From the
p130
Integrins comprise the major class of receptors used by cells to
interact with the extracellular matrix(1, 2) .
Integrin/extracellular matrix protein interactions play a critical role
in a variety of biological processes, including embryonic development,
wound healing, tumor metastasis, and cell growth and
differentiation(2, 3) . It is now evident that integrins
can transduce biochemical signals across the plasma membrane to the
cell interior(3) . Integrins regulate many intracellular signals
including cytoplasmic alkalization, intracellular Ca
In the
present study, we have identified pp130
Using HSF cells, we have previously reported that cell
adhesion to FN, but not to the nonspecific substrate PLL, stimulates
tyrosine phosphorylation of several intracellular proteins including
130- (pp130), 120- (pp120), and 42- (pp42) kDa proteins(16) . We
identified pp120 and pp42 as Fak and MAP kinase, respectively. In the
present study, we have attempted to examine whether pp130 is identical
to Cas. Anti-Tyr(P) immunoblots of Nonidet P-40 lysates from HSF cells
after adherence to FN-coated plates for 30 min demonstrate
significantly higher tyrosine phosphorylation of 120-kDa (Fak) and
broad 130-kDa (pp130) proteins than cells attached to PLL-coated plates (Fig. 1A, lanes1 and 2). The
results are consistent with our previous report(16) , although
pp42 is not obvious in this experiment. From these extracts, Cas was
immunoprecipitated with rabbit anti-Cas serum (anti-Cas2) and
immunoblotted with anti-Tyr(P) (Fig. 1A). Anti-Cas2 (lanes3 and 4) but not normal
rabbit (lane5) sera precipitated a broad 130-kDa
protein whose tyrosine phosphorylation was clearly enhanced by adhesion
to FN. This 130-kDa phosphoprotein comigrated with pp130 in the total
cell extracts. Moreover, anti-Cas2 completely immunodepleted pp130 from
the total extracts of FN-adherent cells, while control serum did not (Fig. 1A, lanes6 and 7),
suggesting that protein(s) immunoprecipitated by anti-Cas2 represent
the vast majority of the pp130 in total cell lysates. To further
confirm the identity of pp130 as Cas, phosphotyrosyl proteins were
immunoprecipitated with anti-Tyr(P) from PLL- and FN-adherent cells and
probed with anti-Cas2 immunoblotting (Fig. 1B). We found
that pp130 in anti-Tyr(P) immunoprecipitates was clearly reactive with
anti-Cas2. These results confirm that pp130 is identical to Cas.
In addition to FN, other adhesive
ligands including vitronectin, laminin, and collagen were also potent
in inducing Cas phosphorylation (Fig. 2), indicating that the
response is not specific to FN. Our findings rather suggest that
tyrosine phosphorylation of Cas is a common event induced by cell
adhesion through various integrins as is the case of adhesion-dependent
Fak phosphorylation.
In the present paper, we have shown that integrin-mediated
adhesion of normal fibroblast cell lines stimulates tyrosine
phosphorylation of Cas in close association with Fak phosphorylation
and actin stress fiber formation. In contrast, extensive
phosphorylation of Cas found in cells transformed by v-Crk and v-Src
occurred independently from cell adhesion and subsequent changes of
cell morphology. Stoichiometrically, the amount of Cas that undergoes
tyrosine phosphorylation after FN-binding by normal fibroblasts was far
less than that in transformed cells. Most of the Cas remained
unphosphorylated in untransformed HSF and 3Y1 cells even after adhesion
to FN. Therefore, one might argue that Cas does not play a major role
in integrin-mediated signal transduction. It is possible, however, that
the phosphorylated Cas, even in a small amount, may interact with
signaling molecules having an affinity for Cas through their SH2
domains. Such multimolecular complexes may amplify and propagate
integrin-mediated signals downstream. Demonstrating interactions
between Cas and Cas-associated molecules and determining their
downstream effects will be necessary to ascertain whether Cas is really
an important player in the integrin-mediated signaling.
