk-inducible 3Y1 cells (clone 21-2-1) were investigated in the absence ({square}) or presence of Dox ({blacksquare}). D, the levels of full-length and cleavage products of CD44 were investigated by anti-CD44cyto Ab. The various cells were cultured on fibronectin uncoating (Lanes 1–5) or coating (Lanes 6–10) dishes. Lanes 1 and 6, 3Y1; Lanes 2 and 7, v-Crk inducible 3Y1 (21-2-1) without Dox; Lanes 3 and 8, v-Crk inducible 3Y1 with Dox; Lanes 4 and 9, v-Crk 3Y1; Lanes 5 and 10, SR-3Y1. Arrow indicates the full-length CD44, and arrowheads indicate the cleavage products of CD44.

 
Because integrin stimulation enhanced cell motility or disclosed the suppression, we examined the levels of CD44 cleavage in v-Crk-induced cells. Correlating with integrin-mediated up-regulation of cell motility, CD44 cleavage was detectable when v-Crk-induced 3Y1 cells were treated with fibronectin (Fig. 5D)Citation . The reduced levels of stress fiber formation were observed in the edge of cells by the fibronectin treatment on v-Crk induction (Fig. 6ACitation , panels f and h). The numbers of focal adhesions including paxillin were also reduced (Fig. 6ACitation , panels j and l). Integrin-stimulation did not alter the levels of v-Crk-induced tyrosine phosphorylation of p130Cas and paxillin (Fig. 6B)Citation , whereas it significantly enhanced v-Crk-induced activation levels of Rho (Fig. 6C)Citation . 3Y1 cells treated with fibronection exhibit the formation of filopodia on the surface and short actin filaments into the cytoplasm (Fig. 6ACitation , panels e and g).



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Fig. 6. Effects of fibronectin on v-Crk-induced actin stress fiber formation, tyrosine phosphorylation, and activation of Rho. A, the effect of v-Crk on the regulation of focal adhesion and actin cytoskeleton with integrin stimulation by fibronectin. The cells were stained by anti-gag Ab (green, a–d), phalloidin (red, e–h), and antipaxillin Ab (green, i–l) in the presence (b, f, and j) or the absence (a, e, and i) of Dox on fibronectin uncoating, and in the presence (d, h, and l) or the absence (c, g, and k) of Dox on fibronectin coating dish. B, the levels of phosphotyrosine, p130Cas, paxillin, v-Crk, and actin in total cell lysates with (right) and without (left) integrin stimulation by fibronectin were examined by immunoblotting. C, pull down assay for active form of Rho in v-Crk inducible cell line with (right) and without (left) integrin stimulation by fibronectin. The cells were cultured with or without Dox.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The integrin connecting to multimolecular machinery recognized as focal adhesion controls reorganization of the cytoskeleton, and plays a pivotal role for cell migration and morphology in response to the ECM. The stimulation of integrin leads to prominent tyrosine phosphorylation of components of focal adhesion such as p130Cas and paxillin, which contain nine and two Crk-SH2 domain-binding motifs, respectively. The tyrosine-phosphorylation levels of p130Cas and paxillin were significantly high in v-Crk-transformed cells, and Crk has been proven to induce these phosphorylations. In this study, to elucidate the mechanisms of Crk-induced tyrosine phosphorylation of p130Cas and paxillin, and corresponding biological effects, we established a conditional expression system of v-Crk using the rat fibroblast cell line 3Y1.

In 3Y1 cells, we found that conditional expression of v-Crk clearly induced actin stress fiber formation within 48 h. v-Crk also enhanced the tyrosine phosphorylation of p130Cas and paxillin, which were localized to the ends of actin stress fibers to generate focal adhesions (data not shown). The association of v-Crk and p130Cas or paxillin in focal adhesions may initiate the reorganization of actin cytoskeleton resulting in stress fiber formation. Because the Rho family of small GTPases is well known to promote actin stress fiber formation, we examined the involvement of Rho in v-Crk-mediated signaling, and directly demonstrated that v-Crk activated Rho by pull down assay. Furthermore, ADP-ribosylase C3, the inhibitor of Rho, was shown to suppress this v-Crk-mediated cytoskeletal response. Considering the reports that v-Crk induced cell flattening by Rho-dependent mechanism in PC12 cells and also that a Crk mutant inhibited growth factor-induced stress fiber formation in rat-1 fibroblasts (42 , 43) , Crk may induce stress fiber formation by a Rho-dependent mechanism in various cells.

