(Received for publication, August 8, 1994; and in revised form, October 6, 1994)
From the
Cell adhesion to extracellular matrix proteins is a dynamic
process leading to dramatic changes in the cell phenotype. Integrins
are one of the major receptor families that mediate cell-matrix
contact. Evidence that integrins can act as signal transducing
molecules has accumulated over the past few years. We report here that
p44 and p42
mitogen-activated protein
(MAP) kinases are rapidly phosphorylated on tyrosine residues upon
adhesion of human skin fibroblasts to fibronectin or upon cross-linking
of
1 integrins with antibody. The tyrosine phosphorylation of both
kinases is associated with increased enzymatic activity. Pretreatment
of the cells with cytochalasin D, which selectively disrupts the
network of the actin filaments, completely inhibits this
adhesion-mediated MAP kinase activation. Thus, our findings indicate
that ligation of
1 integrins induces an increase in both tyrosine
phosphorylation and enzymatic activity of p44
and
p42
MAP kinases, and that the integrity of the actin
cytoskeleton is essential in this process. Since MAP kinase behaves as
a convergence point for diverse receptor-initiated signaling events at
the plasma membrane, this serine/threonine kinase plays a key role and
helps to account for the diversity of integrin-dependent cell
functions.
Integrins are transmembrane, heterodimeric glycoproteins
consisting of an and a
subunit. They function as cell
surface receptors for extracellular matrix proteins (ECM) (
)and also mediate cell-to-cell
interaction(1, 2) . It has been well established that
cell adhesion to ECM or to opposing cells through integrins results in
marked alterations of cell shape, motility, growth, and differentiation (2, 3, 4, 5, 6, 7, 8, 9) .
These facts have strongly suggested that integrins could transmit
signals into the cell interior and hence have urged researchers in
various biological fields to investigate signal transduction pathways
triggered by cell adhesion(2, 3, 10) . Such
efforts have lead to the notion that protein tyrosine phosphorylation
plays a critical role in signal transduction via integrins. A number of
reports (11, 12, 13) have documented that
engagement of
1 integrins stimulated tyrosine phosphorylation of
several proteins in a variety of cell types. Focal adhesion kinase
pp125
, originally identified as a putative substrate for
a retroviral oncogene product,
pp60
, has been shown to be
tyrosine-phosphorylated in response to the ligation of
1 or
3
integrins(14, 15, 16, 17) .
pp125
itself is a protein tyrosine kinase which is
specifically located in the focal adhesion. Recent studies demonstrated
that autophosphorylation of pp125
creates a site having
strong affinity for the Src homology 2 (SH2) domain of Src family
tyrosine kinases such as pp60
and
pp59
(18, 19) . It is proposed
that binding of pp60
and pp59
to pp125
may result in their enzymatic
activation, and additionally provides a mechanism for the recruitment
of these Src family kinases to specific sites within the
cell(19) . Thus, the focal adhesion is highly abundant in
interacting signaling molecules, which would provide a structural and
biochemical basis for the diversity of integrin-dependent cell
function. However, links between biochemical events occurring just
beneath the cell membrane and the nucleus in the integrin-mediated
signaling cascade are still largely unknown(3) .
Mitogen-activated protein kinase (referred to as MAP kinase or extracellular signal-regulated kinase (ERK)) is a serine/threonine protein kinase whose activity is rapidly stimulated by a number of external stimuli through mechanisms mediated by tyrosine kinase-encoded receptors, non-receptor type tyrosine kinases, and G protein-coupled receptors(20, 21, 22) . MAP kinase is activated by phosphorylation on its threonine and tyrosine residues, a process which is carried out by a dual-specificity protein kinase, MAP kinase kinase(23) . MAP kinases have been shown to phosphorylate and thereby activate many well studied regulatory proteins located in diverse cellular compartments, including nuclear transcriptional factors(20, 21, 22, 24, 25) . A function of MAP kinases may therefore be to provide a link between transmembrane signaling and the nucleus.
We report here that two
isoforms of MAP kinase, p44 and p42
,
are activated in response to the adherence of human skin fibroblasts
(HSF) to fibronectin (FN) or by cross-linking of
1 integrins with
antibody. Our findings indicate that MAP kinases may act as a key
molecule connecting cell surface integrins to the expression of genes
associated with a variety of adhesion-dependent cell functions.
