(Received for publication, July 31, 1996, and in revised form, October 15, 1996)
From the Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029
The signaling functions of the oncogenic
protein-tyrosine kinase v-Ros were studied by systematically mutating
the tyrosine residues in its cytoplasmic domain. The carboxyl mutation
of Tyr-564 produces the most pronounced inhibitory effect on v-Ros
autophosphorylation and interaction with phospholipase C. A cluster
of 3 tyrosine residues, Tyr-414, Tyr-418, and Tyr-419, within the PTK
domain of v-Ros plays an important role in modulating its kinase
activity. The mutant F419 and the mutant DI, deleting 6-amino acids
near the catalytic loop, retain wild type protein tyrosine kinase and mitogenic activities, but have dramatically reduced oncogenicity. Both
mutant proteins are able to phosphorylate or activate components in the
Ras/microtubule-associated protein kinase signaling pathway. However,
F419 mutant protein is unable to phosphorylate insulin receptor
substrate 1 (IRS-1) or promote association of IRS-1 with phosphatidylinositol 3-kinase. This tyrosine residue in the context of
the NDYY motif may define a novel recognition site for IRS-1. Both F419
and DI mutants display impaired ability to induce tyrosine phosphorylation of a series of cytoskeletal and cell-cell interacting proteins. Thus the F419 and DI mutations define v-Ros sequences important for cytoskeleton signaling, the impairment of which correlates with the reduced cell transforming ability.
Autophosphorylation of receptor protein-tyrosine kinase (RPTKs)1 is one of the earliest detectable events in response to binding of their cognate ligand. This step has two pivotal roles, one is to activate the intrinsic PTK activity of the receptor and the other is to create site(s) for substrate protein interaction, both of which are essential in initiating pathways of signal transduction mediated by the receptor PTKs (1).
The oncogene v-ros encoded by the avian sarcoma virus UR2 is
a truncated receptor-like PTK (2). The proto-oncogene c-ros codes for a receptor PTK with an extended extracellular domain (3-5).
v-ros differs from c-ros in that all but 21 nucleotides that code for the extracellular domain of c-ros
are truncated and the remaining gene is fused in frame at its 5 end to
viral gag sequences. As a result, v-ros codes for
a gag-Ros transmembrane fusion protein of 68 kilodaltons called
P68gag-ros that is constitutively active (6). In addition,
there are minor alterations in the transmembrane (TM) domain and
carboxyl region of v-Ros in comparison with c-Ros (7). The kinase
domain of Ros is highly homologous with the kinase domains of insulin receptor (IR) and insulin like growth factor I receptor (IGFR) (4). The
PTK domain of Ros has two distinct sequence features in comparison with
members of the Src family and other receptor PTKs (4). One is a cluster
of 3 tyrosine residues consisting of a single tyrosine followed by twin
tyrosines 4 residues downstream. The other is a 6-amino acid insertion
3 amino acids downstream of the predicted PTK catalytic loop defined
from the crystal structure of the kinase domain of IR (8). These
characteristic sequence features of the v-Ros PTK domain are also
shared by its closely related RPTKs, IR, and IGFR (4). Mutation of the
twin tyrosines or all 3 tyrosines in IR or IGFR resulted in dramatic
reduction in their PTK activity (9-12). The importance of these
tyrosine residues was further suggested by the crystal structure of the PTK domain of IR (8). In this study, it was suggested that the middle
tyrosine residue (Tyr-1162) of IR affects gating of the catalytic site
by controlling the accessibility of ATP and substrate binding. Based on
the amino acid sequence homology to IR, it is likely that the triple
tyrosine residues of Ros also play an important role in regulating its
catalytic activity.
The interaction between an activated receptor PTK and some of its
substrates is mediated by a family of src-homology domain 2 or 3 (SH2/SH3)-containing proteins (13). Although RPTKs in general do
not contain SH2 or SH3 motifs, most of their downstream signaling
proteins thus far identified contain the SH2 and/or SH3 domains (1, 13,
14). Their association with RPTKs is facilitated by the tyrosine
phosphorylation of specific sites on the receptors in response to the
binding of ligands to receptors' extracellular domains. For example,
phosphorylation of specific tyrosine residues on platelet-derived
growth factor (PDGF) and epidermal growth factor (EGF) receptors is
required for binding of several of their downstream signaling molecules
including GAP, PI3 kinase, and PLC (reviewed in Cantley (1)). In
contrast, IR and IGFR bind IRS-1 and Shc at an NPXY motif in
the receptors' juxtamembrane domains (15-17).