We have not
yet identified the kinase(s) that phosphorylates Cas in the process of
cell adhesion or transformation. Recently, in an attempt to search
optimal peptide substrates for distinct tyrosine kinases using a
degenerate peptide library, Songyang et al.(24) have
identified the (I/V)YXXP sequence as a good candidate for the
motif that preferentially binds to the kinase domain of the c-Abl
cytoplasmic protein kinase(24) . Intriguingly, the
(I/V)YXXP motif appears a dozen times in Cas(21) . This
would predict that Cas should be an excellent substrate for c-Abl.
c-Abl was also shown to constitutively bind to the SH3 domain of v-Crk
(25), while v-Crk was stably associated with phosphorylated
Cas(21) . Thus, the formation of trimolecular complex between
these signaling molecules may result in the hyperphosphorylation of Cas
in v-Crk-transformed cells. In addition, c-Abl possesses binding
domains in its C-terminal portion for F-actin
cytoskeletons(26) . In this regard, our finding that Cas
phosphorylation was sensitive to disruption of actin stress fibers by
cytochalasin D favors the view that c-Abl may be the responsible
kinase. An alternative candidate for regulating Cas phosphorylation
appears to be c-Src. Our data showed that tyrosine phosphorylation of
Fak and Cas is closely related, suggesting that a common mechanism may
operate in this process. Indeed, the SH2 domain of v-Src tightly binds
to tyrosine-phosphorylated Cas and Fak both in vivo and in
vitro(17, 18, 21) . A binding portion
within Fak for Src-SH2 domain has been identified as Tyr-397, which
corresponds to an autophosphorylation site of this kinase(18) .
Moreover, Schlaepfer et al.(15) have recently
demonstrated that integrin-mediated cell adhesion of NIH3T3 cells
induced autophosphorylation of Fak and subsequent binding of Fak to
c-Src. Fak and c-Src association has been postulated to result in the
activation of Src kinase activity by dissociating a negatively
regulatory C terminus of c-Src from its own SH2
domain(15, 18) . c-Src activated in this fashion may
further phosphorylate Fak (15) and, on the other hand, may
induce Cas phosphorylation. In addition to the (I/V)YXXP
motif, a putative c-Abl substrate, Cas contains two candidate motifs
(YDNV and YDYV) that are suitable for binding to the Src-SH2
domain(21) . In accordance with this, we have detected a trace
amount of c-Src associating with phosphorylated Cas in 3Y1-Crk
cells(21) . So far, however, we have failed to detect c-Src or
any other kinase activities associating with Cas in normal fibroblast
cells. This may be due to the relatively small amount of phosphorylated
Cas contained in normal cells even after adhesion to FN (Fig. 5A). Thus, the kinases responsible for Cas
phosphorylation during the process of integrin ligation remained to be
determined.
In summary, herein we presented a novel aspect of
integrin-mediated signal transduction. Our data further support the
notion that cell adhesion and transformation share common signaling
molecules(10) . This provides a fundamental basis for the
regulation of cell growth and differentiation by the extracellular
matrix protein. Identification of responsible kinases and downstream
elements that bind phosphorylated Cas will be an important issue to
fully understand signaling pathways triggered by integrin ligation.
We thank Dr. David Rothstein (Yale University, New
Haven, CT) for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(Cas) has been recently identified as a
130-kDa protein that is highly phosphorylated on tyrosine residues and
is stably associated with p47
(v-Crk) and p60
(v-Src)
oncogene products in cells transformed by the respective genes. Cas is
a novel signaling molecule having a single Src homology (SH) 3 domain
and a cluster of multiple SH2-binding motifs. While the tight
association of Cas with v-Crk and v-Src is strongly suggestive of a
significant role in regulating cellular transformation, the function of
Cas in normal untransformed cells is totally unknown. We report here
that cell adhesion to fibronectin rapidly promotes tyrosine
phosphorylation of Cas in human and rat fibroblast cell lines. The
response was equally induced by cell adhesion to plates coated with
vitronectin, laminin, and collagen but not by cell attachment to
nonspecific substrate poly-L-lysine. The kinetic profile of
Cas phosphorylation was almost identical with that of tyrosine
phosphorylation of focal adhesion kinase pp125
(Fak),
which is well known to be activated subsequent to integrin-mediated
cell adhesion. Adhesion-dependent Cas phosphorylation was completely
inhibited by treating cells with cytochalasin D, an agent that disrupts
polymerization of actin stress fibers. These results suggest that
tyrosine phosphorylation of Cas is stimulated by normal cell adhesion
in close association with Fak phosphorylation and the formation of
actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the
tyrosine phosphorylation of Cas is markedly increased in an
adhesion-independent manner that is insensitive to treatment with
cytochalasin D. Thus, Cas plays a role in signaling pathways mediated
by cell adhesion as well as by transformation. We propose that Cas may
amplify and propagate integrin-mediated signals by interacting with
SH2-containing molecule(s).