Among the downstream effectors of Rho such as ROCK, mDia, Citron kinase, protein kinase N, Rhophilin, and Rhotekin (28, 29, 30 , 44, 45, 46) , we found that ROCK was involved in v-Crk-induced stress fiber formation because Y27632, a specific inhibitor of ROCK, suppressed the effects of v-Crk on cytoskeleton. These findings indicate that Rho/ROCK functions downstream of v-Crk. Despite the identification of several guanine-nucleotide exchange factors for Rho, none of these could bind to v-Crk, and vice versa, no known effector of v-Crk such as C3G, Sos, Dock180, c-Abl, or Eps15 share homology to Rho-guanine-nucleotide exchange factors. Thus, the elucidation of the mechanism of v-Crk-induced activation of Rho needs additional investigation.

Although analysis of the series of knockout cell lines for tyrosine kinases showed the essential role for Fyn in p130Cas phosphorylation (47) , and also in vitro analysis suggested the sequential phosphorylation of 15 tyrosine residues of p130Cas by the v-Crk/c-Abl complex (48) , the precise mechanism of v-Crk-induced tyrosine phosphorylation of p130Cas has not yet been clarified. In this study, ROCK was suggested to be involved in v-Crk-induced phosphorylation of p130Cas, because the treatment of ROCK inhibitor Y27632 reduced its phosphorylation. Recently Rho-dependent tyrosine phosphorylation of p130Cas and paxillin has been reported, and another group suggested that PI3k leads to Rho-dependent tyrosine phosphorylation of p130Cas (49) . Considering the report by Akagi, et al. (22) indicating that v-Crk, activated PI3k through focal adhesion kinase, we are currently examining an involvement of PI3k in v-Crk-dependent activation of Rho resulting in tyrosine phosphorylation of p130Cas.

Because a serine/threonine kinase ROCK has been shown to be involved in v-Crk-induced stress fiber formation, we analyzed the profile of threonine-phosphorylated proteins using antiphosphothreonine (p-Thr) antibody and found that v-Crk-induced positive bands sized at 72 and 78 kDa in 3Y1 cells. Because we did not observe any positive band around 72–78 kDa in v-Src-transformed 3Y1 cells, an unique signaling pathway was suggested to be evoked by Crk. It should be noted that we did not observed these signals in NIH3T3 mouse fibroblasts expressing v-Crk or in chicken embryonal fibroblasts infected with v-Crk encoding retrovirus CT10. Thus, we cannot exclude the possibility that these bands were not essential for v-Crk-induced biological effects in fibroblasts in species other than rat. Although several reports have shown the phosphorylation on serine/threonine residues of Raf-1 and paxillin by integrin stimulation, we could not detect any threonine-phosphorylated forms of Raf-1 and paxillin with the induction of v-Crk (data not shown). We also failed to detect v-Crk-induced threonine phosphorylation of ERM family of proteins (data not shown).

We have investigated the effects of v-Crk on the cell motility by wound healing and phagokinetic track assays, and found that v-Crk decreased the motility at 48 h after v-Crk induction. The continuous activation of Rho by v-Crk seems to form tight assemblies between induced actin stress fibers and focal adhesions, resulting in the decrease of motility. Furthermore, these suppressions of motility were released by integrin stimulation. In chemotactic assay, we observed that v-Crk enhanced cell motility toward fibronectin. Moreover, the integrin stimulation additionally activated Rho in v-Crk-induced 3Y1 cells but reduced the amount of stress fibers and focal adhesions. Because v-Crk-induced Rho activation did not simply amplify the amount of stress fibers, we hypothesized that higher levels of the active form of Rho may lead to dynamic reorganization of actin cytoskeletons, resulting in the enhancement of cell motility.

