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Endocrinology Vol. 138, No. 11 4950-4958
Copyright © 1997 by The Endocrine Society

ARTICLES

Evidence for Functional Roles of Crk-II in Insulin and Epidermal Growth Factor Signaling in Rat-1 Fibroblasts Overexpressing Insulin Receptors1

Manabu Ishiki, Toshiyasu Sasaoka, Hajime Ishihara, Takeshi Imamura, Isao Usui, Yasumitsu Takata and Masashi Kobayashi

First Department of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930–01, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., First Department of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930–01, Japan. E-mail: tsasaoka-tym{at}umin.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CRK PROTEIN is an Src homology 2 (SH2) and SH3 domain-containing protein, originally isolated as an oncogene product encoded by the avian sarcoma virus CT10 (1). Three cellular homologs of the viral oncogene v-crk have been identified. Crk-II is a 40-kDa protein consisting of one SH2 and two SH3 domains (2). Crk-I is a 21-kDa protein that appears to be an alternatively spliced form of Crk-II and possesses one SH2 and one SH3 domain, missing the C-terminal SH3 domain of Crk-II (3). Crk-L is a 36-kDa protein with one SH2 and two SH3 domains that shares about 60% homology with Crk-II (4, 5). The Crk SH3 domain has been shown to interact with C3G (6, 7), Sos (8, 9), and c-abl (10). As C3G and Sos have guanine nucleotide exchange activity, Crk might affect the p21ras signaling pathway. In addition, the abl oncogene product is a nonreceptor tyrosine kinase, and c-abl has been shown to regulate mitogenesis via its kinase activity toward Crk protein (10). On the other hand, the Crk SH2 domain has been reported to associate with paxillin (11), p130cas (12), and c-cbl (13, 14). As both paxillin and p130cas have been implicated in integrin-mediated signal transduction as well as mitogenic signal transduction (15) and are thought to be substrates of the focal adhesion kinase p125FAK (16, 17), Crk may be involved in cytoskeleton organization, such as actin fiber rearrangement and membrane ruffling. Moreover, as c-cbl is a protooncogene product (18, 19), the Crk-c-cbl complex may play a role in promoting cell cycle progression. Along this line, previous studies have shown that overexpression of the oncogenic form of Crk results in cell transformation and tyrosine phosphorylation of various cellular proteins in chicken embryo fibroblast cells (20), and that overexpression of v-Crk induces cellular differentiation upon EGF and nerve growth factor stimulation in PC12 cells (21). Although these studies have indicated that v-Crk plays a role in signal transduction mediated by tyrosine kinases, little is known about the biological roles of endogenous Crk. As these endogenous Crk proteins lack apparent catalytic domains, their function probably lies in their ability to bind specific proteins via their SH2 and SH3 domains.

Among endogenous Crk members, Rat-1 fibroblasts were found to express Crk-II and Crk-L, but not Crk-I, by Western blot analysis. In the present study, to evaluate the potential role of Crk-II in insulin and EGF signaling, we examined Crk-II interactions with these signal transducing molecules upon insulin and EGF stimulation in Rat-1 fibroblasts overexpressing insulin receptors (HIRc). Furthermore, the functional involvement of Crk-II in insulin- and EGF-induced DNA synthesis and cytoskeletal rearrangement was directly assessed by single cell microinjection analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines
Rat-1 fibroblasts expressing 1 x 106 human insulin receptors/cell (HIRc) were provided by Dr. J. M. Olefsky (University of California-San Diego, CA) and were maintained in DMEM-Ham’s F-12 medium containing 10% FCS as previously described (22). As Rat-1 fibroblasts express 1 x 105 endogenous EGF receptors/cell, HIRc cells are sensitive to insulin and EGF.

Materials
Porcine insulin was a gift from Shimizu Pharmaceutical Co. (Shizuoka, Japan). EGF was purchased from Life Technologies (Grand Island, NY). Electrophoresis reagents were obtained from Bio-Rad (Hercules, CA). Bromodeoxyuridine (BrdU), an anti-BrdU antibody, and enhanced chemiluminescence reagents were obtained from Amersham Corp. (Arlington Heights, IL). A polyclonal anti-C3G antibody was provided by Dr. Hidesaburo Hanafusa (Rockefeller University, New York, NY). A monoclonal anti-Crk antibody, a monoclonal antipaxillin antibody, a monoclonal anti-p130cas antibody, a monoclonal anti-Sos antibody, and monoclonal and polyclonal antiphosphotyrosine antibodies (pY20) were purchased from Transduction Laboratories (Lexington, KY). A polyclonal anti-Crk-II antibody, a polyclonal anti-Crk-L antibody, a polyclonal anti-c-abl antibody, a polyclonal anti-c-cbl antibody, and a polyclonal anti-Sos antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IgG and fluorescein isothiocyanate (FITC)- or rhodamine-conjugated antirat and antimouse IgG antibodies were obtained from Jackson Laboratories (West Grove, NY). Carboxytetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin, deoxyribonuclease I, and other routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Western blotting studies
Cell monolayers were starved for 24 h in serum-free DMEM. The cells were then treated with 17 nM insulin or 160 nM EGF for the indicated times at 37 C. Cells were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM Na3VO4, pH 7.4. The cell lysates were centrifuged to remove insoluble materials. The supernatants were used for immunoprecipitation with the indicated antibodies for 3 h at 4 C. The precipitates were separated by SDS-PAGE and transferred to Immobilon-P using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween-20, and 2.5% BSA, pH 7.5, for 2 h at 20 C. The membranes were then probed with the specified antibodies for 2 h at 20 C. After washing the membranes in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.5, blots were incubated with horseradish peroxidase-linked secondary antibody followed by enhanced chemiluminescence detection using the ECL reagent according to the manufacturer’s instructions (Amersham Corp.) (23, 24).