Recent studies have shown that the engagement of 1
integrins with components of ECM stimulated tyrosine phosphorylation of
several cellular
proteins(11, 12, 13, 15, 16, 27, 28, 29) .
Some of these phosphoproteins have been identified as tensin, paxillin,
and focal adhesion kinase pp125
, whereas others (such as
pp105 and pp130) have not been fully characterized. In an attempt to
determine molecules which are involved in the signaling cascade of
1 integrins, we chose HSF cells, since adhesion-induced tyrosine
phosphorylation could be clearly detected in this cell type. As shown
in Fig. 1A (lanes 1 and 2), culturing
HSF cells in dishes that had been coated with human FN-induced
increased tyrosine phosphorylation of 190-kDa (pp190), 130-kDa (pp130),
120-kDa (pp120), 70-kDa (pp70), and 42-kDa (pp42) proteins. Tyrosine
phosphorylation of these proteins was detectable 5 min after starting
cell culture, and reached maximal levels in 20-40 min (Fig. 1B, lanes 1-4). HSF cells cultured
in dishes coated with PLL failed to exhibited this response, suggesting
that enhanced phosphorylation was not induced by binding to nonspecific
substrates (Fig. 1B, lanes 5-8).
Moreover, pretreatment of cells with soluble anti-
1 integrin
antibody (4B4) inhibited tyrosine phosphorylation as well as cell
adhesion to FN (data not shown). To further verify the involvement of
1 integrin in this response, we cultured HSF cells on dishes
coated with anti-4B4 to cross-link the
1 integrins, and examined
protein tyrosine phosphorylation by anti-phosphotyrosine
immunoblotting. As shown in Fig. 1A (lanes 3 and 4), adhesion of HSF cells to immobilized anti-
1
integrin but not to anti-MHC class I antibodies resulted in enhanced
tyrosine phosphorylation of the same set of proteins found in
FN-adherent cells. These results indicate that engagement of
1
integrins with antibody stimulates signaling pathways closely related
to those triggered by cell adhesion to FN. While this is consistent
with the notion that tyrosine phosphorylation induced by adherence to
FN is at least partially dependent on
1 integrins, we have not
ruled out the possibility that other classes of integrins or other
non-integrin molecules contribute to this response. We examined no
fewer than five strains of HSF cells derived from different donors, and
obtained essentially identical results (data not shown). By
immunoprecipitation using anti-pp125
(2A7), we have
confirmed the identity of pp120 as pp125
(data not
shown).
Figure 1:
Tyrosine phosphorylation induced by
engagement of 1 integrins. A, HSF cells were allowed to
adhere to plates that had been coated with PLL (lane 1), FN (lane 2), anti-MHC class I (lane 3), or anti-
1
integrin (lane 4) antibodies. After 30 min of incubation,
bound cells were lysed and processed for anti-phosphotyrosine
immunoblotting. B, HSF cells were cultured on plates coated
with FN (lanes 1-4) or PLL (lanes 5-8) at
37 °C for indicated periods. Bound cells were lysed and then
processed for anti-phosphotyrosine
immunoblotting.
p44 and p42
MAP kinases are
serine/threonine kinases whose enzyme activity can be stimulated by
phosphorylation on tyrosine as well as threonine residues of this
molecule(20, 21, 22, 23) . Similar
molecular size and a crucial role of MAP kinases in gene regulation
prompted us to examine whether pp42 in the present study was identical
to MAP kinase. We used
Y91 to immunoprecipitate MAP kinase from
lysates prepared from HSF cells plated on FN.
Y91 antiserum was
raised against a synthetic peptide corresponding to residues
307-327 of the amino acid sequence deduced from the p44
cDNA, and has been shown to recognize both p44
and p42
MAP kinases in the presence of 0.15% of
SDS(26) . Immunoprecipitates were then subjected to
immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2A, upper panel, enhanced tyrosine
phosphorylation of 42- and 44-kDa proteins was induced by adherence to
FN in a time-dependent manner. Duplicate filters were analyzed by
immunoblotting with
Y91, to verify that the same amount of the MAP
kinases were loaded (Fig. 2A, lower panel).