The Shc- and Grb2-mediated activation of Ras/MAP kinase pathway is
involved in the mitogenic signaling by growth factors and cytokines (1,
18). Other signaling pathways involving PLC, IRS-1, and PI3 kinase
have also begun to be elucidated. IRS-1 serves as the major adaptor for
recruiting other signaling molecules upon stimulation of IR by insulin
and most likely of IGFR by IGF-1 (19). Tyrosine-phosphorylated IRS-1 is
capable of binding to and activating PI3 kinase (20). Mutation of the
IRS-1 recognition motif NPEY diminishes insulin-induced tyrosine
phosphorylation of IRS-1 and activation of PI3 kinase (17).
Reorganization of cytoskeletal structure and alteration of membrane properties represents an important component of the process of malignant cell transformation. However, the processes involved in cytoskeletal signaling remained unclear. Ever increasing attention has been directed toward understanding the transmission of signals from the interaction of integrins and their extracellular matrix ligands, particularly at adhesion plaques (21). The focal adhesion protein-tyrosine kinase, pp125 FAK, colocalizes with the integrin receptor in cellular focal adhesions and is activated upon engagement of the integrin receptor with its ligand or upon Src transformation (22, 23). Cadherin and its associated catenins constitute other important molecules in cytoskeleton-mediated signaling. Cadherins mediate Ca2+-dependent cell-cell adhesion via homophilic interaction of these cell surface molecules (24).
If specific tyrosine residues of an oncogenic RPTK interact with different intermediate substrates leading to distinctive signal transduction pathways, it may, therefore, be possible to impair specific pathways responsible for different biological effects by mutating those substrates interacting tyrosine residues. To explore the functional role of specific tyrosine residues of the oncogenic v-Ros, particularly with respect to their roles in mitogenic versus transforming activity, we have systematically mutated all of the tyrosine residues in the cytoplasmic domain of v-Ros. In addition, we have also removed the 6-amino acid insertion in the v-Ros catalytic domain. These mutants allowed us to identify the tyrosine residues of v-Ros important for regulating PTK activity and interaction with specific substrates. They also allow the differentiation of signaling pathways leading to mitogenicity versus morphological transformation and anchorage-independent growth.
Chicken embryo fibroblasts (CEF) were prepared from 11-day-old embryos and maintained according to the previously published procedure (6, 7, 25). The CEF were maintained as monolayer culture except in the colony formation assay, where they were suspended in the agar medium. Unless otherwise specified, CEF were maintained in F10 medium containing 5% calf serum and 1% chick serum (6, 7, 25). Molecularly cloned avian sarcoma virus UR2 and its helper virus UR2AV have been described (2, 7).
Biological AssayCell transformation was monitored by morphological change and anchorage-independent growth as described previously (7, 11, 25, 26). For colony formation in methyl cellulose, medium containing 1.3% pure methyl cellulose was used for the top layer on the same platform of bottom layer agar.
AntibodiesAnti-Ros and anti-IRS-1 antibodies were made in
our laboratory and have been described (6, 11). Anti-MAP kinase
polyclonal antibody TR10 was a gift from M. Weber, Anti-annexin II
polyclonal antibody was a gift from T. Hunter. Monoclonal antibodies
for cortactin, tensin, and CAS were gifts from T. Parsons and A. Bouton. Anti-p190 Rho/GAP polyclonal antibody was a gift from S. Parsons. Anti-Grb2 polyclonal antibody was a gift from B. Mayer.
Antibodies for FAK, - and
-catenin and an alkaline
phosphatase-coupled anti-phosphotyrosine (Tyr(P)) antibody RC20 were
purchased from Transduction Lab. Antibodies for PLC
and PI3 kinase
were purchased from Upstate Biotechology Inc. Anti-
1-integrin and
anti-N-cadherin were purchased from Sigma
and Zymed Laboratories Inc., respectively.
The plasmid pUR2H1 contains the
full-length UR2 genome cloned at the HindIII site of pBR322
(2, 7). The UR2 genome in this plasmid is permuted with respect to the
HindIII site at the 3 region of the UR2 genome. To
facilitate DNA transfection and expression of viral genes, a
nonpermuted UR2 plasmid was reconstructed. This was done by isolating
the 2.3-kilobase pair PstI to NruI fragment
containing the entire gag-Ros coding sequence from pUR2H1 and using it
to replace the gag-IGFR sequence in a nonpermuted viral plasmid
pBUIGFR-II constructed previously (26). The resulting plasmid was named
pBUR2 which served as the parental plasmid for mutant construction.
