(Received for publication, May 19, 1995; and in revised form, July 27, 1995)
From the
Expression of oncogenic variants of pp60 leads to dramatic changes in cytoskeletal organization
characteristic of transformation. Activated Src associates with the
cytoskeletal matrix, resulting in tyrosine phosphorylation of specific
cytoskeletal substrates. We have previously shown that stable
association of Src with the cytoskeletal matrix is mediated by the Src
SH2 domain in a phosphotyrosine-dependent interaction. In this report,
we demonstrate that one of the cytoskeletal binding partners of Src is
p80/85 cortactin. The association was observed in lysates of
transformed cells but was not seen in normal fibroblasts. The
interaction could be reconstituted in vitro using transformed
cell extracts and a glutathione S-transferase (GST) fusion
protein containing the Src SH2 domain but not with GST-Src SH3 or with
GST-Src SH2 containing a point mutation in the FLVRES sequence.
Confocal microscopy revealed that cortactin redistributed and
colocalized with v-Src and a Src SH3 deletion mutant in transformed
cells. However, in cells expressing a Src SH2 deletion mutant, the
redistribution of cortactin and colocalization with Src did not occur.
Furthermore, biochemical fractionation of transformed cells indicated
that a significant increase in cortactin distribution to the
cytoskeletal fraction occurred, which correlated with a shift in the
tyrosine-phosphorylated form of the protein. Cortactin fractionated
from cells expressing kinase-defective or myristylation-defective Src
mutants did not exhibit this shift. These data suggest a molecular
mechanism by which tyrosine phosphorylation of cortactin and
association with the Src SH2 domain influence the cytoskeletal
reorganization induced in Src-transformed cells.
The v-Src oncogene mediates cellular transformation by encoding
a tyrosine kinase which exhibits elevated levels of catalytic activity.
Interaction of pp60 with the plasma
membrane is essential for achieving transformation and results in the
tyrosine phosphorylation of a number of membrane and cytoskeletal
proteins. It is thought that tyrosine phosphorylation of key
cytoskeletal substrates leads to the dramatic changes in morphology,
cytoskeletal reorganization, and decreased adhesive properties
characteristic of transformed cells (for review, see (1) and (2) ). Consistent with this hypothesis,
pp60
and its nontransforming
cellular homolog, pp60
differ in
their ability to bind to the cytoskeletal
matrix(3, 4) . When expressed in fibroblasts, v-Src
and oncogenic mutants of c-Src stably associate with the cytoskeletal
matrix, whereas nontransforming Src variants, including wild-type
c-Src, do not. These observations suggest that it is not only the
phosphorylation of cytoskeletal proteins, but also the association of
activated Src with specific cytoskeletal substrates that is important
for the changes observed upon transformation.
We have recently shown that stable association of Src with proteins in the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent interaction(5) . Several proteins, which are tyrosine-phosphorylated in transformed cells, are candidate binding partners for activated Src in the cytoskeletal matrix (6, 7, 8, 9) . One of these potential cytoskeletal binding partners is p80/85 cortactin. Cortactin is an F-actin binding protein that possesses several interesting structural features, including an SH3 domain, a proline-rich region, and a series of tandem internal repeats that serves as the actin binding domain(10, 11) . Cortactin is normally phosphorylated on serine and threonine, but it becomes tyrosine-phosphorylated in response to growth factor stimulation or upon transformation by activated Src(11, 12, 13) . In nontransformed cells, cortactin has been shown to transiently associate with c-Src in response to thrombin activation in platelets (14) and in response to FGF-1 in fibroblasts(15) . In oncogenic cells, the cortactin locus is amplified and overexpressed in certain human breast and squamous cell carcinomas(16) , while in mice the inappropriate expression of cortactin appears to be associated with the transformation of plasma cells(17) . Based on these observations, it has been suggested that cortactin may play an important role in the transduction of mitogenic signals, cytoskeletal organization, and cell adhesion.
