Tyrosine Phosphorylation of p130cas by Bombesin, Lysophosphatidic Acid, Phorbol Esters, and Platelet-derived Growth Factor
SIGNALING PATHWAYS AND FORMATION OF A p130cas-Crk COMPLEX*

(Received for publication, November 14, 1996, and in revised form, January 16, 1997)

Adele Casamassima Dagger and Enrique Rozengurt §

From the Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Treatment of Swiss 3T3 cells with bombesin rapidly induced tyrosine phosphorylation of the p130 Crk-associated substrate (p130cas). Vasopressin, endothelin, bradykinin, lysophosphatidic acid, sphingosylphosphorylcholine, and phorbol 12,13-dibutyrate also stimulated p130cas tyrosine phosphorylation. Bombesin-induced p130cas tyrosine phosphorylation could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. In contrast, cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130cas by bombesin. Platelet-derived growth factor, at low concentrations (1-5 ng/ml), also induced tyrosine phosphorylation of p130cas via a pathway that depended on the integrity of the actin cytoskeleton. The phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 prevented tyrosine phosphorylation of p130cas in response to platelet-derived growth factor but not in response to neuropeptides, lysophosphatidic acid, sphingosylphosphorylcholine, or phorbol 12,13-dibutyrate. All agonists that induced p130cas tyrosine phosphorylation also promoted the formation of a p130cas·Crk complex in intact Swiss 3T3 cells. Thus, our results identified distinct signal transduction pathways that lead to tyrosine phosphorylation of p130cas in the same cells and suggest that p130cas could play a role in mitogen-mediated signal transduction.


INTRODUCTION

Neuropeptides stimulate DNA synthesis and proliferation in cultured cells and are implicated as growth factors in a variety of biological processes, including development, tissue regeneration, and tumorigenesis (1-3). In particular the neuropeptides bombesin, vasopressin, and endothelin are potent mitogens for Swiss 3T3 cells (1-4), a useful model system for the elucidation of signal transduction pathways leading to cell proliferation (4). Tyrosine phosphorylation has recently been implicated in the action of neuropeptides that act as cellular growth factors through G protein-coupled receptors (5). Addition of bombesin, vasopressin, endothelin, and bradykinin to Swiss 3T3 cells stimulates tyrosine phosphorylation of multiple substrates including proteins migrating in the Mr 110,000-130,000 range (6-8). The cytosolic tyrosine kinase p125fak1 (9, 10) and the adaptor protein paxillin (11, 12), which are associated with focal adhesion plaques, have been identified as prominent tyrosine-phosphorylated proteins in neuropeptide-stimulated Swiss 3T3 cells (13-15). Tyrosine phosphorylation of p125fak and paxillin is also induced by bioactive lipids such as LPA and SPC (16, 17), PDGF (18), phorbol esters (14), Pasteurella multocida toxin (19), extracellular matrix proteins (20-24), and transforming variants of p60src (21, 25). Recently, the organization of the actin cytoskeleton and the functional PI3'-kinase have been implicated in mitogen-stimulated tyrosine phosphorylation of p125fak and paxillin (17-19, 26-28).

Addition of bombesin (13), LPA (16), or PDGF (18) also induces tyrosine phosphorylation of the previously reported p130 substrate of pp60v-src (25, 29, 30). Subsequently, this protein has been shown to be closely related or identical to the most prominent tyrosine-phosphorylated protein in v-Crk transformed cells (31-33). The molecular cloning of this p130 v-Crk associated substrate (p130cas) revealed an adaptor protein that contains an SH3 domain, proline-rich regions, and a cluster of 15 putative SH2-binding motifs; 9 of these are YD(V/T)P which corresponds to the binding motif for the Crk SH2 domain (33). The unique structure of p130cas suggests a role of this protein in assembling multiple SH2-containing proteins in signaling complexes. However, neither the signal transduction pathways leading to p130cas tyrosine phosphorylation nor the existence of signaling complexes involving p130cas have been identified in cells stimulated by mitogenic neuropeptides or other growth factors.

In the present study we report that p130cas is rapidly tyrosine-phosphorylated in response to bombesin, other neuropeptides, phorbol esters, bioactive lipids, and PDGF. Our findings indicate that tyrosine phosphorylation of p130cas requires the integrity of the actin cytoskeleton and is stimulated through PI3'-kinase- and PKC-dependent and independent pathways. Furthermore, we also show that neuropeptides, bioactive lipids, and PDGF induce the formation of a complex between tyrosine-phosphorylated p130cas and c-Crk in intact Swiss 3T3 cells.


EXPERIMENTAL PROCEDURES

Cell Culture

Stock cultures of Swiss 3T3 fibroblasts were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO2 and 90% air at 37 °C. For experimental purposes, cells were plated in 100-mm dishes at 6 × 105 cells/dish in DMEM containing 10% fetal bovine serum and used after 6-8 days when the cells were confluent and quiescent.

Immunoprecipitation

Quiescent cultures of Swiss 3T3 cells (1-2 × 106) were washed twice with DMEM, treated with bombesin or other factors in 10 ml of DMEM for the times indicated, and lysed at 4 °C in 1 ml of a solution containing 10 mM Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (lysis buffer). Lysates were clarified by centrifugation at 15,000 × g for 10 min, and the supernatants were transferred to fresh tubes for immunoprecipitation. Proteins were immunoprecipitated at 4 °C for 14 h with anti-mouse IgG-agarose-linked mAb directed against anti-Tyr(P) (Py72), p130cas or c-Crk mAbs as indicated. Immunoprecipitates were washed three times with lysis buffer and extracted for 10 min at 95 °C in 2 × SDS-PAGE sample buffer (200 mM Tris/HCl, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8) and resolved by SDS-PAGE.

Western Blotting

Treatment of quiescent cultures of cells with factors, cell lysis, and immunoprecipitation was performed as described above. After SDS-PAGE, proteins were transferred to Immobilon membranes. Membranes were blocked using 5% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 2 h at 22 °C with either the anti-Tyr(P) mAb (4G10, 1 µg/ml) or the anti-p130cas mAb (1 µg/ml) as indicated. Immunoreactive bands were visualized using 125I-labeled sheep anti-mouse IgG (1:1000) followed by autoradiography. Autoradiograms were scanned using an LKB Ultrascan XL densitometer, and labeled bands were quantified using an Ultrascan XL internal integrator. The values were expressed as percentages of the maximum increase in tyrosine phosphorylation above control values.