pp42 identified in the whole cell lysates was found to comigrate with
p42
in
Y91 immunoprecipitates (data not shown). Fig. 2B shows the results obtained by densitometric
scanning of the transblot bands in Fig. 2A. Tyrosine
phosphorylation of MAP kinases in the immunoprecipitates occurred as
early as 5 min after plating cells onto FN-coated dishes, and the
overall kinetics were identical to that of pp42 in the total cell
lysates (Fig. 1B and Fig. 2, A and B). Maximal response, obtained at 20 min after cell adhesion,
was 4-5-fold increased from the basal levels. Similar results
were obtained after anti-
1 integrin cross-linking (data not
shown). Taken together, tyrosine phosphorylation of both isoforms of
MAP kinase were significantly enhanced by cell adherence to immobilized
FN or anti-
1 integrin antibody.
Figure 2:
Adherence-dependent tyrosine
phosphorylation of MAP kinases. A, MAP kinases were
immunoprecipitated with Y91 antiserum in the presence of 0.15% SDS
from lysates of non-adherent fibroblasts (lane 1) and
fibroblasts adhered to plates coated with FN for indicated periods (lanes 2-5). Immunoprecipitates were separated on
SDS-PAGE and probed with anti-phosphotyrosine antibody (upper
panel). Duplicate filters were probed with
Y91 to show that
the same amount of the MAP kinases were loaded into each lane (lower panel). B, densitometric scanning of transblot
bands, shown in Fig. 2A, of p44 (open circle)
and p42 (closed circle). Data represent the percentage of
control (non-adherent cells, 0 min).
Next, we examined whether
adhesion-induced tyrosine phosphorylation of MAP kinases was associated
with an increase in kinase activity. First, we measured the enzyme
activity of the MAP kinases in whole cell extracts using MBP as a
substrate. Fig. 3A shows the kinetics of MBP kinase
activity detected in cell extracts from HSF cells adhered to FN or PLL.
MBP kinase activity was significantly increased in HSF cells plated on
FN compared with those bound to PLL. The time course was again
comparable to that of tyrosine phosphorylation of MAP kinases described
above ( Fig. 2and 3A). To further verify the activation
of MAP kinases, we performed MAP kinase assays in MBP-containing gels
after SDS-PAGE. Extracts of HSF cells that had adhered to FN or PLL for
30 min were immunoprecipitated with Y91 antiserum. The
immunoprecipitates were then electrophoresed in an SDS-polyacrylamide
gel containing MBP. After denaturation and renaturation, the gel was
incubated with [
-
P]ATP and
Mg
. We observed increased MBP kinase activity
migrating at both 42 and 44 kDa after cell adhesion to FN but not to
PLL (Fig. 3B, lanes 1 and 2). Clear
distinction between these two bands, readily apparent on the original
autographs, is more difficult to resolve after reproduction. Duplicate
filters were immunoblotted with
Y91 to confirm that the same
amounts of MAP kinases were loaded (data not shown). MBP kinase
activity was significantly higher in HSF cells adhered to immobilized
anti-
1 integrin than those attached to anti-MHC class I antibodies (Fig. 3B, lanes 3 and 4).
Densitometric analysis revealed that the MBP kinase activity of both
kinases were increased by 3.5- and 2.4-fold after cell adhesion to
dishes coated with FN and anti-4B4, respectively. These results
indicated that the kinase activities of both p44
and
p42
MAP kinases were increased after cell adhesion to
FN, and that this response could be duplicated by the ligation of
1 integrins with antibody.
Figure 3:
MAP kinase activation induced by cell
adhesion to FN. A, fibroblasts were allowed to adhere to
plates coated with PLL (closed circles) or FN (open
circles) for the indicated time periods. MBP phosphotransferase
activity in the cell extracts was measured. Data represent the average
percentage of control (non-adherent cells) from three independent
experiments (*p < 0.05,**p < 0.01 versus adherence to PLL). B, fibroblasts adhered to PLL (lane 1), FN (lane 2), anti-MHC class I (lane
3), and anti-1 integrin monoclonal antibodies (lane
4) for 30 min were lysed, and the supernatants were
immunoprecipitated with
Y91 in the presence of 0.15% SDS. The
immunoprecipitates were electrophoresed in an SDS-polyacrylamide gel
containing MBP, and kinase assays were performed as described under
``Experimental Procedures.'' Note that the enzyme activity
was detected in both the 42- and 44-kDa bands in the
gel.