The mutants were engineered by a M13-mediated mutagenesis kit (Promega) described previously (11) or polymerase chain reaction using oligonucleleotides containing specific base changes. The sites of mutagenesis was sequenced to confirm the mutation.
Protein AnalysisCellular protein extraction, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and Western blotting have been described elsewhere (6, 26). In vitro assays of PTK, PI3 kinase, and MAP kinase followed those described previously (7, 25).
All the tyrosine
residues in the cytoplasmic domain of P68gag-ros were converted
individually, or in combination to phenylalanines (Fig.
1). The viruses encoding the mutant v-Ros proteins were named according to the positions of the mutated tyrosine residues (2)
with the exception of double and triple mutants F2, F3, and F4. In
addition, a unique 6-amino acid insertion located 3 amino acids
downstream of the predicted catalytic loop was deleted to generate the
mutant DI.
Biological Properties of the v-Ros Mutants
Two or three
independent clones of each mutant v-Ros expression plasmid were
co-transfected with UR2AV helper virus DNA into CEF to assess their
biological activity. Most mutants showed both mitogenic and
transforming activity indistinguishable from that of parental UR2 (Fig.
1). However, the mutant F2 containing the Y418F and Y419F double
mutation had only a residual transforming activity. The triple mutant
F3 had undetectable mitogenic and transforming activity. Mutants F419
and DI showed dramatically reduced transforming ability as reflected in
morphological alteration of the transfected cells (data not shown) and
ability of the cells to form colonies in soft agar (Fig.
2). However, both mutants displayed wild-type mitogenic
activity when infected cells were maintained as monolayer culture in
either 5 or 0.5% serum-containing medium (Fig. 2).
The double mutant F4 displayed a further reduced transforming
capability in comparison with F419 (data not shown) despite the fact
that a single mutation of Tyr-564 produced no detectable difference
from UR2. This result indicates that the effect of the Tyr-564
mutation, which impairs the interaction of v-Ros with PLC (see
below), can only be detected in the background of a weak transforming
protein such as F419.
Tumorigenicity of F419, DI, and the parental UR2 was compared. The result (Table I) shows that both mutants have dramatically reduced tumorigenicity. In fact, none of the chicks succumbed to the tumors induced by the mutants during the 1-month observation period (Table I) and even in an extended period of 2 months in a separate experiment (data not shown). This result is consistent with the impaired ability of the two mutants in promoting anchorage independent growth, but does not correspond to their mitogenic activity in monolayer cultures.
|
The kinase
activities of mutant v-Ros proteins were analyzed by in
vitro auto- and trans-phosphorylation, in vivo tyrosine phosphorylation of the Ros proteins, as well as their ability to
phosphorylate cellular proteins (Fig. 3). Mutation of
both Tyr-418 and Tyr-419 (F2) resulted in a greatly reduced kinase activity, particularly its in vivo autophosphorylation and
ability to phosphorylate cellular substrates. Only a residual in
vitro kinase activity and no detectable in vivo kinase
activity was detected for the F3 protein containing the triple mutation
of Y414F, Y418F, and Y419F. These results suggest that these residues, particularly Tyr-418 and Tyr-419, play a major role in modulating the
PTK activity of v-Ros. Surprisingly, despite a lack of detectable activity in the in vitro autophosphorylation (Fig.