Down-regulation of PKC

Phorbol ester-sensitive PKC isoforms were down-regulated in Swiss 3T3 cells by prolonged pretreatment with PDB (34, 35). In the present study, confluent and quiescent cultures were pretreated with 800 nM PDB for 48 h in conditioned medium, which was depleted of growth-promoting activity.

Materials

Bombesin, vasopressin, endothelin, bradykinin, SPC, LPA, PDB, cytochalasin D, wortmannin, and agarose-linked anti-mouse IgG were obtained from the Sigma. Recombinant PDGF (BB homodimer) and 125I-sheep anti-mouse IgG (15 mCi/mg) were from Amersham, UK. The PKC inhibitor GF 109203X and thapsigargin were obtained from Calbiochem-Novabiochem Ltd. (Nottingham, UK). The 4G10 anti-Tyr(P) mAb was from Upstate Biotechnology Inc. (Lake Placid, New York). The anti-Tyr(P) mAb Py72 was obtained from the hybridoma development unit, Imperial Cancer Research Fund. The mAbs directed against p130cas or c-Crk were from Transduction Laboratories. LY294002 was provided by Dr. S. Cartlidge, Zeneca, UK.


RESULTS

Bombesin, Vasopressin, Endothelin, Bradykinin, LPA, and SPC Induce p130cas Tyrosine Phosphorylation in Swiss 3T3 Cells

Quiescent cultures of Swiss 3T3 cells were stimulated with 10 nM bombesin for 10 min, a concentration and a time that causes maximum stimulation of tyrosine phosphorylation (7, 8, 13), and lysed. The lysates were immunoprecipitated with anti-Tyr(P) mAb, and the resulting immunoprecipitates were analyzed by Western blotting with p130cas mAb. As shown in Fig. 1A, bombesin caused a striking increase in the tyrosine phosphorylation of p130cas (5.6 ± 0.8 fold; n = 12). An increase in tyrosine phosphorylation of p130cas by bombesin was also demonstrated when the cell lysates were first immunoprecipitated with anti-p130cas mAb and then analyzed by anti-Tyr(P) immunoblotting (Fig. 1A). Immunoprecipitation using anti-p130cas mAb followed by Western blotting with the same antibody showed that the recovery of p130cas from cell lysates was not altered by treatment with bombesin.


Fig. 1. Neuropeptides and bioactive lipids stimulate p130cas tyrosine phosphorylation in Swiss 3T3. A, bombesin induces tyrosine phosphorylation of p130cas. Quiescent cells were treated either in the absence (-) or in presence (+) of 10 nM bombesin (BOM) for 10 min at 37 °C and lysed. Lysates prepared from parallel cultures were immunoprecipitated (I.P.) with anti-Tyr(P) mAb or anti-p130cas mAb, and the immunoprecipitates were then analyzed by Western blotting (W.B.) using either anti-p130cas or anti-Tyr(P) mAbs as indicated. B, effect of neuropeptides and bioactive lipids on tyrosine phosphorylation of p130cas. Quiescent Swiss 3T3 cells were incubated for 10 min at 37 °C without (-) or with 10 nM bombesin (BOM), 20 nM vasopressin (VP), 10 nM endothelin (END), 20 nM bradykinin (BK), 5 µM SPC, or 2 µM LPA. The cell lysates were then immunoprecipitated with anti-Tyr(P) mAb, and the immunoprecipitates were analyzed by immunoblotting with the anti-p130cas mAb. The position of p130cas is indicated by arrows. The autoradiograms shown are representative of three independent experiments.
[View Larger Version of this Image (29K GIF file)]


Other mitogenic neuropeptides such as vasopressin, endothelin, and bradykinin, and the bioactive lipids SPC and LPA, elicit a pattern of tyrosine-phosphorylated bands similar to that induced by bombesin, including a broad band migrating in the Mr 110,00-130,000 range (13, 16, 17). To examine the effect of these stimuli on p130cas tyrosine phosphorylation, Swiss 3T3 cells were treated for 10 min with 20 nM vasopressin, 10 nM endothelin, 20 nM bradykinin, 2 µM LPA, or 5 µM SPC and lysed. The cell lysates were immunoprecipitated with anti-Tyr(P) mAb and the immunoprecipitates analyzed by immunoblotting with anti-p130cas mAb. As shown in Fig. 1B, all of these agonists induced a marked increase in p130cas tyrosine phosphorylation.

The effect of bombesin on p130cas tyrosine phosphorylation, as revealed by anti-p130cas immunoblotting of anti-Tyr(P) immunoprecipitates, was concentration- and time-dependent. Half-maximum and maximum effects were achieved at 0.3 and 10 nM, respectively (Fig. 2, left panel). An increase in tyrosine phosphorylation of p130cas could be detected as early as 30 s after addition of 10 nM bombesin, reaching a maximum after 1-2.5 min (Fig. 2, right panel). Thereafter, p130cas tyrosine phosphorylation declined but remained above base-line levels for at least 1 h.


Fig. 2. Dose response and time course of bombesin-induced p130cas tyrosine phosphorylation. Left panel, quiescent Swiss 3T3 were treated at 37 °C for 10 min either in the absence or presence of various concentrations of bombesin as indicated and subsequently lysed. Tyrosine phosphorylation of p130cas was analyzed by immunoprecipitation using anti-Tyr(P) mAb followed by Western blotting with anti-p130cas mAb. The results are representative of five independent experiments. Right panel, quiescent Swiss 3T3 cells were treated at 37 °C with 10 nM bombesin for various times, and cell lysates were further analyzed as described above. The position of p130cas is indicated by the arrow. The results shown are representative of three identical experiments. In all cases, quantification of p130cas tyrosine phosphorylation was performed by scanning densitometry. Values are expressed as percentages of the maximum response after subtraction of the control.
[View Larger Version of this Image (26K GIF file)]


Role of PKC Activation and Ca2+ Mobilization in PDB- and Bombesin-stimulated p130cas Tyrosine Phosphorylation