levels, and induction of gene
expression(3, 4, 5) . Recent evidence indicates
that integrin-mediated signaling pathways also involve a cascade of
tyrosine kinases and phosphorylation events (6-11). Engagement of
cell surface integrins has been shown to rapidly stimulate tyrosine
phosphorylation of several intracellular proteins, including paxillin,
tensin, focal adhesion kinase pp125
(Fak), and
mitogen-activated protein (MAP)
(
)kinases(11, 12, 13, 14, 15, 16) .
Among these, Fak has received the most attention, since it constitutes
a novel family of protein tyrosine kinases and is activated by integrin
clustering on the cell surface(9, 10, 11) . Once
phosphorylated on its tyrosine residues, Fak has been shown to bind to
SH2 domains of Src family proteins and of phosphatidylinositol 3-kinase
(17-19). It is proposed that binding of these kinases to Fak may
result in their enzymatic activation(18, 19) . Moreover,
recent evidence indicates that integrin-mediated cell adhesion also
promotes SH2-mediated association of the Grb2/Ash adaptor protein with
Fak(15) . Since Grb2 is constitutively associated with the Ras
GDP/GTP exchange protein Sos, the formation of Fak
Grb2
Sos
complex may lead to an accumulation of GTP-Ras followed by sequential
activation of a serine/threonine kinase cascade including Raf, MAP
kinase kinase, and MAP kinase(15) . Indeed, we and others have
recently reported that MAP kinase activation occurs in response to
integrin-mediated cell adhesion (14-16). All of these
observations indicate that signal transduction events elicited by
integrin ligation are similar to those stimulated by growth factor
binding to receptor tyrosine kinases, in which protein-protein
interactions through SH2 and SH3 domains are essential (20).
(Cas) as another
molecule that participates in integrin-mediated signaling cascades. Cas
was originally identified as a 130-kDa protein that is highly
phosphorylated on tyrosine residues in cells expressing transforming
gene products p47
(v-Crk) and
p60
(v-Src)(21, 22) . The amino acid sequence
deduced from cDNA encoding this molecule revealed that Cas is a novel
SH3-containing molecule with a cluster of multiple putative SH2-binding
motifs(21) . Tyrosine phosphorylation of Cas occurs after
cellular transformation by v-Crk and v-Src but not by other
transforming genes such as v-K-ras. Cas forms a stable complex
with v-Crk and v-Src in vivo in a phosphorylation-dependent
manner. Moreover, immune complex kinase assays have shown that Cas is a
major substrate component of both v-Crk and v-Src in vitro (21). These results have strongly suggested that Cas plays a role
in signaling pathways of cellular transformation triggered by v-Crk and
v-Src. Little is yet known, however, about physiological stimuli that
promote Cas phosphorylation in normal untransformed cells. Here we
demonstrate that cell adhesion to matrix proteins of normal fibroblast
cells induces a significant increase in the tyrosine phosphorylation of
Cas. Our findings suggest that Cas mediates integrin-mediated signals
by assembling multiple SH2-containing molecules.
Reagents
Human fibronectin (FN), vitronectin,
laminin and type I collagen, and rat FN were all purchased from Telios
(San Diego, CA). Anti-phosphotyrosine antibody (anti-Tyr(P)) (4G10) was
obtained from UBI Laboratories (Lake Placid, NY).
Poly-L-lysine (PLL), and cytochalasin D were obtained from
Sigma. Rabbit anti-p130 protein serum (anti-Cas2) was
developed in our laboratory and described previously (21). Rabbit
anti-serum against FAK was developed by immunizing rabbits with
GST-human Fak C-terminal protein.