3A) and phosphorylation of the exogenously added substrate,
enolase (Fig. 3B), the DI protein, appears to have wild-type
kinase activity intracellularly as reflected in its in vivo
autophosphorylation (Fig. 3A) and phosphorylation of
cellular proteins (Fig. 3C). The apparent paradox of the
in vitro and in vivo kinase activity of the DI
protein will be discussed. As expected, the DI protein exhibited a
slightly faster mobility in the SDS-gel when detected with anti-Ros
antibody (Fig. 3, A and B, bottom
panels). The F564 protein containing the carboxyl-terminal
tyrosine to phenylalanine mutation appears to be underphosphorylated
and to have a faster mobility when detected in Western blotting with
anti-Tyr(P) antibody (Fig. 3 A and
C). However, F564 protein has no detectable
decrease in in vitro kinase activity, although its
phosphorylated products also appear to be down-shifted in gel mobility
in comparison with those of the parental v-Ros (Fig. 3A,
top panel and other data not shown). The mobility
downshifting of in vivo phosphorylated protein was also
apparent for the F4 protein containing the Y419F and Y564F double
mutation (Fig. 3A). Again, no detectable difference of in vitro or in vivo kinase activity was observed
for the F4 protein. The expression levels of the various mutant v-Ros
proteins in transfected cells was comparable, with the exception of F2
and F3, where the expression levels were 4-5-fold lower. More protein lysates from F2- and F3-infected cells were needed in order to normalize the Ros protein in the experiments shown in Fig. 3. Except
for the mutants described above, no effect on the in vitro or in vivo PTK activity was observed for the rest of
mutants.
Phosphorylation and Activation of Specific Signaling Proteins
To identify the tyrosine site(s) required for
interaction of v-Ros with specific substrates, the mutant proteins were
compared for their ability to phosphorylate or activate various
signaling molecules. Our results (Fig. 4) show that with the exception
of the kinase-defective mutants, F2 and F3, all the mutants were capable of inducing tyrosine phosphorylation of Shc and 5C2, an 88-kDa
cellular protein we previously identified to be a prominent substrate
of v-Ros (25). In addition to F2 and F3, the F564 protein was also
unable to cause tyrosine phosphorylation of PLC despite its
wild-type kinase activity level. As expected this is also true for F4.
Therefore, Tyr-564 may serve as PLC
recognition site directly or be
critical for the formation of a PLC
site elsewhere in the protein.
Our observation that F564 has a wild-type transforming activity level
indicates that phosphorylation of PLC
is not essential for this
activity. However, since mutation of Tyr-564 in the background of F419
further reduces its transforming ability, it is likely that
phosphorylation of PLC
may enhance cellular transformation by
v-Ros.
To further explore the biochemical
basis for the reduced transforming activity of F419 and DI, we examined
the ability of these mutants to activate MAP kinase, a downstream
effector of the Ras signaling pathway. Consistent with their ability to
induce tyrosine phosphorylation of Shc, both the F419 and DI proteins are able to promote association of Grb2 with three distinct tyrosine phosphorylated proteins with gel mobilities corresponding to those of
Shc proteins (46, 52, and 66 kDa). The mutants also activate MAP kinase
as efficiently as the wild type v-Ros (Fig. 5). This is
consistent with the observed mitogenic activity of F419 and DI
mutants.
IRS-1:Phosphorylation and Association with PI3 Kinase
We next
examined the signaling molecules IRS-1 and PI3 kinase, which were
previously shown to be phosphorylated and activated by v-Ros (25). Our
result shows that mutation of Tyr-419 specifically decreases the
ability of v-Ros to cause tyrosine phosphorylation of IRS-1 (Fig.
6A). No such effect was observed for any of
the other mutants, with the exception of the kinase inactive ones (data
not shown). Consistent with the reduced phosphorylation of IRS-1, F419
protein also failed to promote association of PI3 kinase with IRS-1 as
reflected in the in vitro PI3 kinase assay (Fig.
6B). This observation was confirmed by reciprocal
immunoprecipitation and Western blotting using anti-p85 and anti-IRS-1
antibodies to detect their physical interaction. Association of IRS-1
with the 85 kDa subunit of PI3 kinase was observed in DI- and UR2-, but
not in F419-infected cells.(data not shown). These results indicate
that phosphorylation of IRS-1 and activation of PI3 kinase are not
essential for promoting the growth of cells on monolayer culture.
Effect of v-Ros Mutations on Cytoskeleton-associated Proteins
Reorganization of cytoskeletal structure is intimately
related to morphological transformation. The effect of cytoskeletal alteration on cell-to-cell and cell-to-matrix interactions could play
an important role in the growth of cells in agar. We compared v-Ros and
its mutant proteins for their ability to cause tyrosine phosphorylation
and interaction of a series of cytoskeletal proteins involved in the
formation of focal adhesion plaques and cell-cell interactions. No
difference was observed among F419, DI, and UR2 PTKs in causing
tyrosine phosphorylation of FAK, cortactin, paxillin, CAS (21), a
Crk-associated protein, and annexin II (27), a cytoskeleton-associated
Ca2+-dependent phospholipid binding protein
(Fig. 7). Greatly increased tyrosine phosphorylation of
annexin II, cortactin, and paxillin was observed in cell infected with
wild-type v-Ros and the mutants. In contrast, the increase in tyrosine
phosphorylation of FAK and CAS is only about 2-fold above the control
CEF. We observed a tyrosine-phosphorylated 190-kDa protein associated
with 1 integrin in UR2-, but much less in F419- and DI-infected
cells. However, no significant tyrosine phosphorylation of integrin was
detected (Fig. 8). In addition, tensin was more
abundantly phosphorylated in UR2- than in the mutant-infected cells.