Activation of PKC and mobilization of Ca2+ from intracellular stores are two prominent early signals elicited by mitogenic neuropeptides and bioactive lipids (17, 36). In addition, direct activation of PKC increases tyrosine phosphorylation of substrates migrating in the Mr 110,000-130,000 range in Swiss 3T3 cells. Consequently, we examined the possibility that these early events mediated bombesin-induced p130cas tyrosine phosphorylation. As shown in Fig. 3 (upper panel), direct activation of PKC by PDB enhanced p130cas tyrosine phosphorylation. An increase was detectable after 1 min, reached a maximum after 5 min, and remained elevated for at least 1 h (not shown). To investigate the involvement of PKC activation in bombesin-induced p130cas tyrosine phosphorylation, PKC was selectively inhibited by pretreatment of quiescent cells with 3.5 µM GF109203X for 1 h (37). Subsequently, the cells were stimulated with either 10 nM bombesin or 200 nM PDB for 10 min. As shown in Fig. 3 (central panel), pretreatment of Swiss 3T3 cells with GF 109203X blocked PDB stimulation of p130cas tyrosine phosphorylation. In contrast, bombesin-induced tyrosine phosphorylation of p130cas was not prevented by pretreatment with 3.5 µM GF109203X. Identical results were obtained when the cells were challenged with bombesin or PDB for 2.5 min instead of 10 min (Fig. 3, central panel, inset). Similarly, down-regulation of phorbol ester-sensitive isoforms of PKC markedly reduced the effect of PDB but did not prevent bombesin stimulation of p130cas tyrosine phosphorylation (data not shown).


Fig. 3. Effect of PDB on p130cas tyrosine phosphorylation. Upper panel, time course of PDB stimulation of p130cas tyrosine phosphorylation. Quiescent Swiss 3T3 cells were incubated at 37 °C with 200 nM PDB for various times, as indicated. The cell lysates were then immunoprecipitated with anti-Tyr(P) mAb and further analyzed by Western blotting with anti-p130cas mAb. Quantification of p130cas tyrosine phosphorylation was performed by scanning densitometry. Values are expressed as percentages of the maximum response. The results shown are representative of three identical experiments. Central panel, role of PKC activity in bombesin-induced p130cas tyrosine phosphorylation. Quiescent Swiss 3T3 cells were preincubated either in the absence (-) or presence (+) of 3.5 µM GF109203X for 1 h. The cells were subsequently stimulated for 10 min with 200 nM PDB or 10 nM bombesin (BOM), lysed, and immunoprecipitated with anti-Tyr(P) mAb and further analyzed by p130cas Western blotting. Quantification of tyrosine phosphorylation was performed by scanning densitometry. Values shown are the mean ± S.E. of three independent experiments and are expressed as percentages of the maximum increase in tyrosine phosphorylation of p130cas above unstimulated control values. The inset shows an autoradiogram of a similar experiment except that the cells were stimulated with PDB or bombesin (BOM) for 2.5 min instead of 10 min. Lower panel, role of intracellular Ca2+ in bombesin-induced p130cas tyrosine phosphorylation. Quiescent Swiss 3T3 cells were preincubated at 37 °C without (-) or with (+) 30 nM thapsigargin (TG) for 30 min as indicated. Cells were subsequently stimulated without (-) or with 10 nM bombesin (BOM) for 10 min and lysed, and the lysates were immunoprecipitated with anti-Tyr(P) mAb followed by anti-p130cas Western blotting. The increase in tyrosine phosphorylation of p130cas was quantified by scanning densitometry. Values shown are the mean ± S.E. of three independent experiments and are expressed as percentages of the maximum increase in tyrosine phosphorylation of p130cas above unstimulated control values. The inset shows an autoradiogram of a similar experiment except that the cells were stimulated with PDB or bombesin (BOM) for 2.5 min instead of 10 min.
[View Larger Version of this Image (25K GIF file)]


Next, we investigated the role of [Ca2+]i in bombesin stimulation of p130cas tyrosine phosphorylation using the tumor promoter thapsigargin. This agent specifically inhibits the endoplasmic reticulum Ca2+-ATPase and thereby depletes Ca2+ from intracellular compartments (38). As shown in Fig. 3 (lower panel), pretreatment of quiescent Swiss 3T3 with 30 nM thapsigargin for 30 min did not have any effect on bombesin-induced tyrosine phosphorylation of p130cas but blocked the increase in [Ca2+]i by bombesin (data not shown). Thus, neither PKC activation nor Ca2+ mobilization is responsible for the rapid bombesin-induced p130cas tyrosine phosphorylation in Swiss 3T3 cells.

The Integrity of the Actin Cytoskeleton Is Necessary for Bombesin-Induced Tyrosine Phosphorylation of p130cas

As p130cas is localized at focal adhesion plaques (39, 40), the distinct sites on the plasma membrane where actin stress fibers emanate, we examined whether disruption of the actin cytoskeleton could interfere with p130cas tyrosine phosphorylation induced by bombesin. Quiescent Swiss 3T3 cells were pretreated for 2 h with increasing concentrations of cytochalasin D and then stimulated with 10 nM bombesin for another 10 min. As shown in Fig. 4, cytochalasin D blocked bombesin stimulation of p130cas tyrosine phosphorylation in a concentration-dependent manner; half-maximum effect was obtained at a concentration of 0.4 µM and a maximum effect was achieved at 1 µM, a concentration that is known to completely disrupt the actin cytoskeleton and the assembly of focal adhesions in Swiss 3T3 cells. The drug also inhibited PDB-induced tyrosine phosphorylation of p130cas (Fig. 4, inset). Thus, the integrity of the actin cytoskeleton is required for PKC-dependent and PKC-independent signal transduction pathways leading to tyrosine phosphorylation of p130cas.


Fig. 4. Effect of cytochalasin D on bombesin-induced tyrosine phosphorylation of p130cas. Upper, quiescent Swiss 3T3 cells were treated for 2 h in the absence (0) or in the presence of different concentrations of cytochalasin D (Cyt D) as indicated and then stimulated without (-) or with 10 nM bombesin (BOM) for a further 10 min. Parallel cultures were incubated for 2 h either in the absence (0) or in the presence of 1.2 µM cytochalasin D and subsequently treated for a further 10 min with 200 nM PDB. The cells were then lysed, and lysates were further analyzed by immunoprecipitation with anti-Tyr(P) mAb followed by anti-p130cas Western blotting. Shown is a representative experiment of three independent experiments. The position of p130cas is shown by the arrow. Lower, the bombesin-induced anti-Tyr(P) immunoreactivity of p130cas obtained after pretreatment with various concentrations of cytochalasin D was quantified by scanning densitometry. Inset, quantification by scanning densitometry of the autoradiogram showing the effect of the cytochalasin D on PDB-induced p130cas tyrosine phosphorylation. The values shown represent percentages of the maximum response after subtraction of the control and are representative of three independent experiments. The maximum phosphorylation was induced by 10 nM bombesin in cells not pretreated with cytochalasin D.
[View Larger Version of this Image (36K GIF file)]