(
)
Cell Culture
Human skin fibroblasts (HSF) were
established as described previously(16) . Cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and
50 µg/ml streptomycin. 3Y1 is a rat fibroblast cell line and is
maintained in 10% fetal calf serum/Dulbecco's modified
Eagle's medium. SR-3Y1 is a 3Y1 cell line transformed by v-Src of
Rous sarcoma virus(21) . 3Y1-Crk is an isolated clone of rat 3Y1
cells transfected with v-crk cDNA of an avian sarcoma
virus, ASV-1, inserted in the pMV-7 expression vector(21) .
Cell Adhesion and Preparation of Cell
Lysates
Preparation of culture dishes coated with adhesive
ligands, PLL, and mAbs was described previously(8, 16) .
Confluent cells were detached by treating with 0.05% trypsin/EDTA,
followed by washing 3 times with serum-free Dulbecco's modified
Eagle's medium. Cells were then plated onto dishes coated with
different reagents as indicated and incubated at 37 °C for the
indicated time periods in serum-free Dulbecco's modified
Eagle's medium. Bound cells were lysed in situ with 1%
Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 mM iodoacetamide, 10 mM NaF, 10 mM sodium pyrophosphate, and 0.4 mM sodium vanadate). After removing insoluble materials by
centrifugation at 14,000 rpm for 10 min, protein concentrations in the
supernatant were determined using micro BCA protein assay kit (Pierce,
Rockford, IL). Cell lysates were stored at -70 °C until use.
Immunoblotting and Immunoprecipitation
Cell
lysates were loaded on 7.5% SDS-polyacrylamide gels in reducing
conditions. Proteins on the gel were electrotransfered to
nitrocellulose membranes. Anti-Tyr(P) immunoblotting was performed
according to methods described previously(16) . For
immunoprecipitations, cell extracts were incubated with a 1/100
dilution of anti-Cas2 or anti-Fak sera for 1 h at 4 °C followed by
additional incubation with protein A-Sepharose beads for 1 h at 4
°C. Beads were then washed 5 times with 1% Nonidet P-40 lysis
buffer to remove unbound proteins. Immune complexes were treated with
sample buffer and subjected to SDS-polyacrylamide gel electrophoresis
for immunoblotting with anti-Tyr(P) or anti-Cas2.
Figure 1:
Tyrosine phosphorylation of Cas is
induced by cell adhesion to FN. A, HSF cells were allowed to
adhere to PLL (P; lanes1 and 3)
and FN (F; lanes2, 4, and 5) for 30 min. Total cell extracts (lanes1 and 2) and immunoprecipitates with anti-Cas2 (lanes3 and 4) or control sera (lane5) were all probed by immunoblotting with anti-Tyr(P).
Supernatants of anti-Cas2 immunoprecipitates (lane6)
and of control anti-serum immunoprecipitates (lane7)
from FN-adherent cell extracts were also subjected to anti-Tyr(P)
blotting. B, phosphotyrosyl proteins were immunoprecipitated
with anti-Tyr(P) from PLL- (P; lane1) and
FN-adherent (F; lane2) HSF cells and then
probed with anti-Cas2. C, anti-Cas2 immunoprecipitates from
HSF cells adhered to PLL (P; lanes1 and 3) and FN (F; lanes2 and 4) for 30 min were immunoblotted with either anti-Tyr(P) (lanes1 and 2) or anti-Cas2 (lanes3 and 4). D, HSF cells were allowed to
adhere to plates coated with anti-MHC class I (lane1) and anti-integrin 1 (lane2)
antibodies for 30 min. Cas was immunoprecipitated from these extracts
and probed with anti-Tyr(P).