In this report, we demonstrate that
v-Src and cortactin form a complex in Rous sarcoma virus-transformed
cells that is dependent on the structural integrity of the Src SH2
domain. The association was most readily observed in the cytoskeletal
matrix-enriched fraction of the cell and correlated with the tyrosine
phosphorylation of cortactin. The interaction of
pp60 and cortactin could be
reconstituted in vitro by incubating cytoskeletal matrix
extracts from transformed cells with GST (
)fusion protein
containing the Src SH2 domain, but it was not observed with a GST Src
SH2 domain containing a point mutation in the FLVRES sequence. Confocal
microscopy revealed that cortactin redistributed from the cell
periphery to colocalize with wild-type v-Src in transformed
fibroblasts. However, in cells expressing a Src SH2 deletion mutant,
the redistribution of cortactin and colocalization with Src did not
occur. Upon transformation, a significant increase in cortactin
distribution to the cytoskeletal fraction was observed that could be
attributed to a shift by the tyrosine-phosphorylated form of the
protein. In contrast, cortactin fractionated from cells expressing a
kinase-defective Src mutant did not exhibit this shift, consistent with
its lack of tyrosine phosphorylation. Taken together, these data
demonstrate a functional role for the tyrosine phosphorylation of
cortactin. Our results provide direct evidence for the interaction of
tyrosine-phosphorylated cortactin with v-Src and suggest a molecular
mechanism by which this association influences the cytoskeletal
reorganization induced in Src-transformed cells.
Figure 1:
Association of p80/85
cortactin with pp60. Normal CEF or
CEF transformed with v-Src were lysed, and equal amounts of cellular
protein were immunoprecipitated with anti-Src polyclonal antibody 873.
The immune complexes were resolved by SDS-PAGE and immunoblotted with
antibody against p80/85 cortactin. Immunoprecipitates were from whole
cell lysates of normal CEF (lane 1) or v-Src expressing cells (lane 2), or from the Triton-insoluble cytoskeleton-enriched
fraction of v-Src transformed cells (lane 3). The
coprecipitation experiment was also performed using pre-immune serum,
and cortactin association was not observed (data not shown). The
migration of molecular size markers is indicated in the margin in
kilodaltons. The band at 50 kDa represents IgG heavy
chain.
Figure 2: Association of cortactin with Src homology domains. 3 µg of GST fusion protein containing various Src homology domains were bound to glutathione-Sepharose beads and then incubated with approximately 500 µg of cellular protein from the cytoskeleton-enriched fraction of v-Src transformed cells. Bound proteins were analyzed by SDS-PAGE and immunoblotting with cortactin antibody. GST fusion proteins consisted of GST alone (lane 1), GST-Src SH3/SH2 (lane 2), GST-Src SH3 (lane 3), GST-Src SH2 (lane 4), or GST-Src SH2(R175K) point mutation (lane 5). As a control, an aliquot of lysate from the cytoskeleton-enriched fraction was run in parallel (lane 6).
In addition, we were able to observe direct association in vitro of purified, phosphorylated cortactin with purified Src SH2 domain. GST-p80 cortactin fusion protein (10) expressed in Escherichia coli was purified, and approximately 5 µg of fusion protein were attached to glutathione-Sepharose beads. The bound GST-cortactin fusion protein was phosphorylated in vitro with purified Src, washed extensively, and then cleaved with thrombin to remove the GST moiety. The phosphorylated cortactin fragment was isolated and tested for its ability to associate with GST-Src SH2. Cortactin protein purified and phosphorylated in this manner was able to associate with the Src SH2 domain, while nonphosphorylated cortactin purified and assayed the same way did not appear to interact. Together, these results indicate that the interaction between Src and cortactin is specific and requires an intact Src SH2 domain.