Bombesin, Vasopressin, Endothelin, LPA, and SPC Induce the Formation of a p130cas·c-Crk Complex in Swiss 3T3 Cells

p130cas has a cluster of 15 potential SH2-binding motifs, nine of these are YDXP sequences that are expected to have a preferential affinity for Crk-SH2 domain (33). Consequently, we examined whether bombesin-induced tyrosine phosphorylation of p130cas could lead to the formation of a complex between endogenous c-Crk and p130cas in intact Swiss 3T3 cells. Anti-p130cas Western blotting of c-Crk immunoprecipitates revealed that bombesin stimulated an association of p130cas with c-Crk (Fig. 5A). We verified that similar amounts of c-Crk were recovered from lysates of cells treated without or with bombesin. The association of p130cas with c-Crk reached a maximum after 1 min of bombesin stimulation then declined, remaining at about 50% of the maximum level for the next 10 min (Fig. 5B). Treatment of Swiss 3T3 cells with vasopressin, endothelin, LPA, SPC, or PDB, all of which induced tyrosine phosphorylation of p130cas (Fig. 1), also induced complex formation between p130cas and c-Crk (Fig. 5C).


Fig. 5. Neuropeptides and bioactive lipids induce association of p130cas with endogenous c-Crk. A, formation of complex between p130cas and Crk in Swiss 3T3 cells. Cultures of these cells (6 × 106) were treated in the absence (-) or in the presence (+) of 10 nM bombesin (BOM) for 1 min and lysed. The lysates were immunoprecipitated with anti-Crk mAb. The resulting immunocomplexes were analyzed by immunoblotting with anti-Crk mAb or anti-p130cas mAb. The positions of Crk and p130cas are indicated by the arrows. B, time-dependent formation of a p130cas·cCrk complex. Cultures of quiescent Swiss 3T3 cells (6 × 106) were treated for various times as indicated with 10 nM bombesin and lysed. The lysates were then immunoprecipitated with anti-Crk mAb and analyzed by immunoblotting with the anti-p130cas mAb. The inset shows a representative experiment of two independent experiments. Quantification of p130cas tyrosine phosphorylation was performed by scanning densitometry. Values are expressed as percentages of the maximum response. C, neuropeptides, bioactive lipids, and PDB induce p130cas·cCrk complex formation. Quiescent Swiss 3T3 cells (6 × 106) were incubated for 10 min at 37 °C without (-) or with 10 nM bombesin (BOM), 20 nM vasopressin (VP), 10 nM endothelin (END), 2 µM LPA, 5 µM SPC, or 200 nM PDB. The cell lysates were then immunoprecipitated with anti-Crk mAb and the immunoprecipitates analyzed by immunoblotting with the anti-p130cas mAb. The position of p130cas is indicated by the arrow. The results shown are representative of three independent experiments. D, cytochalasin D prevents the formation of the p130cas·cCrk complex. Quiescent Swiss 3T3 cells (6 × 106) were preincubated for 2 h in the absence (-) or presence of 1.2 µM cytochalasin D (Cyt. D). Cells were subsequently incubated without (-) or with (+) 10 nM bombesin (BOM) for 10 min and then lysed. Lysates were immunoprecipitated with anti-Crk mAb and analyzed by immunoblotting with anti-p130cas mAb. The position of p130cas is indicated by the arrow. The results shown are representative of three independent experiments. Identical results were obtained when the cultures were stimulated with bombesin for 1 min instead of 10 min.
[View Larger Version of this Image (27K GIF file)]


To assess whether the complex formation between p130cas and Crk depended on the integrity of the actin cytoskeleton, quiescent Swiss 3T3 cells were pretreated for 2 h with or without 1.2 µM cytochalasin D and then stimulated with 10 nM bombesin. Fig. 5D shows that treatment of Swiss 3T3 cells with cytochalasin D, at a concentration shown in Fig. 4 to inhibit p130cas tyrosine phosphorylation, prevented the association of p130cas with c-Crk induced by bombesin.

Thus, mitogenic neuropeptides and bioactive lipids induce rapid association of tyrosine-phosphorylated p130cas with c-Crk in Swiss 3T3 cells.

PDGF Induces Tyrosine Phosphorylation of p130cas and Formation of a p130cas·cCrk Complex

Next, we examined the regulation of tyrosine phosphorylation of p130cas by PDGF BB, which binds to the PDGF receptor a and b chains in Swiss 3T3 cells (41, 42). PDGF caused a time- and dose-dependent increase in the tyrosine phosphorylation of p130cas (Fig. 6A). Maximum response was observed at 5 ng/ml PDGF. At higher concentrations of PDGF the tyrosine phosphorylation of p130cas was drastically reduced. PDGF, at 5 ng/ml, also induced the formation of a complex between p130cas and c-Crk (Fig. 6B). PDGF, at low concentrations, is known to stimulate the accumulation of actin into membrane ruffles, and at higher concentrations PDGF induced actin disorganization and focal adhesion disassembly (18). Thus, the bell-shaped dose-response curve of PDGF on tyrosine phosphorylation of p130cas could be explained by the ability of PDGF (at high concentrations) to disrupt actin organization and focal adhesion formation in Swiss 3T3 cells. In accord with this interpretation, disruption of actin cytoskeleton by cytochalasin D prevented the increase in p130cas tyrosine phosphorylation induced by 5 ng/ml PDGF (Fig. 6A, inset). Treatment with cytochalasin D also inhibited the formation of a complex between p130cas and c-Crk (Fig. 6B).