Correlating to enhanced cell motility, integrin stimulation induced CD44 cleavage in v-Crk-induced 3Y1 cells. v-Crk may regulate the CD44 cleavage by activating the Rho family of proteins leading to enhancement of the transcription levels of matrix metalloproteinase. Considering the report suggesting that Rho suppressed CD44 cleavage and Rac activated it, we could not conclude that only v-Crk-dependent activation of Rho was necessary for enhancement of motility. One of the downstream effectors of Crk such as Dock180-Rac signaling may also be involved in the regulation of v-Crk-mediated regulation of cell motility. Because the requirement of C3G-dependent Rap-1 activation for cell adhesion was demonstrated by using C3G null mouse fibroblasts (50) , regulation of Rap-1 activity may also contribute to the cell motility.

In conclusion, we found that v-Crk activated Rho, and induced Rho/ROCK-dependent formation of actin stress fibers and tyrosine phosphorylation of p130Cas and paxillin. The additional activation of Rho and the cleavage of CD44 were also found in v-Crk-inducible 3Y1 cells with fibronectin stimulation. These biological responses may coordinately regulate the focal adhesions and cell motility. Furthermore, we found that v-Crk induced threonine-phosphorylated proteins specifically in 3Y1 cells. Identification of these proteins might be a key step in elucidating the additional mechanisms of Crk-dependent reorganization of actin cytoskeleton and its biological function.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Antibodies.
Rat fibroblasts 3Y1 cells (JCRB0734) were maintained in DMEM containing 10% of Tet system-approved fetal bovine serum (Clontech; complete medium) in a humidified atmosphere of 5% CO2 at 37°C. The SR-3Y1 cell line was established in the Hanafusa laboratory at The Rockefeller University, New York, NY. The mAb against viral gag protein fused to v-Crk (mAb 3C2) has been described (51) . The Abs for phosphotyrosine (PY20 and RC20H), p130Cas, and paxillin were purchased from Transduction Laboratories (Lexington, KY); phosphothreonine was from Cell Signalling Technology (Beverly, MA); RhoA (sc-179) was from Santa Cruz Biotechnology (Santa Cruz, CA); actin for immunoblotting was from Chemicon International (Temecula, CA). The phalloidin-594 for actin staining was purchased from Molecular Probes (Eugene, OR). Anti-CD44cyto antibody was a generous gift from Hideyuki Saya (Kumamoto University, Kumamoto, Japan).

Plasmids.
For establishment of the tetracycline-inducible system, we purchased the Tet-on kit from Clontech including pTet-on for the regulatory element of rtTA, pTRE, for expression of target protein, and pTK-hygro for drug selection. v-Crk was amplified from pCT10 (6) by PCR and after sequencing it was subcloned into Notl/Xbal sites of the pTRE vector, which was driven by the rtTA-responsible promoter. pGEX-rhotekin-RBD was generous gift from Toshinori Iwahara (OBI, Osaka, Japan).

Transfection of DNAs and Establishment of v-Crkinducible 3Y1 Cells.
3Y1 rat fibroblasts were cultured on 10-cm-diameter dishes with DMEM containing 10% fetal bovine serum, and at 70% cell confluence, 1 µg of pTet-on plasmid was transfected into the cells using Effectene transfection reagent (Qiagen). Forty-eight hours after transfection, cells were treated with 400 µg/ml of G418 (Calbiochem), and drug-resistant colonies were isolated by cloning cylinders. In isolated colonies, activities of rtTA were examined by luciferase assay, and the clones expressing functional rtTA were additionally cotransfected with 1 µg of pTRE-v-Crk and pTK-hygro, and were treated with 200 µg/ml of hygromycin (Calbiochem). The drug-resistant colonies were isolated and examined for induction levels of v-Crk in the presence of Dox (2 and 10 µg/ml).

Immunoblotting.
Cells were lysed with buffer composed of 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (Sigma), and 1 mM sodium orthovanadate (Na3 VO4) for 10 min on ice, and were centrifuged at 15,000 rpm for 10 min at 4°C. Supernatants were subjected to SDS-PAGE, and separated proteins were analyzed by immunoblotting using polyvinylidene difluoride filter (Immobilon, Millipore) as described elsewhere (52) . Signals were detected by enhanced chemiluminescence Western blotting reagents (Amersham) and quantified using a Lumino Image Analyzer (LAS1000; Fuji Film, Tokyo, Japan).

