(Received for publication, November 14, 1996, and in revised form, January 16, 1997)
From the Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
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.
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.
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.
ImmunoprecipitationQuiescent 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 BlottingTreatment 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 PKCPhorbol 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.
MaterialsBombesin, 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.
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.
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.
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).
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 p130casAs 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.
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).
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 ComplexNext, 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).
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 p130casPDGF, 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%.
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.
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.
We thank Dr. T. Seufferlein and J. Sinnet-Smith for valuable discussions.