Fig. 6. A, effect of PDGF on tyrosine phosphorylation of p130cas. Upper, quiescent Swiss 3T3 cells were incubated for various times with 5 ng/ml PDGF or for 10 min at 37 °C with the indicated concentrations of PDGF and then lysed. Tyrosine phosphorylation of p130cas was analyzed by immunoprecipitation with an anti-Tyr(P) mAb and immunoblotting with anti-p130cas mAb. This panel shows a representative of three independent experiments. The position of p130cas is indicated by the arrow. Lower, anti-Tyr(P) immunoreactivity of p130cas bands generated in response to various concentrations of PDGF was quantified by scanning densitometry. Values shown are expressed as the percentage of the maximum response after subtraction of the control. The results shown are representative of three independent experiments. Inset, effect of cytochalasin D pretreatment on PDGF-induced tyrosine phosphorylation of p130cas. Quiescent Swiss 3T3 cells were preincubated in absence (-) or in presence (+) of 1.2 µM cytochalasin D (Cyt. D) for 2 h at 37 °C. The cells were subsequently incubated with 5 ng/ml PDGF for 10 min and then lysed. Lysates were immunoprecipitated with anti-Tyr(P) mAb and analyzed by immunoblotting with anti-p130cas mAb. The PDGF-induced tyrosine phosphorylation of p130cas in the presence of cytochalasin D was quantified by scanning densitometry. The values shown are expressed as the percentage of the maximum increase in p130cas tyrosine phosphorylation after subtraction of the control and are representative of three independent experiments. B, PDGF induces the formation of a complex between tyrosine-phosphorylated p130cas and c-Crk. Cultures of quiescent Swiss 3T3 cells (6 × 106) were preincubated for 2 h in the absence (-) or presence of 1.2 µM cytochalasin D (Cyt. D). Cells were subsequently incubated without (-) or with (+) 5 ng/ml PDGF for 10 min and then lysed. Lysates were immunoprecipitated with anti-Crk mAb and analyzed by immunoblotting with anti-p130cas mAb. The position of p130cas is indicated by the arrow. The results shown are representative of three independent experiments. C, effect of PDGF on bombesin-induced tyrosine phosphorylation of p130cas. Quiescent Swiss 3T3 cells were incubated for 20 min at 37 °C without (-) or with (+) 10 nM bombesin (BOM), 5 or 30 ng/ml PDGF, or 10 nM bombesin in the absence (-) or presence of 5 or 30 ng/ml PDGF, as indicated. Tyrosine phosphorylation of p130cas was analyzed by immunoprecipitation with anti-Tyr(P) mAb and immunoblotting with anti-p130cas mAb quantified by scanning densitometry. The values were expressed as the percentage of the maximum increase in p130cas tyrosine phosphorylation above control, unstimulated values, induced by bombesin and are the mean ± the range of two independent experiments.
[View Larger Version of this Image (29K GIF file)]


Since high concentrations of PDGF disrupt the actin cytoskeleton (18), we examined the effect of PDGF at 30 ng/ml on bombesin-induced tyrosine phosphorylation of p130cas. Tyrosine phosphorylation of p130cas was determined in cells treated with 10 nM bombesin for 20 min either in the absence or presence of 5 ng/ml or 30 ng/ml PDGF. PDGF at 30 ng/ml but not at 5 ng/ml completely inhibited p130cas tyrosine phosphorylation by bombesin (Fig. 6C).

Effect of Wortmannin and LY294002 on PDGF- and Bombesin-stimulated Tyrosine Phosphorylation of p130cas

PDGF, at low concentrations, induces the recruitment of actin into membrane ruffles through a PI3'-kinase-dependent signaling pathway (43-45). Consequently, we examined the role of PI3'-kinase in PDGF-stimulated tyrosine phosphorylation of p130cas in Swiss 3T3 cells. Quiescent Swiss 3T3 cells were treated for 20 min with wortmannin, which binds to and inhibits the catalytic (110 kDa) subunit of PI3'-kinase (46, 47), at concentrations (0-40 nM) that inhibit PI3'-kinase activity and actin cytoskeleton reorganization in PDGF-treated Swiss 3T3 cells (27). The cells were then stimulated with 3 ng/ml PDGF for a further 10 min. As shown in Fig. 7A), wortmannin induced a striking dose-dependent inhibition of the tyrosine phosphorylation of p130cas in response to 3 ng/ml PDGF. At 40 nM, wortmannin inhibited PDGF-stimulated p130cas tyrosine phosphorylation by 90%.


Fig. 7. A, effect of wortmannin on PDGF- and bombesin-induced tyrosine phosphorylation of p130cas. Quiescent Swiss 3T3 cells were preincubated for 20 min at 37 °C with increasing concentrations (0-40 nM) of wortmannin as indicated. Cells were subsequently incubated with either 3 ng/ml PDGF or 10 nM bombesin for another 10 min, lysed, and immunoprecipitated with the anti-Tyr(P) mAb. Immunoprecipitates were analyzed by immunoblotting with anti-p130cas mAb. The position of p130cas is shown by the arrows. The autoradiograms obtained were quantified by scanning densitometry to determine the tyrosine phosphorylation of p130cas induced by 3 ng/ml PDGF (closed circles) or 10 nM bombesin (open circles) in cells pretreated with various concentration of wortmannin. The values were expressed as a percentage of the maximum response induced by either 10 nM bombesin or 3 ng/ml PDGF. The results shown are representative of two independent experiments. B, effect of wortmannin on vasopressin-, endothelin-, LPA-, and SPC-stimulated tyrosine phosphorylation of p130cas in Swiss 3T3 cells. Quiescent Swiss 3T3 cells were preincubated for 20 min without (-) or with (+) 40 nM wortmannin (Wort.). Cells were subsequently incubated for 10 min in the absence (-) or presence of 3 ng/ml PDGF, 20 nM vasopressin (VP), 10 nM endothelin (END), 2 µM LPA, or 5 µM SPC and then lysed. The cell lysates were then immunoprecipitated with an anti-Tyr(P) mAb. The immunoprecipitates were analyzed by immunoblotting with anti-p130cas mAb. The position of p130cas is shown by the arrow. The results shown are representative of three experiments. C, effect of LY94002 on PDGF and bombesin-induced tyrosine phosphorylation of p130cas. Quiescent Swiss 3T3 cells were preincubated for 2 h at 37 °C with increasing concentration (0-10 µM) of LY 94002 as indicated. Cells were subsequently incubated with either 3 ng/ml PDGF or 10 nM bombesin for another 10 min, lysed, and immunoprecipitated with the anti-Tyr(P) mAb. Immunoprecipitates were analyzed by immunoblotting with anti-p130cas mAb. The position of p130cas is shown by the arrows. The autoradiograms obtained were quantified by scanning densitometry to determine the tyrosine phosphorylation of p130cas induced by 3 ng/ml PDGF (closed circles) or 10 nM bombesin (open circles) in cells pretreated with various concentration of LY 94002. The values were expressed as the percentage of the maximum response induced by either 10 nM bombesin or 3 ng/ml PDGF. The results shown are representative of two independent experiments.
[View Larger Version of this Image (41K GIF file)]


In contrast to PDGF, bombesin acts through a seven transmembrane receptor that does not stimulate PI3'-kinase in Swiss 3T3 cells (7, 48, 49). Hence, bombesin-induced tyrosine phosphorylation of p130cas should not be inhibited by wortmannin in these cells. As shown in Fig. 7A, the tyrosine phosphorylation of p130cas stimulated by 10 nM bombesin was not inhibited by preincubation of the cells with wortmannin up to 40 nM. Wortmannin, therefore, differentially inhibited PDGF-stimulated tyrosine phosphorylation of p130cas, while having no effect on bombesin-stimulated tyrosine phosphorylation of this substrate.