Similarly, the p190 Rho/GAP was more highly phosphorylated in UR2- than
in the mutant-infected cells, particularly in comparison with the F419
cells.
For the proteins involved in cell-cell interaction, we observed
significantly more abundant tyrosine phosphorylation of - and
-catenin, as well as more association between cadherin and
-catenin, in UR2-, than in F419- and DI-infected cells(Fig.
9). Association of
-catenin with cadherin was
confirmed by Western blotting of the anti-cadherin immunoprecipitates
with an anti-
-catenin antibody (data not shown). Neither
-catenin
nor cadherin was significantly tyrosine phosphorylated by any of our
v-Ros proteins. These results indicate that F419 and DI proteins are
either incapable or less effective in promoting tyrosine
phosphorylation and interaction between various proteins involved in
the formation of focal adhesion plaques and cell-cell interactions.
This study identifies several sequences in v-Ros that play
important roles in regulating PTK activity and cell transforming functions. The kinase positive and transformation-negative
(K+T) or attenuated
(K+T±) mutants are useful in that they may
allow identification of the signaling components essential for cell
growth and transformation. We previously generated a mutant called TM1
by deleting 3 amino acids in the TM domain of v-Ros (Fig. 1) (25). This
mutation has no effect on the kinase activity, but impairs both the
mitogenic and transforming activities of v-Ros. The kinase-positive,
mitogenicity positive but transformation-defective mutants
(K+M+T±), represented by F419 and
DI in this study, are thus particularly useful since they are
selectively impaired in signaling pathways leading to distinct
biological properties. Our results with F419 and DI indicate that
activation of the Ras/MAP kinase pathway is not sufficient for cell
transformation. Our data also suggest that the cytoskeletal
protein-mediated signaling may be more closely related to morphological
transformation and anchorage independent growth of cells. Numerous
site-specific deletion mutants within the N-terminal region of v-Src
have been reported to affect its ability to induce morphological
transformation and promote colony formation in soft agar (28). However,
it is not clear how those mutants affect the growth of cells on
monolayer culture. Our F419 and DI mutants resemble a recently reported
tyrosine 807 mutant in v-fms, which retains mitogenic but
not morphological transforming activity (29).
Our data show that all the tyrosine residues in the cytoplasmic domain of gag-Ros, except for Tyr-419 and Tyr-564 are not individually required for Ros's biochemical and biological properties. Our finding of the effect of the triple tyrosine mutation cluster on v-Ros PTK activity is consistent with those of other RPTKs including IR (9) and IGFR (10, 11), which also contain such a tyrosine cluster. However, mutation of Tyr-418 of v-Ros, which corresponds to Tyr-1162 of IR, suggested to be the "gate-keeper" of its catalytic site (8), did not yield any detectable biochemical or biological effect on v-Ros. Instead, mutation of the third tyrosine Tyr-419 in the cluster resulted in the impairment of v-Ros-transforming ability and substrate specificity. This result is consistent with our previous observation on the mutation of the corresponding tyrosine residue Tyr-1136 of an oncogenic gag-IGFR fusion PTK except that the mutation in that case resulted in dramatic decrease of both mitogenic and transforming activity (11). Deletion of the 6-amino acid insertion near the catalytic loop of v-Ros resulted in the loss of in vitro kinase activity, but produces little effect on the in vivo tyrosine phosphorylation of the mutant DI protein. Moreover, the DI protein is able to induce tyrosine phosphorylation of the array of cellular substrates with a pattern indistinguishable from that of the wild-type v-Ros. The simplest explanation for this observation is that the deletion results in an enzyme whose conformation is relatively unstable and is easier to be inactivated during cellular protein extraction and in vitro processing. However, the possibility that the mutant DI protein is phosphorylated by other endogenous PTK(s) and becomes activated in vivo cannot be ruled out. If so, the active DI protein is apparently inactivated again during the protein extraction and processing since neither auto- nor trans-phosphorylation activity could be detected in vitro.