In
normal fibroblast cells of rat (3Y1) and mouse (NIH3T3) origin, Cas has
been detected as two bands at 115 and 125 kDa (Cas-A and Cas-B,
respectively)(21) . Cellular transformation by v-Crk or v-Src,
however, is associated with a decrease in Cas-A, and the simultaneous
appearance of a broad 130 kDa band (Cas-C). Since tyrosine
phosphorylation is found mostly in Cas-C, Cas-C appeared to be a
modified form of Cas-A or Cas-B with a retarded gel motility secondary
to extensive phosphorylation at multiple sites(21) . Most of the
Cas in HSF is also present as 115- and 125-kDa proteins (Cas-A and
Cas-B), as shown in Fig. 1C, lanes3 and 4. However, cells adherent to FN exhibit a faint
broad band at 130 kDa that is hardly detectable in cells adherent to
PLL. This band corresponds to tyrosine phosphorylated forms of Cas
(Cas-C) (Fig. 1C, lanes1 and 2). In contrast to v-Crk- or v-Src-transformants(21) ,
HSF cell adhesion did not result in a significant change in the amounts
of Cas-A/B. This suggests that only a small proportion of Cas is
tyrosine phosphorylated upon FN adherence by normal untransformed
cells. Tyrosine phosphorylation of Cas was similarly induced by HSF
cell adhesion to culture plates coated with anti-1 integrin
antibody (anti-4B4) but not with anti-MHC class I antibody (W6/32) (Fig. 1D, upperpanel). Duplicate
filters were blotted with anti-Cas2 (Fig. 1D, lowerpanel). A faint band representing Cas-C was again
detectable in cells adherent to anti-4B4-coated plates. Moreover,
pretreatment of cells with soluble anti-
1 integrin antibody
inhibited tyrosine phosphorylation as well as cell adhesion to FN (data
not shown). These results suggest that Cas phosphorylation induced by
adherence to FN is at least partially dependent on
1 integrins.
Taken together, our results indicate that cell adhesion to FN and
ligation of
1 integrins by immobilized antibody stimulates
tyrosine phosphorylation of Cas.
Figure 2:
Tyrosine phosphorylation of Cas is induced
by HSF adhesion to vitronectin, laminin, and collagen. Cas was
immunoprecipitated with anti-Cas2 from lysates of HSF cells adhered to
PLL (lane1), vitronectin (lane2),
laminin (lane3), and collagen (lane4), followed by anti-Tyr(P)
immunoblotting.
We next examined kinetics of Cas
phosphorylation induced by HSF adhesion to FN. Tyrosine phosphorylation
of Cas was detectable 10 min after starting cell culture on FN-coated
plates and reached maximal levels in 40-80 min (Fig. 3A, upperpanel). Duplicate
filters were probed with anti-Cas2 to confirm that the same amount of
Cas was loaded in each lane (data not shown). We compared kinetics of
tyrosine phosphorylation of Cas and Fak (Fig. 3A, lowerpanel) induced by FN adherence and present the
results in Fig. 3B by densitometric analysis of the
bands obtained by immunoblotting. The overall kinetics of
phosphorylation of both proteins were quite similar, suggesting that
phosphorylation of Fak and Cas are related in cell adhesion-mediated
signaling pathways.
Figure 3:
Adhesion-dependent tyrosine
phosphorylation of Cas and Fak. A, Cas (upperpanel) and Fak (lowerpanel) were
immunoprecipitated from lysates of nonadherent HSF cells and cells
adhered to plates coated with FN for indicated periods.
Immunoprecipitates were then immunoblotted with anti-Tyr(P). B, densitometric scanning of adhesion-dependent tyrosine
phosphorylation of Cas (opencircles) and Fak (closedcircles). Data represent the percentage of
control (nonadherent cells, 0 min).
Following attachment to substrata, cells alter
their shape and spread by developing actin stress fibers(23) .
Such morphological changes are generally preceded by tyrosine
phosphorylation of proteins including Fak(11, 16) .
Meanwhile, cytochalasin D that disrupts actin polymerization has been
shown to inhibit adhesion-dependent tyrosine phosphorylation of Fak or
MAP kinase(14, 16) . Thus, protein tyrosine
phosphorylation induced by cell adhesion is closely linked with
cytoskeletal organization. In the previous study(16) , we showed
that adhesion-dependent tyrosine phosphorylation of pp130 in HSF cells
was completely abrogated by treating cells with cytochalasin D. In
accordance with this, the adhesion-dependent increase of Cas
phosphorylation, determined by anti-Tyr(P) blotting of anti-Cas2
immunoprecipitates, was inhibited by cytochalasin D in a dose-dependent
manner (Fig. 4). Duplicate filters were blotted with ant-Cas2,
showing that cytochalasin D did not affect the amount of Cas (data not
shown). Thus, cytoskeleton organization is required for
adhesion-induced tyrosine phosphorylation of Cas as well as of Fak and
MAP kinase.