The association of integrin subunits
with cytoskeletal proteins is thought to contribute to the formation of
focal adhesions and actin stress fiber
organization(2, 3, 30) . In accordance with
this model, cytochalasin D, an agent which disrupts actin
polymerization, has been shown to prevent adhesion-dependent tyrosine
phosphorylation(28, 29) . Therefore, we next examined
whether cytochalasin D inhibited the MAP kinase activation induced by
adhesion of HSF cells to FN. 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. Bound cells were lysed with Nonidet P-40 lysis buffer,
and processed for anti-phosphotyrosine immunoblotting or MAP kinase
assays. As shown in Fig. 4A, the adherence-induced
tyrosine phosphorylation of pp130, pp120, and pp42 was inhibited by
treatment with cytochalasin D in a dose-dependent manner. Three
µM cytochalasin D, which was capable of suppressing
tyrosine phosphorylation to basal levels (Fig. 4A, lane 6), was also sufficient to depolymerize the network of
actin filaments in HSF cells and thereby inhibit cell spreading (data
not shown). Along with the failure of tyrosine phosphorylation and cell
spreading, adherence-induced MAP kinase activation detected by the
kinase assay in MBP-containing gels was completely prevented by
treating cells with 3 µM of cytochalasin D (Fig. 4B). Immunoblotting of duplicate filters with
Y91 confirmed that the same amount of MAP kinases were loaded in
each lane (data not shown). These results indicate that the integrity
of actin fibers is essential for adhesion-induced MAP kinase activation
as well as tyrosine phosphorylation of other proteins.
Figure 4:
Inhibition of MAP kinase activation by
cytochalasin D. A, Cells bound to PLL (lane 1) or FN (lane 2) in the absence of cytochalasin D, and cells bound to
FN (lane 3-6) in the presence of the indicated
concentrations of cytochalasin D were lysed, and the supernatants were
subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibody. B, extracts from cells adhered to PLL (lane
1), and extracts from cells adhered to FN in the absence (lane
2) and in the presence (lane 3) of cytochalasin D (3
µM) were immunoprecipitated with Y91. The
immunoprecipitates were subjected on SDS-polyacrylamide gels containing
MBP, and MBP kinases assays were performed in the
gel.
There is much
evidence that MAP kinases may be involved in cell growth and
differentiation by phosphorylating and thereby activating nuclear
transcriptional
factors(20, 21, 22, 24, 25) .
Meanwhile, there are many compelling examples of gene expression
induced by adhesive interactions with
ECM(6, 7, 8, 9) . Thus, MAP kinase
may play a pivotal role in adhesion-dependent gene activation through
the integrin-mediated signaling pathways. The fact that MAP kinase is
activated by both growth factor- and integrin-mediated signals suggests
that these two signaling pathways converge at this or upstream points.
In this regard, it is interesting to note that pp125 which was the molecule discovered to undergo tyrosine
phosphorylation after integrin-mediated signals, has now been found to
also undergo tyrosine phosphorylation after stimulation with several
growth factors(31) . However, resolving the nature and degree
of interactions between integrin- and growth factor-mediated signaling
pathways, and their relative contributions, must await a more complete
definition and understanding of integrin-mediated signal transduction
pathways.
Schaller et al.(19) have recently
identified Tyr-397 as the major autophosphorylation site on
pp125 both in vivo and in vitro. Of
special interest is that tyrosine phosphorylation of this site results
in the stable binding of pp125
to the SH2 domain of Src
family tyrosine kinases such as pp60
and
pp59
(18, 19) . They proposed that the
association of pp60
and pp59
with
pp125
may lead to their enzymatic
activation(19) . If this proposed model actually operates in
the cell adhesion process, Src family kinases might be a strong
candidate in the
1 integrin signal transduction pathway leading to
the MAP kinase activation. So far, there has been no direct evidence
that Src kinases are activated by cell adhesion. In this regard,
further characterization of pp130 whose tyrosine phosphorylation is
enhanced by cell adhesion (Fig. 1) would be important, since
pp60
has been shown to be tightly associated
with a phosphotyrosyl protein with an apparent molecular weight of 130
kDa(18, 32) . We are currently investigating these
possibilities. Such efforts will shed more light on the mechanism of
the integrin-mediated signal transduction.