To extend the results presented above we examined the effect of wortmannin on p130cas tyrosine phosphorylation in response to other neuropeptides and bioactive lipids. Fig. 7B shows that in contrast to the result obtained with PDGF, the increase in tyrosine phosphorylation of p130cas induced by vasopressin, endothelin, LPA, and SPC was not affected by pretreatment with 40 nM wortmannin.

To substantiate the results obtained with wortmannin, we examined if a structurally unrelated compound, LY294002 (a flavonoid related to quercetin), which inhibits PI3'-kinase by a distinct mechanism (50) also prevents PDGF-stimulated p130cas tyrosine phosphorylation in a selective manner. Quiescent Swiss 3T3 cells were preincubated for 2 h with increasing concentrations of LY94002 (0-10 µM) and then stimulated with either 3 ng/ml PDGF or 10 nM bombesin for further 10 min. As shown in Fig. 7C, pretreatment with LY294002 inhibited p130cas tyrosine phosphorylation induced by PDGF in a dose-dependent fashion. In contrast, LY294002 did not affect p130cas tyrosine phosphorylation in response to bombesin. Thus, tyrosine phosphorylation of p130cas can be stimulated through a PI3'-kinase-dependent and -independent pathway in Swiss 3T3 cells.


DISCUSSION

The findings presented here demonstrate that p130cas is a prominent tyrosine-phosphorylated protein in Swiss 3T3 cells stimulated by bombesin, vasopressin, endothelin, and bradykinin. The rapidity of bombesin-induced p130cas tyrosine phosphorylation suggests that this event may be functionally important in the action of this neuropeptide. In addition, the bioactive lipids LPA and SPC that are thought to act through G protein-coupled receptors (51, 52) also induce p130cas tyrosine phosphorylation.

At present very little is known about the signaling pathways that link the bombesin receptors to tyrosine phosphorylation of p130cas. Bombesin and other neuropeptides are known to induce the rapid hydrolysis of polyphosphoinositides to generate the intracellular second messengers diacylglycerol and inositol 1,4,5-trisphosphate, which activate PKC and mobilize Ca2+, respectively. As shown in the present study, direct activation of PKC by addition of PDB also stimulated p130cas tyrosine phosphorylation. Thus, PKC activation is a potential signaling pathway that could mediate bombesin stimulation of p130cas tyrosine phosphorylation. However, our results indicate that bombesin stimulates p130cas tyrosine phosphorylation through a signal transduction pathway that is independent of either PKC activation or Ca2+ mobilization.

Bombesin, endothelin, LPA, and SPC have been shown to induce a rapid increase in stress fibers and in focal adhesions (17, 53), an effect apparently mediated by Rho (54, 55), which belongs to the Ras-related small G protein superfamily (56). Focal adhesions that form at the termini of actin stress fibers are thought to play a central role in the processes that regulate cell adhesion and motility (57). In view of the localization of p130cas in the focal adhesions (39) and recent findings that integrin activation also leads to tyrosine phosphorylation of this protein (39, 40, 58, 59), we examined whether the actin cytoskeleton played a role in bombesin stimulation of p130cas tyrosine phosphorylation. Our results with cytochalasin D indicate that the maintenance of cytoskeletal organization is essential for the stimulation of p130cas tyrosine phosphorylation.

PDGF, at low concentrations (5 ng/ml), also induced tyrosine phosphorylation of p130cas through a pathway that is critically dependent on the integrity of actin cytoskeleton and focal adhesions. The structurally unrelated PI3'-kinase inhibitors wortmannin (46, 47) and LY294002 (50) prevented PDGF-mediated stimulation of p130cas tyrosine phosphorylation, implicating a PI3'-kinase-dependent pathway in the tyrosine phosphorylation of p130cas. It has been demonstrated that PI3'-kinase activation is required for the formation of membrane ruffles and that the small G protein Rac lies downstream of PI3'-kinase (49, 60). Activated Rac has been shown to direct the formation of membrane ruffles and the assembly of focal adhesions (55). Taken together, these findings suggest the existence of a linear signal transduction pathway in the action of PDGF involving PI3'-kinase and Rac that leads to the tyrosine phosphorylation of p130cas. Our results also demonstrate that the increase in the tyrosine phosphorylation of p130cas induced by bombesin, vasopressin, endothelin, LPA, and SPC is not prevented by either wortmannin or LY294002 at concentrations that profoundly inhibited p130cas tyrosine phosphorylation induced by PDGF. We conclude that there is a PI3' kinase-dependent and a PI3'-kinase-independent signal transduction pathway stimulating the tyrosine phosphorylation of p130cas in the same cells.

Similar to the data reported here, bombesin-induced p125fak and paxillin tyrosine phosphorylation also occurs through a PKC, Ca2+, and PI3' kinase-independent pathway, which is critically dependent on the integrity of the actin cytoskeleton (14, 15, 27, 53). Furthermore, the activation of PI3'-kinase has been recently identified as an important step in the signal transduction pathway that links the PDGF receptor with the tyrosine phosphorylation of p125fak and paxillin (27). In addition, bombesin and PDGF stimulated p130cas tyrosine phosphorylation at concentrations that parallel those required to induce p125fak and paxillin tyrosine phosphorylation (14, 15). We conclude that tyrosine phosphorylation of p130cas, p125fak, and paxillin are coordinately regulated. In agreement with this conclusion, it has been demonstrated that p130cas associates directly with p125fak (61, 62) and that the major site of p125fak autophosphorylation (Tyr-397) binds the SH2 domain of Src (63), a kinase implicated in the phosphorylation of p130cas (33, 58) that is rapidly and transiently activated by bombesin, vasopressin, and bradykinin in Swiss 3T3 cells (64).

