From the Tumor Biology Program, Department of
Biochemistry, and ¶ Department of Pediatrics, Mayo Clinic,
Rochester, Minnesota 55905
Received for publication, May 4, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Mutations in the epidermal growth
factor receptor have been identified in several human tumor
types, including gliomas. These receptor mutants have deletions in
their extracellular ligand-binding domains and are, therefore, no
longer regulated by ligand, resulting in constitutive activation of the
receptor kinase. These mutants have been proposed to transduce
oncogenic signals via ligand-independent signaling pathways. Avian
viral homologues of these oncogenic epidermal growth factor receptors
exhibit structurally homologous deletions and form tumors in chickens.
One such mutant, S3v-ErbB, transforms fibroblasts in vitro,
and transformation has been correlated with the formation of a novel
tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and
transformation have been shown to occur independently of Ras
activation. The major aims of this study are to further characterize
this ligand-independent v-ErbB oncogenic signaling pathway. Here we
show that both v-ErbB-mediated phosphoprotein complex formation and
transformation are inhibited by a dominant negative mutant of Rho. This
inhibition is specific for dominant negative Rho; dominant negative
mutants of Rac and Cdc42 have no effect on transformation or on
tyrosine phosphorylation of the phosphoprotein complex. Based on these
observations, we propose that S3v-ErbB stimulates a
Rho-dependent tyrosine kinase, resulting in complex
formation and ultimately oncogenic transformation.
An avian viral mutant of the
EGFR,1 S3v-ErbB, transforms
fibroblasts in vitro, and in vivo expression
results in the development of fibrosarcomas and hemangiosarcomas (1).
S3v-ErbB transformation of fibroblasts results in the loss of
anchorage-dependent cell growth. This growth pattern has
been correlated with specific cytoskeletal changes in
v-ErbB-transformed fibroblasts (2, 38).
Specifically, stress fibers are disassembled, and myosin light chain
kinase activity is reduced (2, 38). In addition, two cytoskeletal
associated proteins, i.e. caldesmon and p21-activated kinase, are uniquely tyrosine-phosphorylated in S3v-ErbB-transformed cells (2, 4, 38). Interestingly, Ras activation, a key aspect of
ligand-dependent EGFR mitogenic signaling, is not required for S3v-ErbB-mediated transformation or for these cytoskeletal changes
(5). Together, these studies suggest that ligand-independent oncogenic
signaling occurs through a pathway that is distinct from the well
characterized ligand-dependent mitogenic signaling pathway
of EGFR.
Rho, Rac, and Cdc42 are small GTP-binding proteins
important in the dynamic reorganization of the cytoskeleton of the
cell. Rho is a crucial regulator of such cytoskeletal events and
functions by controlling stress fiber assembly and focal adhesion
formation (6). In contrast, Rac is important for membrane ruffling,
lamellipodia formation, and focal complex formation (7-10), whereas
Cdc42 is required for filopodia formation (11). The coordinated
interplay of the activities of these proteins results in the control of complex biological events such as cell movement, cell-cell
communication, cell growth, and cell death. When the regulatory
activity of Rho, Rac, or Cdc42 is altered, dramatic changes in the
actin-based cytoskeleton, as well as deregulation of these biological
functions, occurs, resulting in pathologic effects such as malignant transformation.
Because we previously have shown that Ras activity is not
required for S3v-ErbB transformation, in this study we have examined the role of Rho, Rac, and Cdc42 in S3v-ErbB-mediated primary fibroblast transformation. Our results demonstrate that Rho activity is essential for S3v-ErbB-mediated fibroblast transformation, whereas the loss of
Rac or Cdc42 activity does not inhibit transformation. In support of
this observation, expression of a dominant negative RhoA mutant inhibits the formation of a previously described
transformation-associated tyrosine phosphoprotein complex. These data
implicate Rho, as well as downstream mediators of Rho signaling, as
important mediators of S3v-ErbB-mediated complex formation and
transformation in fibroblasts. These results have important
implications for ligand-independent signaling by receptor tyrosine
kinases and may be particularly relevant to the development of novel
therapeutics capable of uniquely targeting oncogenic signaling pathways.