Microinjection
Cells were grown on glass coverslips and rendered quiescent by starvation for 24 h in serum-free DMEM. Antibodies were solubilized in microinjection buffer consisting of 5 mM NaPO4 and 100 mM KCl, pH 7.4, and then microinjected using glass capillary needles. Approximately 1 x 10-14 liters buffer were introduced into each cell. The injection included about 1 x 106 molecules of IgG. Two hundred and fifty to 300 cells/coverslip were injected (25). Immunofluorescent staining of the injected cells, as described below, indicated that about 75% of the cells were successfully microinjected.

BrdU incorporation
Two hours after microinjection, cells were incubated with BrdU plus vehicle, 1.7 or 17 nM insulin, 160 nM EGF, or 10% FCS for 16 h at 37 C. The cells were fixed with 3% formaldehyde in PBS for 20 min at 22 C. The fixed cells were permeabilized with 0.5% Nonidet P-40 in PBS and blocked with a solution containing 5% BSA and 0.5% Nonidet P-40 in PBS. The cells were incubated with rat polyclonal anti-BrdU antibody in a buffer containing 10 mM MgCl2 and deoxyribonuclease I for 1 h at 22 C. The cells were then stained by incubation with rhodamine-labeled donkey antirat IgG antibody and FITC-labeled donkey antimouse IgG antibody for 1 h at 22 C (25). After the coverslips were mounted, the cells were analyzed with a Microphot-FXA fluorescence microscope (Nikon, Tokyo, Japan).

Fluorescent labeling of actin filaments
Two hours after microinjection, cells were incubated with 17 nM insulin for the indicated times, then fixed with 3% formaldehyde in PBS for 20 min at 22 C. The fixed cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 3 min at 22 C. After rinsing twice with PBS, the cells were blocked with a solution containing 0.1% BSA in PBS for 15 min at 20 C. Immunofluorescent labeling was carried out by incubation with TRITC-conjugated phalloidin and FITC-labeled donkey antimouse IgG antibody for 1 h at 22 C (26). After the coverslips were mounted, the cells were analyzed with the Microphot-FXA fluorescence microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Crk association with C3G and Sos
Previous investigations indicated that Crk associates with both C3G and Sos in PC12 cells (9). As both C3G and Sos have guanine nucleotide exchange activities (6), Crk interaction with C3G and/or Sos might mediate mitogenic signaling by insulin and EGF. To address this issue, insulin- and EGF-stimulated Crk-II association with C3G and Sos was examined in HIRc cells by immunoblotting anti-Crk immunoprecipitates with anti-C3G or anti-Sos antibody. The anti-Crk antibody used immunoprecipitated about 95% of the total cellular Crk-II in Rat 1 fibroblasts (data not shown). In contrast to the findings in PC12 cells, no apparent Crk-II association with either C3G or Sos was detected in either basal or stimulated states. To confirm this finding, we first immunoprecipitated with anti-Sos or anti-C3G antibody, and the precipitates were immunoblotted with anti-Crk-II antibody. The results again demonstrated no apparent Crk-II association with either C3G or Sos (data not shown).

Electrophoretic mobility shift of C3G and Sos
Serine-threonine phosphorylation causes decreased electrophoretic mobility of Sos on SDS-PAGE (27). In accordance with a previous report (27), insulin and EGF stimulation induced a Sos mobility shift in a time-dependent fashion in HIRc cells (data not shown). If C3G is also involved in insulin and EGF signaling in HIRc cells, serine-threonine phosphorylation of C3G might be seen. To address this possibility, we examined the electrophoretic mobility shift of C3G after insulin and EGF stimulation. In contrast to the results with Sos, neither insulin nor EGF stimulation induced an electrophoretic mobility shift of C3G in HIRc cells (data not shown).

c-abl phosphorylation and association with Crk
c-abl is a cellular homolog of the v-abl oncogene product (28). c-abl has been reported to associate with the Crk SH3 domain in vitro (10). In HIRc cells, c-abl was tyrosine phosphorylated in the basal state, and treatment with either insulin or EGF did not affect the tyrosine phosphorylation state of c-abl (data not shown). Consistent with the basal tyrosine phosphorylation, a small amount of c-abl was associated with Crk-II in the basal state. Insulin and EGF did not change the association of c-abl with Crk-II (data not shown).

p130cas dephosphorylation and dissociation from Crk
It has been reported that Crk-associated substrate (p130cas) participates in cytoskeleton signaling in addition to mitogenic signaling (29, 30, 31, 32). When v-Src or v-Crk is overexpressed (29, 30, 31) or integrin is bound to extracellular matrix ligands (29, 30), p130cas becomes tyrosine phosphorylated and forms a complex with v-Crk. As p130cas was not efficiently immunoprecipitated with anti-p130cas antibody, we examined tyrosine phosphorylation of p130cas by immunoprecipitation with antiphosphotyrosine antibody and immunoblotting with anti-p130cas antibody. In the basal state, p130cas was tyrosine phosphorylated in HIRc cells. Both insulin and EGF treatment induced tyrosine dephosphorylation of p130cas. As shown in Fig. 1Go, A and C, dephosphorylation of p130cas occurred at 1 min and persisted through 20 min of insulin or EGF stimulation, although insulin treatment led to greater dephosphorylation of p130cas than EGF treatment. p130cas was complexed with Crk-II in the basal state and dissociated from Crk-II after both insulin and EGF stimulation (Fig. 1BGo). The time course of p130cas dissociation from Crk-II correlated with the kinetics of p130cas dephosphorylation, as shown in Fig. 1DGo.