For detection of CD44 cleavage products, cultured cells were incubated with 10 µM MG132 for 14 h, lysed with SDS-PAGE sample buffer containing 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.1% 2-ME, and 0.02% bromophenol blue, and boiled for 10 min. Lysates were then subjected to immunoblotting using anti-CD44cyto Ab.

Confocal Laser Scanning Microscopical Study.
For analysis of the subcellular localization of v-Crk, cells (1 x 104/chamber) on eight-chamber slides (Nunc, Napervilole, IL) were fixed with 3% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 4 min at room temperature, and then refixed with 70% methanol for 5 min at -20°C. The cells were washed and incubated with 1% goat serum to block the nonspecific binding of Abs. Subsequently, the cells were incubated with anti-gag mAb, 3C2 (1:50 dilution) at 4°C overnight. The cells were additionally incubated with Alexa 488-conjugated goat antimouse immunoglobulin antibody (1:200 dilution; Molecular Probes) for 1 h at room temperature in light shielding. Control cells were stained only with Alexa 488-labeled secondary Ab. Finally, for the staining of actin, the cells were exposed to 1 unit of phalloidin-594 for 40 min at room temperature in light shielding. The samples were observed using a confocal laser-scanning microscope (MRC-1024; Bio-Rad Microscience Division, Watford, United Kingdom) equipped with a computer.

Rho Pull Down 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% NP40, 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 5000 x g at 4°C for 5 min, and the supernatants were incubated with 10 µg of glutathione S-transferase-rhotekin Rho binding domain fusion protein preconjugated with glutathione beads at 4°C for 1 h. The beads were washed three times with lysis buffer and subjected to SDS-PAGE with a 12% gel. Bound RhoA was detected by immunoblotting using anti-RhoA Ab.

Wound Healing Assay and Phagokinetic Track Assay.
The methods for the wound healing assay and the phagokinetic track assay were described previously (53 , 54) . Briefly, for the wound healing assay, after the initial plating of cells for 48 h on uncoating or 10 µg/ml fibronectin-coating culture dishes, cells were scraped off/wounded using a yellow tip. Subsequently at 12, 24, 36, and 48 h, the numbers of moved cells from the base line were measured. For the phagokinetic track assay, 1 x 103 cells were inoculated on the 24 x 24-mm cover slides coated with 1% BSA and gold particles in the absence or presence of Dox, Y27632, and C3. After 12 h, the areas that the cells moved were measured using image gauge analyzer (Fuji Film).

Chemotactic Assay toward Fibronectin.
DMEM containing 50 µg/ml fibronectin or 0.1% BSA as control for measuring random motility was poured into the lower chamber of Transwell cell culture chamber (Costar, Cambridge, MA). Subsequently, 2 x 104 cells suspending in DMEM containing 0.1% BSA were inoculated into the upper chamber of Transwell. The cells were incubated for 6 h in humidified atmosphere of 5% CO2 at 37°C. After 6 h, the cells were fixed with 5% glutaraldehyde-containing PBS for 30 min, and stained with Giemsa solution for 20 min. The cells on the filter membrane were wiped out; only the cells of inside or under the filter were counted.


    Acknowledgments
 
We thank Michiyuki Matsuda (Osaka University, Osaka, Japan), Tsuyoshi Akagi (OBI, Osaka, Japan), and Takaharu Yamamoto (Hokkaido University, Sapporo, Japan) for useful discussions; Toshinori Iwahara (OBI, Japan) for plasmids; Hideyuki Saya (Kumamoto University, Kumamoto, Japan) for antibody; William W. Hall (University College Dublin, Dublin, Ireland) for critical reading of the manuscript; and Sumie Oikawa and Misato Yamada for technical assistance.


    Footnotes
 
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.

1 Supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N 15, W 7, Kita-ku, Sapporo, 060-8638, Japan. Phone: 81-11-706-7808; Fax: 81-11-706-7808; E-mail: sitanaka{at}patho2.med.hokudai.ac.jp. Back

3 The abbreviations used are: ECM, extracellular matrix; GTPase, guanosine triphosphatase; ROCK, Rho-associated kinase; PI3k, phosphatidylinositol 3'-kinase; Dox, doxycycline; ERM, ezrin, radixin, and moesin; mAb, monoclonal antibody; Ab, antibody. Back

Received for publication 8/21/01. Revision received 12/27/01. Accepted for publication 2/12/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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