Figure 3: Association of GST-Src fusion proteins with cortactin from v-Src-transformed and normal CEF. GST fusion proteins containing GST alone (lanes 1 and 5), GST-Src SH3 (lanes 2 and 6), or GST-Src SH2 (lanes 3 and 7) were incubated in parallel with lysates from normal CEF (lanes 1-3) or Src-transformed CEF (lanes 5-7). Bound proteins were separated by SDS-PAGE and immunoblotted with anti-cortactin antibody. Aliquots of lysate from normal (lane 4) and transformed cells (lane 8) were run alongside as controls.
Figure 4: Competition of Src SH2-cortactin binding with pYEEI peptide. GST-Src SH2 fusion protein bound to glutathione beads was incubated with cellular protein from transformed cells without peptide (lane 1), or in the presence of 25 µM (lane 2) and 75 µM (lane 3) pYEEI peptide.
Figure 5:
Interaction of cortactin with other SH2
domain-containing proteins. Approximately 500 µg of protein from
the cytoskeleton-enriched fraction of Src-transformed cells was
incubated with 5 µg of GST fusion protein containing the SH2
domains of Src, Abl, Crk, Fps, Nck, phosphatidylinositol 3-kinase (PI3K) (C-terminal), and phospholipase C- (PLC)
(N-terminal). An aliquot of lysate from Src-transformed cells was run
alongside as a control. Bound proteins were analyzed by SDS-PAGE and
immunoblotting with anti-cortactin
antibody.
Figure 6: Localization of cortactin in cells expressing Src mutants. Cells were double stained for the detection of cortactin (A, C, E, and G) and Src (B, D, F, and H), and visualized by confocal microscopy. Identical fields are shown on the left and right panels. Normal CEF (A and B) or CEF expressing wild-type v-Src (C and D), Src SH2 deletion mutant (E and F), or Src SH3 deletion mutant (G and H) were grown overnight on coverslips and processed as described under ``Materials and Methods.'' Cortactin and Src were detected using a mixture of anti-cortactin monoclonal antibody 4F11/Texas Red-conjugated anti-mouse, and anti-Src 873/FITC-conjugated anti-rabbit antibodies.
Figure 7: Fractionation of cortactin. Normal CEF, as well as CEF expressing wild-type v-Src, the kinase-defective Src mutant K295M, Src SH2 deletion mutant, R175K point mutant, and Src SH3 deletion mutant were incubated in a 1% Triton X-100 buffer and fractionated into detergent-soluble (S), and detergent-resistant (R) fractions. The fractions were immunoprecipitated with anti-cortactin antibody and then analyzed by SDS-PAGE and Western blotting. A, the blot was probed with anti-cortactin antibody. B, the identical blot was then stripped and probed with anti-phosphotyrosine antibody.
The immunoblot was stripped and reprobed with anti-phosphotyrosine antibodies to determine whether the increase in cytoskeletal distribution was attributable to tyrosine phosphorylation. Cortactin from normal CEF or from CEF expressing the kinase-defective Src contained no phosphotyrosine (Fig. 7B). As expected, cortactin isolated from v-Src-transformed cells, as well as from cells expressing a Src SH3 deletion mutant, did contain phosphotyrosine, and this population fractionated almost exclusively with the cytoskeletal fraction (Fig. 7B). Although the vast majority of cortactin from cells infected with the Src SH2 domain mutants was found in the soluble fraction, there were low levels of tyrosine-phosphorylated cortactin that remained in the cytoskeletal fraction. A similar result was obtained with the nonmyristylated v-Src mutant NY315, with tyrosine-phosphorylated cortactin fractionating with the cytoskeletal matrix. Taken together, these results strongly suggest that Src-dependent phosphorylation of cortactin results in redistribution of cortactin to the detergent-insoluble cytoskeletal matrix.