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  		Figure 1. Tyrosine dephosphorylation of p130cas and its dissociation from Crk. Serum-starved cells were treated with 17 nM insulin or 160 nM EGF for the indicated times. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody (A) or anti-Crk antibody (B). The immunoprecipitates were analyzed by immunoblotting with anti-p130cas antibody. Representative results are shown. The molecular mass of p130cas (130 kDa) is shown by an arrow. The amount of p130cas dephosphorylation (C) and p130cas dissociation from Crk (D) were quantitated by densitometry. Results are the mean ± SE of three separate experiments.

 
Paxillin dephosphorylation and association with Crk
Paxillin is involved in focal adhesion signaling and becomes tyrosine phosphorylated when stimulated by growth factors such as platelet-derived growth factor (33, 34). To examine tyrosine phosphorylation of paxillin, cell lysates were immunoprecipitated with antiphosphotyrosine antibody, and the precipitates were immunoblotted with antipaxillin antibody. In HIRc cells, paxillin was tyrosine phosphorylated in the basal state. Both insulin and EGF treatment induced tyrosine dephosphorylation of paxillin in a time-dependent manner, as shown in Fig. 2AGo. These results are quantitated in Fig. 2CGo. Insulin induced faster and greater dephosphorylation of paxillin than EGF. Insulin- and EGF-induced tyrosine dephosphorylation of paxillin was also confirmed by immunoblotting antipaxillin precipitates with antiphosphotyrosine antibody (data not shown). In contrast to the relationship between p130cas dephosphorylation and p130cas dissociation from Crk-II, as shown in Fig. 1Go, both insulin and EGF stimulated paxillin association with Crk-II in a time-dependent fashion (Fig. 2BGo). Insulin stimulation induced greater association of paxillin with Crk-II than EGF, as shown in Fig. 2DGo. The identity of the 68-kDa band as paxillin was confirmed by immunoblotting with antipaxillin antibody.



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  		Figure 2. Tyrosine dephosphorylation of paxillin and its association with Crk. Serum-starved cells were treated with 17 nM insulin or 160 nM EGF for the indicated times. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody (A) or anti-Crk antibody (B). The immunoprecipitates or the supernatants were analyzed by immunoblotting with antipaxillin antibody. Representative results are shown. The molecular mass of paxillin (68 kDa) is shown by an arrow. The amount of paxillin dephosphorylation (C) and paxillin association with Crk (D) were quantitated by densitometry. Results are the mean ± SE of four separate experiments.

 
c-cbl phosphorylation and association with Crk
The c-cbl protooncogene product (c-cbl) was identified as the cellular homolog of the viral transforming protein of the murine Cas NS-1 retrovirus that caused pre-B cell lymphomas and myeloid leukemias (18, 35). Thus, previous studies revealed that c-cbl was involved in hematopoietic cell signaling (18, 35). To examine the involvement of c-cbl in insulin and EGF signaling, we investigated insulin- and EGF-stimulated tyrosine phosphorylation of c-cbl in HIRc cells. After insulin or EGF stimulation, cell lysates were first immunoprecipitated with antiphosphotyrosine antibody, and the precipitates were immunoblotted with anti-c-cbl antibody. This approach was used because of the low precipitation efficiency of the anti-c-cbl antibody. EGF stimulated tyrosine phosphorylation of c-cbl in HIRc cells. After EGF stimulation, maximal phosphorylation was observed at 1 min and gradually declined thereafter. In contrast, insulin treatment did not stimulate any apparent tyrosine phosphorylation of c-cbl, as shown in Fig. 3AGo. In accordance with the kinetics of tyrosine phosphorylation of c-cbl, EGF treatment led to an increased association of Crk-II with c-cbl. In contrast, we could not detect any significant association of Crk-II with c-cbl after insulin stimulation (Fig. 3BGo).



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  		Figure 3. Tyrosine phosphorylation of c-cbl and its association with Crk. Serum-starved cells were treated with 17 nM insulin or 160 nM EGF for the indicated times. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody (A) or anti-Crk antibody (B). The immunoprecipitates were analyzed by immunoblotting with anti-c-cbl antibody. The molecular mass of c-cbl (120 kDa) is shown by an arrow. Results are representative of three separate experiments.

 
Effect of microinjection of anti-Crk antibody on DNA synthesis
The functional importance of Crk-II on insulin- and EGF-induced DNA synthesis was assessed by microinjection studies (Fig. 4Go). BrdU incorporation was monitored to study the importance of Crk-II. Comparison of BrdU incorporation between injected (5.1 ± 0.6%) and uninjected (5.8 ± 0.3%) quiescent cells indicated that the injection of anti-Crk antibody had no effect on the basal rate of BrdU incorporation. In addition, the anti-Crk antibody was not toxic, as the stimulatory effect of serum was not inhibited by microinjection of the anti-Crk antibody (data not shown). Insulin (1.7 and 17 nM) and EGF (160 nM) stimulated BrdU incorporation in 41.5 ± 2.2%, 59.0 ± 2.5%, and 39.8 ± 1.6% of total cells, respectively. Microinjection of control IgG did not show any inhibitory effect on either insulin- or EGF-induced DNA synthesis. In addition, microinjection of anti-Crk antibody had no inhibitory effect on insulin stimulation of BrdU incorporation. In contrast, the injection effectively inhibited EGF-induced DNA synthesis by 59.7 ± 2.9% of the control value. The IGF-I receptor has functional similarities compared with the insulin receptor (36). Rat-1 fibroblasts have 1.3 x 105 IGF-I receptors/cell (36), and 13 nM IGF-I stimulated BrdU incorporation in 49.0 ± 1.2% of the total cells. Therefore, the effect of the anti-Crk antibody microinjection on IGF-I-induced BrdU incorporation was also examined. As with insulin, microinjection of the anti-Crk antibody did not affect IGF-I-stimulated BrdU incorporation (data not shown).