Cells and Viruses--
Primary chicken embryo fibroblasts (CEF)
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 2% chick serum at 37 °C.
RCAN-based retroviral vectors were used for coinfection studies
(12). E1v-ErbB (a nontransforming, but constitutively active, form of
v-ErbB) and S3v-ErbB were cloned into RCAN BH env subgroup A,
and dominant negative Rho (N19RhoA, a gift from A. Hall), dominant
negative Rac (N17Rac, a gift from A. Ridley), and dominant negative
Cdc42 (N17Cdc42, a gift from M. Symons) were cloned into RCAS BH
env subgroup B.
Retroviral Coinfection--
Low passage CEF were infected with
RCAS (B)-N19RhoA, N17Rac1, N17Cdc42, or vector only for 3 days. Cells
were passaged 1:3 and were subsequently infected with RCAN
(A)-E1v-ErbB, S3v-ErbB, or vector in the presence of 2 mg/ml polybrene
for 4 days as described previously (5).
Western Blot Analysis--
Coinfected CEF were
plated at 1 × 106 cells per 100-mm2 plate
and were lysed in buffer containing 1% Triton X-100, 50 mM
HEPES (pH 7.5), 5 mM EDTA, 50 mM NaCl, 10 mM NaPPi, 50 mM NaF, 0.5% deoxycholate, 4 mM diisopropyl fluorophosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate. Ten micrograms of total
protein were separated by SDS-PAGE followed by transfer to a
polyvinylidene difluoride membrane. The membrane was blocked in
Tris-buffered saline, 0.1% Tween 20, and 5% nonfat dry milk
for 1 h at room temperature and incubated with antibodies (diluted 1:500) against EGFR (13), Rho (Santa Cruz
Biotechnology), or Myc tag (Upstate Biotechnology) for 1 h
at room temperature. Membranes were washed three times for 5 min each
in Tris-buffered saline, 0.1% Tween 20 at room temperature,
followed by incubation in anti-mouse IgG horseradish peroxidase and
anti-rabbit IgG horseradish peroxidase (Amersham Pharmacia Biotech) at
a dilution of 1:2000 in blocking buffer for 1 h at room
temperature. Membranes were washed again as above and incubated with
chemiluminescence reagents (Pierce) for 5 min, followed by analysis
using a lumi-imager (Gel Expert, Nucleotech).
pp75 Phosphoprotein Complex Formation--
Equal amounts of
lysates (500 µg) from CEF coexpressing v-ErbB and DNRho, DNRac, or
DNCdc42 were immunoprecipitated with an anti-Shc antibody (1 µg/ml)
for 1 h at 4 °C. Protein A/G-agarose beads were added to the
lysates for 30 min at 4 °C, and immunoprecipitates were then washed
and analyzed as described previously.
Soft Agar Colony Formation--
CEF coexpressing v-ErbB and
DNRho, DNRac, and DNCdc42 were plated as previously described (14).
Plates were supplemented with a few drops of media every 3 days,
for a total culture time of 3 weeks. Colonies (>25 cells/colony) were
counted by bright field light microscopy. Coinfected cultures of cells
were maintained throughout the soft agar colony incubation. The average
number of colonies for S3v-ErbB CEF was normalized to 100 for
comparison between replicates. Each experiment consisted of four
replicates and was repeated four times.
