(Received for publication, June 5, 1995)
From the
We show in this report that two v-src substrate
proteins, p130 and cortactin, become
tyrosine-phosphorylated during integrin-mediated cell adhesion to
extracellular matrix substrata and upon cell attachment onto
immobilized anti-integrin antibodies. This tyrosine phosphorylation
does not occur when cells attach to polylysine or through antibodies
against major histocompatibility complex. It also does not take place
when adhesion-mediated reorganization of the actin cytoskeleton is
inhibited with cytochalasin D. Tyrosine phosphorylation of p130
and cortactin coincides with tyrosine phosphorylation of focal
adhesion kinase during integrin-mediated cell adhesion but is
independent of cell adhesion in v-src-transformed cells. The
tyrosine-phosphorylated sites in p130
and cortactin may
serve as binding sites for proteins containing Src homology 2 domains,
as is the case with two other integrin-regulated docking proteins,
focal adhesion kinase and paxillin. Thus, these results suggest that
ligand binding of integrins regulates the tyrosine phosphorylation
state of multiple docking proteins. These proteins may mediate
anchorage dependence of growth; their misregulation in
v-src-transformed and other tumorigenic cells may be
responsible for the anchorage independence of such cells.
Integrins are a family of heterodimeric transmembrane proteins
that function as receptors for proteins of the extracellular matrix
(ECM), such as fibronectin, vitronectin, collagens, and
laminin(1, 2) . Upon ligand binding, integrins form
clusters at sites of close cell-substrate contact termed focal
adhesions, providing a linkage between the ECM and the cytoskeleton (3) . This linkage is thought to arise from the interaction of
the cytoplasmic domains of the integrins with cytoskeletal proteins
such as
-actinin and talin(4, 5) . Focal
adhesions are important not only as structural links between the ECM
and the cytoskeleton, but also as sites of signal transduction from the
ECM.
Elevated phosphotyrosine has been noted in focal adhesions in cells adhered on ECM, indicating that tyrosine kinase(s) are present and active at these sites(6) . One of the kinases present in the focal adhesions is the focal adhesion protein-tyrosine kinase FAK(7, 8, 9) . Upon coupling of integrins to the ECM, FAK becomes activated and tyrosine-phosphorylated(8, 10, 11, 12, 13, 14) , creating a binding site for the Src homology 2 (SH2)-domains of Src-family tyrosine kinases(15, 16, 17, 18) . Src phosphorylates several other sites in FAK, which in turn can function as binding sites for other proteins containing SH2-domains (19) . Association of FAK with C-terminal Src kinase (20) and Grb2 (18) has been reported, suggesting a role for FAK in connecting integrin ligand binding to downstream signaling pathways, such as activation of the mitogen-activated protein kinases(18, 21, 22, 23) . Two other focal adhesion proteins besides FAK, namely paxillin and tensin(12, 24) , have been shown to contain elevated phosphotyrosine in response to cell adhesion. These proteins may also serve as a connection to the cytoskeleton and to recruit additional SH2-bearing signaling molecules to focal adhesions.
In this report,
we have examined tyrosine phosphorylation of proteins in response to
cell adhesion to ECM substrata. We show that two recently described
signaling molecules, the v-src substrate proteins p130(25) and cortactin(26, 27) , become
tyrosine-phosphorylated upon cell adhesion to ECM proteins and to
immobilized anti-integrin antibodies in normal fibroblastic cells.
Figure 1:
Tyrosine phosphorylation of
p130 and cortactin in response to cell adhesion on
fibronectin or anti-integrin antibodies. REF cells were either held in
suspension (lanes 1, 3, and 7), or plated on
dishes coated with polylysine (lanes 4 and 8),
fibronectin (lanes 2, 5, and 9), or
anti-
5
1 antibodies (lanes 6 and 10).
Phosphotyrosine (lanes 1 and 2), p130
(lanes 3-6) and cortactin (lanes
7-10) immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by
anti-phosphotyrosine immunoblot (upper panel). The blot was
stripped and reprobed with anti-p130
(lanes
3-6) and anti-cortactin (lanes 7-10) to
confirm equal loading (lower panel). The molecular masses of
marker proteins are indicated in kilodaltons, and Ig indicates
immunoglobulins.