The molecular cloning and sequencing of p130cas revealed a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding sites for Crk and Src. This suggests that tyrosine-phosphorylated p130cas may serve to promote the assembly of multiple SH2-containing molecules (33). Indeed, recent reports have shown that integrin-dependent cell adhesion can induce a SH2-mediated association of c-Crk with p130cas (58). Our results demonstrate that mitogenic neuropeptides, bioactive lipids, and PDGF induce the formation of a p130cas·Crk complex in intact Swiss 3T3 cells that is dependent on the integrity of the actin cytoskeleton. The interaction between p130cas and c-Crk may be important in regulating the subcellular distribution of Crk or the activity of new downstream effectors in neuropeptide signal transduction pathways.

Crk binds to a number of signaling proteins through its SH3 domain including C3G (65, 66), a guanine nucleotide exchange factor for Rap-1 (67), a small GTP-binding protein that induces mitogenesis in Swiss 3T3 cells (68). Interestingly, constitutive activation of Rap-1 has been suggested to lead to tumors in patients with tuberous sclerosis (69). The possibility that tyrosine phosphorylation of p130cas plays a role in mitogenic signaling is attractive and warrants further experimental work.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Supported by the Associazione Italiana Ricerca sul Cancro and a grant from the Human Capital and Mobility program.
§   To whom all correspondence should be addressed. Tel.: 44-171-269-3455; Fax: 44-171-269-3417.
1   The abbreviations used are: p125fak, p125 focal adhesion kinase; LPA, 1-oleoyl-lysophosphatidic acid; PDGF, platelet-derived growth factor; p130cas, p130 Crk-associated substrate; SH2, src homology 2; PI3' kinase, phosphatidylinositol 3'-kinase; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; anti-Tyr(P), anti-phosphotyrosine; mAb, monoclonal antibody; PKC, protein kinase C; PDB, phorbol 12,13-dibutyrate; SPC, sphingosylphosphorylcholine; SH3, src homology 3.

ACKNOWLEDGEMENTS

We thank Dr. T. Seufferlein and J. Sinnet-Smith for valuable discussions.