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  		Figure 4. Inhibition of DNA synthesis by microinjection of anti-Crk antibody. Serum-starved cells were microinjected with 4 mg/ml anti-Crk antibody or control preimmune IgG. After stabilization for 2 h, cells were stimulated with the indicated concentrations of insulin or EGF for 16 h at 37 C. BrdU incorporation into anti-Crk antibody-injected cells (hatched bars), preimmune IgG-injected cells (solid bars), and uninjected cells (open bars) on the same coverslip was determined as described in Materials and Methods. Results are expressed as a percentage of the total cells and are the mean ± SE of four separate experiments.

 
Effect of microinjection of anti-Crk antibody on cytoskeletal reorganization
It has been shown that insulin promotes membrane ruffling and actin fiber rearrangement (37, 38, 39, 40, 41). We studied the role of Crk-II in insulin-induced membrane ruffling using microinjection analysis. Membrane ruffling was visualized by immunofluorescence microscopy using TRITC-labeled phalloidin, and injected cells were identified by FITC-labeled antimouse IgG antibody. As shown in Fig. 5Go, A and B, insulin stimulated membrane ruffling and actin fiber breakdown. Microinjection of control preimmune IgG did not inhibit insulin stimulation of membrane ruffling (Fig. 5Go, C and D). In contrast, microinjection of anti-Crk antibody clearly inhibited insulin stimulation of membrane ruffling (Fig. 5Go, E and F), indicating an important role of Crk-II in insulin-stimulated cytoskeletal reorganization. The data for membrane ruffling are summarized in Fig. 6Go. In the basal state, 1.7 ± 0.2% of the cells revealed membrane ruffling, and microinjection of anti-Crk antibody did not affect membrane ruffling basally. After insulin stimulation, 83.1 ± 2.4% of the cells revealed membrane ruffling. Microinjection of anti-Crk antibody, but not control IgG, inhibited insulin-stimulated membrane ruffling by 84.9 ± 2.8%. We also studied membrane ruffling after EGF stimulation. However, EGF stimulation did not induce membrane ruffling clearly enough to be detected by immunofluorescence staining in HIRc cells (data not shown).



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  		Figure 5. Effect of microinjection of anti-Crk antibody on insulin-induced cytoskeletal rearrangement in HIRc cells. Serum-starved cells were stimulated without (A) or with (B) 17 nM insulin for 5 min. For microinjection analysis, serum-starved cells were microinjected with 4 mg/ml anti-Crk antibody or control preimmune IgG. After stabilization for 2 h, cells were treated with 17 nM insulin for 5 min at 37 C (C–F). The effect on membrane ruffling in injected cells (E and F) and control preimmune IgG-injected cells (C and D) was determined as described in Materials and Methods. Resulting cytoskeletal changes were visualized by staining actin filaments with TRITC-conjugated phalloidin (A, B, D, and F), and identification of injected cells was performed by staining injected IgG with FITC-conjugated anti-IgG (C and E).

 


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  		Figure 6. Inhibition of membrane ruffling by microinjection of anti-Crk antibody. Serum-starved cells were microinjected with anti-Crk antibody (hatched bars) or preimmune control IgG (open bars). After stabilization for 2 h, cells were treated without or with 17 nM insulin for 5 min at 37 C. The cells were fixed and stained with TRITC-conjugated phalloidin and scored for the membrane ruffles as described in Materials and Methods. Results are expressed as a percentage of the total cells and are the mean ± SE of four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been demonstrated that p21ras is an important molecule for insulin and EGF stimulation of mitogenesis (42, 43). p21ras becomes active as a signaling molecule when it is converted from the GDP-bound to the GTP-bound form (44). Insulin and EGF increase p21ras-GTP formation primarily by stimulation of guanine nucleotide exchange factor (GEF) activity (43, 45, 46). As Crk, via its SH3 domain, interacts with two p21ras GEFs, namely C3G and Sos (7, 9), one can speculate that Crk-II may activate the p21ras pathway. However, this was not the case with insulin-induced DNA synthesis in Rat-1 fibroblasts. Our studies showed that neither C3G nor Sos was detected in anti-Crk immunoprecipitates, and that insulin induced an electrophoretic mobility shift in Sos, but not C3G. Crk has also been shown to interact with two transformation-related proteins, c-cbl (13, 47) and c-abl (10). Therefore, Crk-II may transmit mitogenic signaling via these proteins. However, our results revealed that insulin did not induce either tyrosine phosphorylation of c-cbl or its association with Crk-II. Furthermore, basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin stimulation (data not shown). Taken together with the fact that microinjection of anti-Crk antibody did not inhibit insulin-induced BrdU incorporation, Crk-II does not appear to play an important role in insulin-stimulated cell cycle progression in Rat-1 fibroblasts. Like insulin, EGF also did not elicit any apparent association of Crk-II with either C3G or Sos and did not change the constitutively bound Crk-II-c-abl complex (data not shown). In contrast to that stimulated by insulin, EGF-stimulated DNA synthesis was inhibited by microinjection of the anti-Crk antibody, indicating that Crk-II plays a key role in the stimulation of mitogenesis by EGF, but not insulin. The exact mechanism by which EGF, but not insulin, stimulates mitogenic signaling through Crk-II is not clear. However, our results demonstrated that EGF, but not insulin, induced tyrosine phosphorylation of c-cbl and its association with Crk-II. The c-cbl protein has initially been identified as a major target for antigen receptor-induced tyrosine phosphorylation in both T and B cells (48, 49, 50). Recently, EGF treatment has been reported to induce tyrosine phosphorylation of c-cbl and its association with EGF receptors (51, 52, 53, 54). In addition, c-cbl associates with Grb2, which is known to interact with Sos (47, 55). Taken together, Crk-II may function to translocate or redistribute the Grb2-Sos complex via c-cbl to facilitate stimulation of membrane-bound p21ras. Alternatively, by interacting with Crk-II, c-cbl might affect the function of Sos associated with it, leading to EGF-induced mitogenesis. Consistent with this hypothesis, microinjection of the Crk protein into PC12 cells induces neurite outgrowth of the cells in a p21ras-dependent manner, suggesting that the Crk functions to regulate p21ras GEF (56). However, our results do not exclude the possibility that a small amount of Crk-II could interact with either C3G or Sos, which might affect their GEF activity, but that we failed to detect its interaction because the association was unstable or the interaction was below the limit of detection by Western blot analysis.