The IRS-1 and Shc recognition site on IR has been identified as the NPEY motif in the juxtamembrane region of IR, in which the N, P, and Y residues are important for the interaction (16). v-Ros is capable of inducing tyrosine phosphorylation of IRS-1 and Shc (25). However, there is no corresponding NPXY sequence in v-Ros. The Y419F mutation specifically decreases the tyrosine phosphorylation of IRS-1, but not of Shc. Therefore, the NDYY sequence of v-Ros likely defines an alternative recognition site for IRS-1. Shc may interact with v-Ros at another site. Alternatively, the presence of either of the twin tyrosines in NDYY may be sufficient for Shc recognition.
The Tyr-564 is the only residue that upon single mutation results in a
pronounced reduction of intracellular autophosphorylation of v-Ros.
Tryptic mapping of the in vitro autophosphorylated v-Ros proteins of UR2, F2 (Y418F/Y419F), and F564 revealed that several tryptic spots were missing in F564, but not in F2, protein in comparison with those of UR2 v-Ros protein (data not shown). These observations suggest that Tyr-564 is the major phosphorylation site of
v-Ros in vitro and in vivo. Mutation of Tyr-564
also indicates that it is important for recognition of PLC. The
interaction site for PLC
maps to the carboxyl tyrosine residues of a
number of RPTKs including EGF receptor (30), PDGF receptor (31), IGFR
(11), and Met (32). Activation or overexpression of PLC
has been
implicated in stimulating DNA synthesis and promoting cell
transformation by EGF and PDGF receptors (33). Some other reports,
however, concluded that PLC
was not important for PDGF-induced DNA
synthesis (34). Our results shows that PLC
plays only a minor role
in v-Ros-mediated transformation of CEF.
Activation of PI3 kinase has been implicated in diverse functions including mitogenesis (35, 36), GLUT4 translocation/glucose transport (35, 37), membrane ruffling (38), and activation of p70 S6 kinase that is involved in stimulating protein synthesis (35). Our result with F419 and DI suggests that PI3 kinase could play a significant role in v-Ros induced cell transformation, but its activation is insufficient for morphological transformation and anchorage-independent growth and is not important for growth in monolayer cultures.
Our data suggest that signaling involving cytoskeletal proteins and cell-cell interactions may play an important role in morphological transformation and anchorage independent growth. The Rho family of GTP-binding/GTPase proteins including, Rho, Rac, and CDC42 are key players in regulating the cytoskeletal structure and membrane properties and are also important in mediating Ras-induced cell transformation (39, 40). In this regard, it is intriguing that p190 Rho/GAP, a regulator of Rho, is underphosphorylated in the F419- and DI-infected, in comparison with the UR2-infected cells (Fig. 8). EGF-dependent actin cytoskeleton disassembly is modulated by expression of c-Src and correlates with increased tyrosine phosphorylation of p190 Rho/GAP (41). This phenomenon could be explained by increased activity of Rho/GAP resulting in diminished abundance of Rho/GTP needed to promote the formation of actin stress fibers.
Our observation of the increased tyrosine phosphorylation of tensin and
a 1 integrin-associated 190-kDa protein, as well as cadherin-catenin
complex involved in cell-cell interaction in UR2-, but not in F419- or
DI-infected cells is also intriguing. It raises a possibility that
those proteins are involved in mediating morphological transformation
and anchorage-independent growth of UR2. Further work is required to
elucidate the identity of the 190-kDa protein which we know is not
p190Rho/GAP.
The cytoplasmic region of cadherin interacts with -,
- and
-catenins, which may serve as the bridge for interaction with actin,
as well as signaling effectors (24). In v-Src-transformed cells,
-catenin is tyrosine-phosphorylated, and although cadherins are
expressed on the cell surface, they are functionally inactive (42). The
homology between
-catenin and a segment polarity gene in
Drosophila called armadillo raises the
possibility that
-catenin has a similar role in developmental
signaling (43). Thus, catenins may play a dual role in cell-cell
interaction and in signaling. The increased tyrosine phosphorylation of
catenins and their enhanced association with cadherin in v-Ros
transformed cells may not only affect the function of cadherin, but
also modulate the cytoplasmic pool of the catenin involved in
signaling.