REFERENCES

  1. Rozengurt, E. (1992) Curr. Opin. Cell Biol. 4, 161-165 [Medline] [Order article via Infotrieve]
  2. Rozengurt, E. (1991) Eur. J. Clin. Invest. 21, 123-134 [Medline] [Order article via Infotrieve]
  3. Zachary, I., Woll, P. J., and Rozengurt, E. (1987) Dev. Biol. 124, 295-308 [Medline] [Order article via Infotrieve]
  4. Rozengurt, E. (1986) Science 234, 161-166 [Medline] [Order article via Infotrieve]
  5. Rozengurt, E. (1995) Cancer Surv. 24, 81-96 [Medline] [Order article via Infotrieve]
  6. Leeb-Lundberg, L. M. F., and Song, X.-H. (1991) J. Biol. Chem. 266, 7746-7749 [Abstract/Free Full Text]
  7. Zachary, I., Gil, J., Lehmann, W., Sinett-Smith, J., and Rozengurt, E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4577-4581 [Abstract]
  8. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1991) J. Biol. Chem. 266, 24126-24133 [Abstract/Free Full Text]
  9. Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5192-5196 [Abstract]
  10. Hanks, S. K., Calalb, M. B., Harper, M. C., and Patel, S. K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8487-8491 [Abstract]
  11. Turner, C. E., and Miller, J. T. (1994) J. Cell Sci. 107, 1583-1591 [Abstract/Free Full Text]
  12. Salgia, R., Li, J.-L., Lo, S. H., Brunkhorst, B., Kansas, G. S., Sobhany, E. S., Sun, Y., Pisick, E., Hallek, M., Ernst, T., Tantravahi, R., Chen, L. B., and Griffin, J. D. (1995) J. Biol. Chem. 270, 5039-5047 [Abstract/Free Full Text]
  13. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1992) J. Biol. Chem. 267, 19031-19034 [Abstract/Free Full Text]
  14. Sinnett-Smith, J., Zachary, I., Valverde, A. M., and Rozengurt, E. (1993) J. Biol. Chem. 268, 14261-14268 [Abstract/Free Full Text]
  15. Zachary, I., Sinnett-Smith, J., Turner, C. E., and Rozengurt, E. (1993) J. Biol. Chem. 268, 22060-22065 [Abstract/Free Full Text]
  16. Seufferlein, T., and Rozengurt, E. (1994) J. Biol. Chem. 269, 9345-9351 [Abstract/Free Full Text]
  17. Seufferlein, T., and Rozengurt, E. (1995) J. Biol. Chem. 270, 24343-24351 [Abstract/Free Full Text]
  18. Rankin, S., and Rozengurt, E. (1994) J. Biol. Chem. 269, 704-710 [Abstract/Free Full Text]
  19. Lacerda, H. M., Lax, A. J., and Rozengurt, E. (1996) J. Biol. Chem. 271, 439-445 [Abstract/Free Full Text]
  20. Kornberg, L., Earp, H. S., Parsons, J. T., Schaller, M., and Juliano, R. L. (1992) J. Biol. Chem. 267, 23439-23442 [Abstract/Free Full Text]
  21. Guan, J. L., and Shalloway, D. (1992) Nature 358, 690-692 [CrossRef][Medline] [Order article via Infotrieve]
  22. Burridge, K., Turner, C. E., and Romer, L. H. (1992) J. Cell Biol. 119, 893-903 [Abstract]
  23. Lipfert, L., Haimovich, B., Schaller, M. D., Cobb, B. S., Parsons, J. T., and Brugge, J. S. (1992) J. Cell Biol. 119, 905-912 [Abstract]
  24. Vuori, K., and Ruoslahti, E. (1993) J. Biol. Chem. 268, 21459-21462 [Abstract/Free Full Text]
  25. Kanner, S. B., Reynolds, A. B., Vines, R. R., and Parsons, J. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3328-3332 [Abstract]
  26. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399 [Medline] [Order article via Infotrieve]
  27. Rankin, S., Hooshmand-Rad, R., Claesson-Welsh, L., and Rozengurt, E. (1996) J. Biol. Chem. 271, 7829-7834 [Abstract/Free Full Text]
  28. Seckl, M. J., Morii, N., Narumiya, S., and Rozengurt, E. (1995) J. Biol. Chem. 270, 6984-6990 [Abstract/Free Full Text]
  29. Kanner, S. B., Reynolds, A. B., Wang, H.-C. R., Vines, R. R., and Parsons, J. T. (1991) EMBO J. 10, 1689-1698 [Abstract]
  30. Reynolds, A. B., Kanner, S. B., Wang, H.-C., and Parsons, J. T. (1989) Mol. Cell. Biol. 9, 629-638 [Medline] [Order article via Infotrieve]
  31. Matsuda, M., Mayer, B. J., Fukui, Y., and Hanafusa, H. (1990) Science 248, 1537-1539 [Medline] [Order article via Infotrieve]
  32. Birge, R. B., Fajardo, J. E., Mayer, B. J., and Hanafusa, H. (1992) J. Biol. Chem. 267, 10588-10595 [Abstract/Free Full Text]
  33. Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y., and Hirai, H. (1994) EMBO J. 13, 3748-3756 [Abstract]
  34. Olivier, A. R., and Parker, P. J. (1992) J. Cell Physiol. 152, 240-244 [Medline] [Order article via Infotrieve]
  35. Rodriguez, P. A., and Rozengurt, E. (1986) EMBO J. 5, 77-83 [Abstract]
  36. Rozengurt, E. (1991) Cancer Cells 3, 397-398 [Medline] [Order article via Infotrieve]
  37. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781 [Abstract/Free Full Text]
  38. Thastrup, O., Linnebjerg, H., Bjerrum, P. J., Knudsen, J. B., and Christensen, S. B. (1987) Biochim. Biophys. Acta 927, 65-73 [CrossRef][Medline] [Order article via Infotrieve]
  39. Petch, L. A., Bockholt, S. M., Bouton, A., Parsons, J. T., and Burridge, K. (1995) J. Cell Sci. 108, 1371-1379 [Abstract/Free Full Text]
  40. Vuori, K., and Ruoslahti, E. (1995) J. Biol. Chem. 270, 22259-22262 [Abstract/Free Full Text]
  41. Seifert, R. A., Hart, C. E., Phillips, P. E., Forstrom, J. W., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1989) J. Biol. Chem. 264, 8771-8778 [Abstract/Free Full Text]
  42. Heidaran, M. A., Pierce, J. H., Yu, J.-C., Lombardi, D., Artrip, J. E., Fleming, T. P., Thomason, A., and Aaronson, S. A. (1991) J. Biol. Chem. 266, 20232-20237 [Abstract/Free Full Text]
  43. Wennström, S., Siegbahn, A., Yokote, K., Arvidisson, A.-K., Heldin, C.-H., Mori, S., and Claesson-Walsh, L. (1994) Oncogene 9, 651-660 [Medline] [Order article via Infotrieve]
  44. Wennström, S., Hawkins, P., Cooke, F., Hara, K., Yonezawa, K., Kasuga, M., Jackson, T., Claesson-Welsh, L., and Stephens, L. (1994) Curr. Biol. 4, 385-393 [Medline] [Order article via Infotrieve]
  45. Wymanin, M., and Arcaro, A. (1994) Biochem. J. 298, 517-520 [Medline] [Order article via Infotrieve]
  46. Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567 [Abstract/Free Full Text]
  47. Yano, H., Nakanishi, S., Kimura, K., Hanai, N., Saitoh, Y., Fukui, Y., Nonomura, Y., and Matsuda, Y. (1993) J. Biol. Chem. 268, 25846-25856 [Abstract/Free Full Text]
  48. Jackson, T. R., Stephens, L. R., and Hawkins, P. T. (1992) J. Biol. Chem. 267, 16627-16636 [Abstract/Free Full Text]
  49. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell Sci. 108, 225-233 [Abstract/Free Full Text]
  50. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J. Biol. Chem. 269, 5241-5248 [Abstract/Free Full Text]
  51. Van der Bend, R., Bruner, J., Jalink, K., Van Corven, E. J., Moolenaar, W. E., and Van Blitterswijk, W. J. (1992) EMBO J. 11, 2495-2501 [Abstract]
  52. Seufferlein, T., and Rozengurt, E. (1995) J. Biol. Chem. 270, 24334-24342 [Abstract/Free Full Text]
  53. Rankin, S., Morii, N., Narumiya, S., and Rozengurt, E. (1994) FEBS Lett. 354, 315-319 [CrossRef][Medline] [Order article via Infotrieve]
  54. Miura, Y., Kikuchi, A., Musha, T., Kuroda, S., Yaku, H., Sasaki, T., and Takai, Y. (1993) J. Biol. Chem. 268, 510-515 [Abstract/Free Full Text]
  55. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekman, D., and Hall, A. (1992) Cell 70, 401-410 [Medline] [Order article via Infotrieve]
  56. Hall, A. (1990) Science 249, 635-640 [Medline] [Order article via Infotrieve]
  57. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Biol. 7, 337-374 [CrossRef]
  58. Vuori, K., Hirai, H., Aizawa, S., and Ruoslahti, E. (1996) Mol. Cell. Biol. 16, 2607-2613
  59. Nojima, Y., Morino, N., Mimura, T., Hamasaki, K., Furuya, H., Sakai, R., Sato, T., Tachibana, K., Morimoto, C., Yazaki, Y., and Hirai, H. (1995) J. Biol. Chem. 270, 15398-15402 [Abstract/Free Full Text]
  60. Hawkins, P. T., Eguinoa, A., Qui, R.-G., Stokoe, D., Cooke, F. T., Walters, R., Wennström, S., Claesson-Welsh, L., Evans, T., Symons, M., and Stephens, L. (1995) Curr. Biol. 5, 393-403 [Medline] [Order article via Infotrieve]
  61. Polte, T. R., and Hanks, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10678-10682 [Abstract]
  62. Harte, M. T., Hildebrand, J. D., Burnham, M. R., Bouton, A. H., and Parsons, J. T. (1996) J. Cell Biol. 271, 13649-13655
  63. Schaller, M. D., and Parsons, J. T. (1994) Curr. Opin. Cell Biol. 6, 705-710 [Medline] [Order article via Infotrieve]
  64. Rodriguez-Fernandez, J. L., and Rozengurt, E. (1996) J. Biol. Chem. 271, 27895-27901 [Abstract/Free Full Text]
  65. Matsuda, M., Hashimoto, Y., Muroya, K., Hasegawa, H., Kurata, T., Tanaka, S., Nakamura, S., and Hattori, S. (1994) Mol. Cell. Biol. 14, 5495-5500 [Abstract]
  66. 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]
  67. 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]
  68. Yoshida, Y., Kawata, M., Miura, Y., Musha, T., Sasaki, T., Kikuchi, A., and Takai, Y. (1992) Mol. Cell. Biol. 12, 3407-3414 [Abstract]
  69. Wienecke, R., Konig, A., and DeClue, J. E. D. (1995) J. Biol. Chem. 270, 16409-16414 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.