c-abl is a cellular homolog of the v-abl oncogene product (28). Although c-abl overexpression does not lead to cellular transformation, the chromosomal translocation of c-abl to the bcr gene in Philadelphia chromosome-positive human leukemia produces chimeric Bcr-Abl proteins (57), suggesting a role for c-abl in mitogenic signaling. Recently, it has been suggested that the normal function of c-abl is to regulate cell growth in a negative fashion, as cells that overexpress c-abl underwent growth arrest, and dominant-negative c-abl enhanced fibroblast transformation (58). There is a tyrosine phosphorylation site at residue 221 between the two SH3 domains of Crk-II that is phosphorylated by c-abl tyrosine kinase (10). Intramolecular interaction of the Crk SH2 domain with phosphorylated Tyr221 is suggested to be a regulatory mechanism by c-abl. Along this line, v-Crk and Crk-I, which lack this tyrosine phosphorylation site, lead to cell transformation when overexpressed (2, 20). However, basal tyrosine phosphorylation of c-abl and the amount of basal c-abl-Crk-II complex were not affected by either insulin or EGF stimulation in HIRc cells (data not shown). These results suggest that c-abl is not involved in Crk-II-related insulin and EGF signal transduction in Rat-1 fibroblasts.

Paxillin was originally identified as a factor involved in focal adhesion contacts, where the actin cytoskeleton is linked to the extracellular matrix (59, 60). In addition, paxillin is known to be a substrate of p125FAK, which plays a key role in cytoskeletal reorganization (16, 61). Therefore, paxillin has been suggested to be involved in cytoskeletal rearrangement, such as actin stress fiber breakdown and membrane ruffling (62). p130cas was identified as a tyrosine-phosphorylated protein in fibroblasts transformed by v-Crk as well as the nonreceptor tyrosine kinase v-Src (29, 30, 31). Although it is devoid of kinase activity, p130cas has an SH3 domain and clusters of tyrosine phosphorylation sites (12). In addition to the role in cell transformation, p130cas is located at the region of focal adhesions and associates with p125FAK both in vivo and in vitro (63). Several studies have shown adhesion-induced tyrosine phosphorylation of p130cas, suggesting that p130cas plays a role in cytoskeletal rearrangement (29, 32, 64). Our results revealed that microinjection of anti-Crk antibody effectively inhibited membrane ruffling. The results indicate that Crk-II plays an important role in insulin-induced cytoskeletal rearrangement in Rat-1 fibroblasts. Although the exact mechanism by which Crk-II contributes to insulin-induced cytoskeletal rearrangement is unknown, Crk-II interaction with paxillin and/or p130cas may play a role.

Our results showed that insulin stimulated tyrosine dephosphorylation of paxillin and p130cas. It has been reported that insulin induced tyrosine dephosphorylation of p125FAK in Rat-1 fibroblasts and Chinese hamster ovary cells, respectively (26, 65). As the extent of tyrosine phosphorylation of p125FAK is closely related to the phosphorylation state of paxillin and p130cas, our results are consistent with these previous reports (59, 63, 66). Like insulin, EGF also induced tyrosine dephosphorylation of both paxillin and p130cas. Correlating with p130cas dephosphorylation, p130cas dissociated from Crk-II upon both insulin and EGF stimulation, whereas both insulin and EGF induced paxillin association with Crk-II in a time-dependent manner. Although it is possible to speculate that the extent of tyrosine phosphorylation of p130cas and paxillin is not always correlated with association with Crk-II, these results are in contrast to those of previous studies showing that p125FAK-dependent tyrosine phosphorylation of paxillin creates binding sites for Crk (61). As paxillin association with Crk appears to depend on the interaction of the SH2 domain of Crk with the tyrosine-phosphorylated motifs of paxillin (11), our results shown in Fig. 2Go are somewhat surprising. Therefore, to confirm the findings presented in Fig. 2Go, we first immunoprecipitated the cell lysates with antipaxillin antibody and subsequently immunoblotted with anti-Crk-II antibody. Again, we found the Crk-II association with paxillin after insulin and EGF stimulation (data not shown). Although the reason for the differences between our results and those of the previous report remains to be elucidated, they may be due to tissue variation in signal transduction. Along this line, it has been reported that in the basal state, paxillin and p130cas were already heavily tyrosine phosphorylated in Chinese hamster ovary cells (67), whereas only mild phosphorylation of paxillin and p130cas was seen in PC12 cells (21, 68). Alternatively, it may due to the specificities of different Crk isoforms, as previous findings were mainly based on the results of v-Crk (11). The binding affinity of c-Crk to tyrosine-phosphorylated cellular proteins such as EGF receptors was much lower than that of v-Crk despite the fact that both proteins contain identical SH2 domains (69).