Although tyrosine
phosphorylation of cortactin is dependent on Src kinase activity (Fig. 7B), it is not known whether cortactin is a
direct Src substrate. Purified c-Src can phosphorylate a GST-cortactin
fusion protein in vitro (data not shown), but the sites of
phosphorylation have not been mapped and compared with the sites
phosphorylated in vivo. Alternatively, another tyrosine kinase
may phosphorylate cortactin. pp60 may
phosphorylate and thereby activate another tyrosine kinase, which then
phosphorylates cortactin and enables association with Src. In fact, a
tyrosine kinase different from Src is associated with cortactin, but it
appears inactive in nontransformed cells. (
)Determination of
the kinase directly responsible for cortactin tyrosine phosphorylation,
as well as identification of the phosphorylation sites themselves,
awaits further study.
What does tyrosine-phosphorylated cortactin bind to
in the matrix? Our data suggest that one of the binding partners is the
v-Src SH2 domain. Confocal microscopy revealed that cortactin does not
relocalize in cells expressing Src SH2 deletion or point mutants.
However, the v-Src SH2 domain is probably not the only binding partner
for cortactin. Cortactin contains nearly 30 tyrosine residues, some of
which, if phosphorylated, would lie within potential
``consensus'' sequences for binding to Src, Nck, or 3BP2 SH2
domains(22, 23) . In vitro binding assays
with the SH2 domains of other proteins suggest that cortactin can also
associate with other tyrosine kinases like Abl or with bridging
proteins like Crk or Nck (Fig. 5). Yamanashi et al. recently showed that HS1, a cortactin-like protein, becomes
tyrosine-phosphorylated and binds to the Lyn SH2 domain(32) .
In addition, the presence of both an SH3 domain and a proline-rich
region in cortactin further suggests that cortactin plays an important
role in creating a nucleation site for the clustering of other proteins
in the cytoskeletal matrix. Interestingly, it has also recently been
demonstrated that tyrosine phosphorylation of 1 integrin results
in an altered subcellular localization as compared with the
nonphosphorylated form(33) . Taken together, these results
suggest that the tyrosine phosphorylation of cytoskeletal proteins may
serve as a general means of regulating subcellular localization by
promoting association with SH2 domain-containing proteins.
One can question whether podosome association (detergent resistance) of cortactin reflects a general feature of transformation or is the result of interaction between v-Src and cortactin. In the first instance (Model 1), cortactin localization to podosomes would be independent of v-Src localization in podosomes. In Model 2, redistribution of cortactin into podosomes would require the presence of v-Src in podosomes. Our data, summarized in Table 1, support the second model. Cells expressing SH2 domain mutants of Src are partially transformed yet do not exhibit redistribution of cortactin to podosomes, emphasizing the importance of Src localization for this event. The cortactin redistribution is dependent on tyrosine phosphorylation of cortactin and an intact v-Src SH2 domain. Tyrosine phosphorylation of cortactin alone, however, is not sufficient for cortactin redistribution as illustrated by the NY315 Src mutant. In all instances, the bulk of the cortactin redistributes to the podosomes only when v-Src is also present in podosomes.
p80/85 cortactin is
not the only protein that interacts with Src in the cytoskeletal
matrix. Previous reports have established that activated forms of Src
stably interact with a 110-kDa protein (35, 36) that
is associated with actin stress filaments(37) . Recently, it
was demonstrated that activated c-Src also stably interacts with
pp125(38) , a tyrosine kinase associated with
focal adhesions(31) . Unlike cortactin, however, the
association of activated Src with pp110 and pp125
does
not appear to be dependent on Src kinase
activity(37, 38) . pp125
isolated from
nontransformed cells is autophosphorylated on tyrosine and is able to
efficiently bind to the Src SH2 domain(38) . It is therefore
possible that Src is initially recruited to the cytoskeletal matrix
through an interaction with pp125
. As the functional
significance of such Src-cytoskeletal associations are determined, it
will become possible to elucidate the mechanism by which Src mediates
the various cellular changes characteristic of transformation.