Figure 4:
Inhibition of adhesion-dependent Cas
phosphorylation by treating cells with cytochalasin D. HSF cells in
suspension were pretreated for 5 min with the indicated concentrations
of cytochalasin D prior to being added to FN-coated dishes. The
viability of HSF cells was not affected over this dose range of
cytochalasin D. Cells were then allowed to adhere to FN for 30 min in
the continuous presence of cytochalasin D (lanes3-6). Untreated cells were also cultured in PLL-
and FN-coated plates in the absence of cytochalasin D (lanes1 and 2, respectively). Bound cells were lysed
with Nonidet P-40 lysis buffer, immunoprecipitated with anti-Cas2, and
immunoblotted with anti-Tyr(P).
Cas was originally identified as a protein highly
phosphorylated on tyrosyl residues in v-Crk- and v-Src-transformed
cells(21) . We examined, therefore, whether Cas phosphorylation
was dependent on cell adhesion in these transformants. Following
attachment to FN-coated plates, wild-type 3Y1 cells and 3Y1 cells
transformed by v-Crk (3Y1-Crk) gradually developed membrane processes
on the substrata (data not shown). After culturing for 30 min, more
than 90% of cells changed their shape by spreading on the FN-coated
plates, while cells plated on PLL maintained a rounded shape although
they tightly bound to the substrata. These morphological changes
suggest that cells are forming focal adhesions upon adhesion to FN,
thereby developing actin stress fibers. In contrast, 3Y1 cells
transformed by v-Src (SR-3Y1) poorly developed membrane processes even
when cultured on FN-coated plates (data not shown). Most cells remained
to have a rounded appearance, and the relatively weak cell spreading
occurred in less than 20% of this transformant. Thus, morphological
responses induced by cell adhesion were different between two types of
transformants. Cas was immunoprecipitated from lysates (1 mg of total
protein) of wild-type and transformed cells before (in suspension) and
after adhesion to FN. Immunoprecipitates were then probed with
anti-Tyr(P) immunoblotting. Like in HSF cells, tyrosine phosphorylation
of Cas is significantly enhanced by adhesion to FN by wild-type 3Y1
cells (Fig. 5, lanes1 and 2). In both
transformants, however, Cas was highly phosphorylated even when cells
were in suspended conditions (lanes3 and 5). FN adherence did not affect levels of phosphorylation of
Cas (lanes4 and 6). Anti-Cas2 immunoblots
of the same sets of immunoprecipitates (data not shown) revealed that
Cas in these transformants is mostly present as Cas-C and Cas-B with
decreased amounts of Cas-A, as described previously(21) . The
relative amounts of the different forms of Cas did not change after FN
binding. Moreover, treating cells with 3 µM cytochalasin
D, capable of suppressing adhesion-dependent Cas phosphorylation in
normal 3Y1 cells to the basal levels, had no effect on the increased
phosphorylation of Cas in the transformed cells (Fig. 5B). These results indicate that tyrosine
phosphorylation of Cas is constitutively enhanced in these
transformants, independent from cell adhesion, actin organization, and
morphological alterations.
Figure 5:
Cas is constitutively tyrosine
phosphorylated in v-Src- and v-Crk-transformants in an
adhesion-independent manner. A, wild-type 3Y1, SR-3Y1, and 3Y1-Crk were allowed to adhere to FN for
30 min. Cas was immunoprecipitated from extracts of these adherent
cells (F; lanes2, 4, and 6) and from cells in suspension (S; lanes1, 3, and 5) and then probed with
anti-Tyr(P) immunoblotting. B, cells adherent to FN in the
absence (opencolumns) or in the presence (closedcolumns) of 3 µM cytochalasin D were
immunoprecipitated with anti-Cas2 and probed with anti-Tyr(P)
immunoblotting. These bands were analyzed by a densitometric scanner,
and data were presented as the percentage of controls (in the absence
of cytochalasin D; 100%) in each cell line.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.