We have not detected either apparent actin fiber rearrangement or membrane ruffling upon EGF stimulation. It is uncertain why cytoskeletal rearrangement was induced by insulin but not by EGF in Rat-1 fibroblasts. There are several possibilities that may explain the phenomenon. First, it may simply reflect decreased numbers of receptors for EGF compared with insulin in HIRc cells. Along this line, it has been reported that EGF induced membrane ruffling in Swiss 3T3 and KB cells, which have abundant EGF receptors (37, 40). Second, our results showed that the extent of dephosphorylation of paxillin and p130cas and their interaction with Crk-II were only mildly affected by EGF stimulation compared with insulin stimulation. The quantitative differences in these interactions between insulin and EGF may explain the different signal specificity for cytoskeletal rearrangement in Rat-1 fibroblasts. Third, EGF-induced, but not insulin-induced, tyrosine phosphorylation of c-cbl and its association with Crk-II may also affect the specific signal transduction leading to cytoskeletal rearrangement, although the precise mechanism is unknown. Clarification of the involvement of Crk-II in EGF-induced membrane ruffling requires investigation with cell lines in which EGF can induce membrane ruffling. Therefore, we studied the effect of Crk-II on EGF-induced membrane ruffling using Swiss 3T3 cells. Microinjection of anti-Crk antibody inhibited EGF-induced membrane ruffling by 81.3 ± 1.3% in Swiss 3T3 cells (data not shown). This finding indicates that Crk also plays an important role in EGF-induced membrane ruffling.

In summary, the qualitative characteristics of Crk-II association with C3G, Sos, c-abl, p130cas, and paxillin were similar for both insulin and EGF signaling, although the extent of dephosphorylation of paxillin and p130cas on their Crk-II interaction was more affected by insulin than by EGF stimulation. EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. Furthermore, Crk-II was found to play an important role in insulin-induced cytoskeletal rearrangement. In contrast, Crk-II is involved in EGF-induced, but not insulin-induced, cell cycle progression. These results suggest that Crk-II acts as a multifunctional adaptor molecule that links tyrosine kinase receptors to downstream effector pathways of insulin and EGF signaling.


   Acknowledgments
 
We thank Dr. W. John Langlois (University of Toronto, Toronto, Canada) for his helpful comments and discussion, and Dr. Hidesaburo Hanafusa (Rockefeller University, New York, NY) for the anti-C3G antibody.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, and Culture (to T.S.), a grant from the Japan Diabetes Foundation (to T.S.), and a Grant-in-Aid from the Ministry of Education, Science, and Culture (to M.K.). Back