To investigate the
possibility that two v-src substrates, p130(25) and cortactin, an 80/85 kDa
protein(26, 27) , would be among the unidentified
phosphoproteins, samples from suspended and adherent REF cells were
immunoprecipitated with antibodies against p130
and
cortactin, and they were analyzed by immunoblotting with an
anti-phosphotyrosine antibody (Fig. 1, upper panel).
When immunoprecipitated from suspended cells, p130
and
cortactin (lanes 3 and 7) exhibited very low levels
of tyrosine phosphorylation. Cell adhesion to polylysine, to which
cells can adhere in a nonspecific fashion, had no effect on the
tyrosine phosphorylation of p130
and cortactin (lanes
4 and 8). Similarly, no increase in tyrosine
phosphorylation was observed when cells were allowed to adhere on
dishes coated with anti-MHC antibodies (not shown). An increase in
tyrosine phosphorylation was seen in cells plated on fibronectin or on
anti-
5
1 antibodies (lanes 5, 6, 9,
and 10). The amounts of p130
and cortactin were
essentially the same in all samples as shown when the same plot was
stripped and reprobed with anti-p130
and anti-cortactin
antibodies (Fig. 1, lower panel). As it has been
previously reported, p130
was found to migrate as
multiple bands, presumably due to differences in post-translational
modifications(25) . Increased tyrosine phosphorylation of
p130
and cortactin was also observed when cells were
plated on dishes coated with vitronectin or anti-
v
3
antibodies (not shown).
Figure 2:
Time course of tyrosine phosphorylation of
FAK, p130, and cortactin, and inhibition of the tyrosine
phosphorylation by cytochalasin D. REF cells were plated on fibronectin
for various times; time of adhesion is shown in minutes. FAK,
p130
, and cortactin immunoprecipitates were prepared and
subjected to phosphotyrosine analysis. Cyto D denotes samples
that had been treated with cytochalasin D as described under
``Experimental Procedures.'' The blots were stripped and
reprobed to confirm that equal amounts of the immunoprecipitated
proteins were present in each lane (not
shown).
Figure 3:
Tyrosine phosphorylation of p130 and cortactin is independent of cell adhesion in
v-src-transformed cells. NIH 3T3 cells (lanes 1 and 2), v-src-transformed 3T3 cells (lanes 3 and 4), and c-src-transfected 3T3 cells (lanes 5 and 6) were either held in suspension (lanes 1, 3, and 5) or plated on fibronectin for 45 min (lanes 2, 4, and 6). Anti-p130
and anti-cortactin immunoprecipitates were prepared from the cell
lysates and subjected to phosphotyrosine analysis (upper
panel). The blots were stripped and reprobed to confirm equal
loading (lower panel).
Several proteins are tyrosine-phosphorylated in response to
cell adhesion and spreading on extracellular substrata. Three of these
proteins have previously been identified as FAK, paxillin, and tensin (8, 10, 11, 12, 13, 24, 37) .
In this study, we report on elevated phosphotyrosine on two additional
proteins, p130 and cortactin, as cells adhere to ECM
substrates or to immobilized anti-integrin antibodies. We also show
that this tyrosine phosphorylation coincides with tyrosine
phosphorylation of FAK, requires organization of actin cytoskeleton,
and is independent of cell adhesion in v-src-transformed
cells.
p130 was originally identified as a highly
tyrosine-phosphorylated protein during cellular transformation by
v-src(34, 38) , as well as by
v-crk(39, 40) , that forms stable complexes
with these oncoproteins. Recent molecular cloning of p130
identified it as a novel SH3-containing signaling molecule with a
cluster of multiple putative SH2-binding motifs(25) . The
unique structure of p130
indicates the possible role of
p130
in assembling signals from multiple SH2-containing
molecules, including Src and Crk. Therefore, p130
may
serve as a docking protein that tethers other proteins to a
multicomponent complex, and integrin-mediated tyrosine phosphorylation
of p130
observed here may function to regulate such
protein-protein interactions. Recently, FAK has been shown to interact
with Src, Grb2, C-terminal Src kinase, and phosphatidylinositol
3-kinase in an adhesion and/or tyrosine phosphorylation-dependent
manner(15, 16, 17, 18, 20, 41) ,
while paxillin forms complexes with FAK, Src, C-terminal Src kinase,
and Crk (20, 36, 42, 43, 44) .
Thus, ligand binding of integrins seems to regulate multiple putative
docking proteins that connect to downstream signaling pathways.