Received March 31, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mayer BJ, Hamaguchi M, Hanafusa H 1988 A novel viral oncogene with structural similarity to phospholipase C. Nature 332:272–275[CrossRef][Medline]
  2. Matsuda M, Tanaka S, Nagata S, Kojima A, Kurata T, Shibuya M 1992 Two species of human CRK cDNA encode proteins with distinct biological activities. Mol Cell Biol 12:3482–3489[Abstract/Free Full Text]
  3. Reichman CT, Mayer BJ, Keshav S, Hanafusa H 1992 The product of the cellular crk gene consists primarily of SH2 and SH3 regions. Cell Growth Differ 3:451–460[Abstract]
  4. ten Hoeve J, Morris C, Heisterkamp N, Groffen J 1993 Isolation and chromosomal localization of CRKL, a human crk-like gene. Oncogene 8:2469–2474[Medline]
  5. ten Hoeve J, Kaartinen V, Fioretos T, Haataja L, Voncken J-W, Heisterkamp N, Groffen J 1994 Cellular interactions of CRKL, an SH2-SH3 adaptor protein. Cancer Res 54:2563–2567[Abstract/Free Full Text]
  6. Tanaka S, Morishita T, Hashimoto Y, Hattori S, Nakamura S, Shibuya M, Matuoka K, Takenawa T, Kurata T, Nagashima K, Matsuda M 1994 C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins. Proc Natl Acad Sci USA 91:3443–3447[Abstract/Free Full Text]
  7. Knudsen BS, Feller SM, Hanafusa H 1994 Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first src homology 3 domain of Crk. J Biol Chem 269:32781–32787[Abstract/Free Full Text]
  8. Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW, Campbell ID, Waterfield MD 1993 The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75:25–36[CrossRef][Medline]
  9. Matsuda M, Hashimoto Y, Muroya K, Hasegawa H, Kurata T, Tanaka S, Nakamura S, Hattori S 1994 CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells. Mol Cell Biol 14:5495–5500[Abstract/Free Full Text]
  10. Feller SM, Knudsen B, Hanafusa H 1994 c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J 13:2341–2351[Medline]
  11. Birge RB, Fajardo JE, Reichman C, Shoelson SE, Songyang Z, Cantley LC, Hanafusa H 1993 Identification and characterization of a high-affinity interaction between v-crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. Mol Cell Biol 13:4648–4656[Abstract/Free Full Text]
  12. Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y, Hirai H 1994 A novel signaling molecule, p130, forms stable complexes in vivo with v-crk and v-src in a tyrosine phosphorylation-dependent manner. EMBO J 13:3748–3756[Medline]
  13. Ribon V, Hubbell S, Herrera R, Saltiel AR 1996 The product of the cbl oncogene forms stable complexes in vivo with endogenous crk in a tyrosine phosphorylation-dependent manner. Mol Cell Biol 16:45–52[Abstract]
  14. Reedquist KA, Fukazawa T, Panchamoorthy G, Langdon WY, Shoelson SE, Druker BJ, Band H 1996 Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G. J Biol Chem 271:8435–8442[Abstract/Free Full Text]
  15. Clark EA, Brugge JS 1995 Integrins and signal transduction pathways: the road taken. Science 268:233–239[Abstract/Free Full Text]
  16. Bellis SL, Miller JT, Turner CE 1995 Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J Biol Chem 270:17437–17441[Abstract/Free Full Text]
  17. Polte TR, Hanks SK 1995 Interaction between focal adhesion kinase and crk-associated tyrosine kinase substrate p130cas. Proc Natl Acad Sci USA 92:10678–10682[Abstract/Free Full Text]
  18. Blake TJ, Shapiro M, Morse III HC, Langdon WY 1991 The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene 6:653–657[Medline]
  19. Fukazawa T, Reedquist KA, Trub T, Soltoff S, Panchamoorthy G, Druker B, Cantley L, Shoelson SE, Band H 1995 The SH3 domain-binding T cell tyrosyl phosphoprotein p120. J Biol Chem 270:19141–19150[Abstract/Free Full Text]
  20. Mayer BJ, Hanafusa H 1990 Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation. J Virol 64:3581–3589[Abstract/Free Full Text]
  21. Hempstead BL, Birge RB, Fajardo JE, Glassman R, Mahadeo D, Kraemer R, Hanafusa H 1994 Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways. Mol Cell Biol 14:1964–1971[Abstract/Free Full Text]
  22. McClain DA, Maegawa H, Lee J, Dull TJ, Ulrich A, Olefsky JM 1987 A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem 262:14663–14671[Abstract/Free Full Text]
  23. Sasaoka T, Draznin B, Leitner JW, Langlois WJ, Olefsky JM 1994 Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation. J Biol Chem 269:10734–10738[Abstract/Free Full Text]
  24. Sasaoka T, Langlois WJ, Leitner JW, Draznin B, Olefsky JM 1994 The signaling pathway coupling epidermal growth factor receptors to activation of p21ras. J Biol Chem 269:32621–32625[Abstract/Free Full Text]
  25. Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM 1994 Evidence for a functional role of shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem 269:13689–13694[Abstract/Free Full Text]
  26. Knight JB, Yamauchi K, Pessin JE 1995 Divergent insulin and platelet-derived growth factor regulation of focal adhesion kinase (pp125 FAK) tyrosine phosphorylation, and rearrangement of actin stress fibers. J Biol Chem 270:10199–10203[Abstract/Free Full Text]
  27. Langlois WJ, Sasaoka T, Saltiel AR, Olefsky JM 1995 Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation. J Biol Chem 270:25320–25323[Abstract/Free Full Text]
  28. Goff SP, Gilboa E, Witte ON, Baltimore D 1980 Structure of the abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA. Cell 22:777–785[CrossRef][Medline]
  29. Vuori K, Ruoslahti E 1995 Tyrosine phosphorylation of p130cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix. J Biol Chem 270:22259–22262[Abstract/Free Full Text]
  30. Kanner SB, Reynolds AB, Wang H-CR, Vines RR, Parsons JT 1991 The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J 10:1689–1698[Medline]
  31. Matsuda M, Mayer BJ, Fukui Y, Hanafusa H 1990 Binding of transforming protein, p47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science 248:1537–1539[Abstract/Free Full Text]
  32. Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H, Sakai R, Sato T, Tachibana K, Morimoto C, Yazaki Y, Hirai H 1995 Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs. J Biol Chem 270:15398–15402[Abstract/Free Full Text]
  33. Zachary I, Sinnett-Smith J, Turner CE, Rozengurt E 1993 Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells. J Biol Chem 268:22060–22065[Abstract/Free Full Text]
  34. Rankin S, Rozengurt E 1994 Platelet-derived growth factor modulation of focal adhesion kinase (p125FAK) and paxillin tyrosine phosphorylation in swiss 3T3 cells. J Biol Chem 269:704–710[Abstract/Free Full Text]
  35. Langdon WY, Hartley JW, Klinken SP, Ruscetti SK, Morse III HC 1989 v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc Natl Acad Sci USA 86:1168–1172[Abstract/Free Full Text]
  36. Sasaoka T, Ishiki M, Sawa T, Ishihara H, Takata Y, Imamura T, Usui I, Olefsky JM, Kobayashi M 1996 Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways. Endocrinology 137:4427–4434[Abstract]
  37. Ridley AJ, Hall A 1992 The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389–399[CrossRef][Medline]
  38. Li S-L, Miyata Y, Yahara I, Fujita-Yamaguchi Y 1993 Insulin-induced circular membrane ruffling on Rat 1 cells expressing a high number of human insulin receptors: circular ruffles caused by rapid actin reorganization exhibit high density of insulin receptors and phosphotyrosines. Exp Cell Res 205:353–360[CrossRef][Medline]
  39. Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H, Ando A, Chavanieu A, Calas B, Grigorescu F, Nishiyama M, Waterfield MD, Kasuga M 1994 Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J 13:2313–2321[Medline]
  40. Kadowaki T, Koyasu S, Nishida E, Sakai H, Takaku F, Yahara I, Kasuga M 1986 Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. J Biol Chem 261:16141–16147[Abstract/Free Full Text]
  41. Izumi T, Saeki Y, Akanuma Y, Takaku F, Kasuga M 1988 Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. J Biol Chem 263:10386–10393[Abstract/Free Full Text]
  42. Jhun BH, Meinkoth JL, Leitner JW, Draznin B, Olefsky JM 1994 Insulin and insulin-like growth factor-I signal transduction requires p21ras. J Biol Chem 269:5699–5704[Abstract/Free Full Text]
  43. Buday L, Downward J 1993 Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts. Mol Cell Biol 13:1903–1910[Abstract/Free Full Text]
  44. Satoh T, Nakafuku M, Kaziro Y 1992 Function of ras as a molecular switch in signal transduction. J Biol Chem 267:24149–24152[Free Full Text]
  45. Medema RH, Vries-Smits AMM, Zon GCM, Maassen JA, Bos JL 1993 Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras. Mol Cell Biol 13:155–162[Abstract/Free Full Text]
  46. Draznin B, Chang L, Leitner JW, Takata Y, Olefsky JM 1993 Insulin activates p21ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors. J Biol Chem 268:19998–20001[Abstract/Free Full Text]
  47. Buday L, Khwaja A, Sipeki S, Farago A, Downward J 1996 Interactions of Cbl with two adaptor proteins, Grb2 and Crk, upon T cell activation. J Biol Chem 271:6159–6163[Abstract/Free Full Text]
  48. Donovan JA, Wange RL, Langdon WY, Samelson LE 1994 The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol Chem 269:22921–22924[Abstract/Free Full Text]
  49. Panchamoorthy G, Fukazawa T, Miyake S, Soltoff S, Reedquist K, Druker B, Shoelson S, Cantley L, Band H 1996 p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem 271:3187–3194[Abstract/Free Full Text]
  50. Cory GOC, Lovering RC, Hinshelwood S, MacCarthy-Morrogh L, Levinsky RJ, Kinnon C 1995 The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton’s tyrosine kinase. J Exp Med 182:611–615[Abstract/Free Full Text]
  51. Galisteo ML, Dikic I, Batzer AG, Langdon WY, Schlessinger J 1995 Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growrh factor (EGF) receptor upon EGF stimulation. J Biol Chem 270:20242–20245[Abstract/Free Full Text]
  52. Tanaka S, Neff L, Baron R, Levy JB 1995 Tyrosine phosphorylation and translocation of the c-cbl protein after activation of tyrosine kinase signaling pathways. J Biol Chem 270:14347–14351[Abstract/Free Full Text]
  53. Meisner H, Czech MP 1995 Coupling of the proto-oncogene product c-cbl to the epidermal growth factor receptor. J Biol Chem 270:25332–25335[Abstract/Free Full Text]
  54. Bowtell DDL, Langdon WY 1995 The protein product of the c-cbl oncogene rapidly complexes with the EGF receptor and is tyrosine phosphorylated following EGF stimulation. Oncogene 11:1561–1567[Medline]
  55. Smit L, Horst G, Borst J 1996 Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors. J Biol Chem 271:8564–8569[Abstract/Free Full Text]
  56. Tanaka S, Hattori S, Kurata T, Nagashima K, Fukui Y, Nakamura S, Matsuda, M 1993 Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells. Mol Cell Biol 13:4409–4415[Abstract/Free Full Text]
  57. Daley GQ, Van Etten RA, Baltimore D 1990 Induction of chronic myelogeneous leukemia in mice by the p210bcr/abl gene of the philadelphia chromosome. Science 247:824–830[Abstract/Free Full Text]
  58. Sawyers CL, McLaughlin J, Goga A, Havlik M, Witte O 1994 The nuclear tyrosine kinase c-abl negatively regulates cell growth. Cell 77:121–131[CrossRef][Medline]
  59. Turner CE, Glenney Jr JR, Burridge K 1990 Paxillin: a new vinculin-binding protein present in focal adhesion. J Cell Biol 111:1059–1068[Abstract/Free Full Text]
  60. Glenney Jr JR, Zokas L 1989 Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol 108:2401–2408[Abstract/Free Full Text]
  61. Schaller MD, Parsons JT 1995 pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol Cell Biol 15:2635–2645[Abstract]
  62. Burridge K, Turner CE, Romer LH 1992 Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 119:893–903[Abstract/Free Full Text]
  63. Harte MT, Hildebrand JD, Burnham MR, Bouton AH, Parsons JT 1996 p130cas, a substrate associated with v-src and v-crk, localizes to focal adhesions and binds to focal adhesion kinase. J Biol Chem 271:13649–13655[Abstract/Free Full Text]
  64. Petch LA, Bockholt SM, Bouton A, Parsons JT, Burridge K 1995 Adhesion-induced tyrosine phosphorylation of the p130 SRC substrate. J Cell Sci 108:1371–1379[Abstract]
  65. Pillay TS, Sasaoka T, Olefsky JM 1995 Insulin stimulates the tyrosine dephosphorylation of pp125 focal adhesion kinase. J Biol Chem 270:991–994[Abstract/Free Full Text]
  66. Konstantopoulos N, Clark S 1996 Insulin and insulin-like growth factor-1 stimulate dephosphorylation of paxillin in parallel with focal adhesion kinase. Biochem J 314:387–390
  67. Tobe K, Sabe H, Yamamoto T, Yamauchi T, Asai S, Kaburagi Y, Tamemoto H, Ueki K, Kimura H, Akanuma Y, Yazaki Y, Hanafusa H, Kadowaki T 1996 Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins. Mol Cell Biol 16:4765–4772[Abstract]
  68. Ribon V, Saltiel AR 1996 Nerve growth factor stimulates the tyrosine phosphorylation of endogenous Crk-II and augments its association with p130cas in PC-12 cells. J Biol Chem 271:7375–7380[Abstract/Free Full Text]
  69. Fajardo JE, Birge RB, Hanafusa H 1993 A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins. Mol Cell Biol 13:7295–7302[Abstract/Free Full Text]



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