Cortactin is an 80/85-kDa SH3-domain containing protein, that
becomes phosphorylated on tyrosine in v-src-transformed cells
and in cells stimulated with certain growth
factors(26, 27) . Cortactin binds to F-actin and is
enriched in cortical cell structures such as lamellipodia(27) .
It has therefore been suggested that cortactin may be important for
microfilament-membrane interactions and that it may also transduce
signals from the cell surface to the cytoskeleton(27) . Thus,
one possibility is that the integrin-mediated tyrosine phosphorylation
of cortactin observed here may play a role in actin remodeling during
cell adhesion to ECM substrata. Previous results, however, indicate
that tyrosine phosphorylation of cortactin does not influence F-actin
binding(27) . It is also possible that, similar to paxillin,
FAK and p130, phosphorylation of cortactin may promote
the binding of cortactin to cellular proteins containing SH2 domains,
allowing cortactin to contribute to the assembly of signaling molecule
complexes.
Our finding that p130 and cortactin become
tyrosine-phosphorylated following cell adhesion to ECM substrates, but
not to polylysine, is consistent with the tyrosine phosphorylation
being mediated by integrins. This is further supported by the
observation that cell adhesion to two different anti-integrin
antibodies also results in elevated tyrosine phosphorylation of
p130
and cortactin. These findings also suggest that
ligand binding to different integrins can activate the same tyrosine
kinase(s) (the one(s) responsible for phosphorylating p130
and cortactin).
In addition to REF and NIH 3T3 cells, we have
also analyzed tyrosine phosphorylation of p130 and
cortactin in human 293 embryonic kidney carcinoma cell line, and in
mouse embryonic and Rat-1 fibroblasts (not shown). Elevated tyrosine
phosphorylation of p130
in response to cell adhesion was
seen in each of the cell lines we have tested so far. However, we have
not been able to detect elevated tyrosine phosphorylation levels of
cortactin in some of the cell lines tested, including chick embryo
fibroblasts and human skin fibroblasts. Whether this truly reflects
cell type specificity, or whether there are changes that are below
detection limits of our antibodies is not known.
We found that
tyrosine phosphorylation of p130 and cortactin coincides
with tyrosine phosphorylation of FAK. It has been reported that
paxillin and FAK become coordinately phosphorylated on tyrosine during
cell spreading on fibronectin(12) , and that paxillin is a
substrate for FAK kinase activity in vitro and in
vivo(36, 45) . It remains to be seen whether
tyrosine phosphorylation of p130
and cortactin requires
integrin-mediated FAK activation, or whether it is a result of a
separate, integrin-activated, but FAK-independent kinase pathway.
Tyrosine phosphorylation of p130 and cortactin
requires the presence of intact cytoskeleton, since the
adhesion-induced tyrosine phosphorylation of these proteins can be
prevented by cytochalasin D treatment. A similar situation has been
observed with FAK and tensin(24) . Because no direct
association between integrins and kinases has been observed, it is
possible that kinases associate with integrins through interactions
with cytoskeletal complexes induced by the cross-linking of
integrins(9) . Thus, intact, functional cytoskeleton may be
required to bring together the various components of this signaling
complex, resulting in tyrosine phosphorylation of cellular proteins.
While this work was in progress, Petch et al.(46) reported on the adhesion-induced tyrosine
phosphorylation of p130. They demonstrated that, as in
the case of FAK, paxillin, and tensin, p130
is also found
in focal contacts. The focal contact colocalization of the proteins
connected to integrin signaling emphasizes the role of these sites in
the signal transduction from the ECM into the cell.
It has recently
been demonstrated that tyrosine phosphorylation of FAK in
v-src-transformed cells is independent of cell adhesion (18) , and we show here that this is also the case with
p130 and cortactin. Based on the apparent correlation
between cellular transformation and elevated levels of protein tyrosine
phosphorylation in v-src transformed cells, it is believed
that physiologically important Src substrates will be proteins normally
involved in the control of cellular proliferation(47) . Since
it seems that a number of v-src substrates are in the integrin
signal transduction pathway, it is equally possible that these proteins
and integrin signals mediated by them are responsible for
anchorage-dependence, which is lost in transformed cells.
Identification of protein-protein interactions resulting from
integrin-dependent tyrosine phosphorylation of cellular proteins and
characterization of signaling pathways that become activated following
these interactions may lead to better understanding of anchorage
dependence of growth.