Rho Activity Assay--
The ratio of GTP- to GDP-bound Rho was
determined as described previously (15). Briefly, CEF and N19RhoA
expressing CEF were serum-starved for 24 h, metabolically labeled
with 1 mCi of [32P]orthophosphoric acid/4 ml of
phosphate-free media for 4 h at 37 °C, and stimulated
with 50 nM transforming growth factor- Coexpression of S3v-ErbB with N19Rho, N17Rac, or
N17Cdc42--
Dominant negative mutants of Rho (N19RhoA), Rac
(N17Rac), or Cdc42 (N17Cdc42) were subcloned into the
replication-competent avian retrovirus, RCAS, and these recombinant
viruses were used to infect primary CEF. A coinfection strategy for CEF
has previously been developed such that sequential infection of CEF
with two distinct envelope subtypes of RCAS allows for coexpression of two different genes of interest. Fig. 1
illustrates representative Western blot analyses of fibroblasts
coexpressing a nontransforming but constitutively active form of
v-ErbB, E1v-ErbB, or the transforming S3v-ErbB and each of these
dominant negative mutants. The expression of the v-ErbB mutants does
not alter the endogenous expression levels of Rac, Cdc42, or Rho (Fig.
1).
N19RhoA Blocks the Formation of the Tyrosine
Phosphoprotein Complex--
CEF expressing S3v-ErbB form a tyrosine
phosphoprotein complex that coprecipitates with anti-Shc antibodies;
this complex includes the proteins Grb2, Shc, and caldesmon (2). CEF
lysates from cells coinfected with dominant negative mutants of Rho,
Rac, or Cdc42 were immunoprecipitated with an anti-Shc antibody.
Immunoprecipitates were resolved by SDS-PAGE followed by Western blot
analysis with an anti-phosphotyrosine antibody (4G10, Upstate
Biotechnology). Fig. 2 shows that in the
presence of vector alone, N17Rac, or N17Cdc42, the formation of this
complex is not inhibited. In contrast, in CEF coexpressing N19RhoA and
S3v-ErbB the formation of this complex is completely inhibited, and the
tyrosine phosphorylation of Shc also is significantly reduced although
the levels of immunoprecipitated Shc remained constant (data not shown
and Fig. 2).
N19RhoA Inhibits S3v-ErbB-mediated Soft Agar Colony
Formation--
S3v-ErbB-expressing CEF form colonies in soft agar, a
characteristic consistent with the ability of transformed cells to grow in the absence of cell adhesion. To determine whether N19RhoA, N17Rac,
or N17Cdc42 expression would affect S3v-ErbB-induced
anchorage-independent cell growth, coinfected CEF were plated in soft
agar as previously described.2 After 3 weeks of growth,
colonies were counted, and the number of colonies found in S3v-ErbB CEF
was defined as 100% colony formation. As shown in Fig.
3, N19RhoA completely inhibits
S3v-ErbB-mediated soft agar colony formation. Interestingly, N17Rac and
N17Cdc42 also modestly reduce the number of soft agar colonies formed, but never to a statistically significant extent. The reduction in soft
agar colony formation is not the result of a reduction or inhibition of
cell growth. Cultures of coinfected cells grown in parallel with the
soft agar plates did not exhibit altered growth rates when compared
with control cells (data not shown).
Rho, but Not Rac, Is Constitutively Active in S3v-ErbB-transformed
Cells--
To determine whether S3v-ErbB constitutive tyrosine kinase
activity results in constitutive activation of Rho, CEF expressing E1v-ErbB or S3v-ErbB were plated, followed by serum starvation for
24 h. Cells were then stimulated with 50 nM
TGF S3v-ErbB has been shown to mediate fibroblast
transformation through a Ras-independent signaling mechanism (5). In
this study we have performed experiments to identify components of this
ligand-independent oncogenic signaling pathway that might connect
S3v-ErbB to its tyrosine-phosphorylated downstream mediators, such as
caldesmon and p21-activated kinase (2-4). Our results suggest the
existence of a ligand-independent oncogenic signaling pathway from
S3v-ErbB to the small GTP-binding protein Rho. The activation of Rho
apparently leads to the stimulation of at least one tyrosine kinase
that phosphorylates components of a previously described
transformation-associated phosphoprotein complex.
Here, we demonstrate that Rho activity is required for
S3v-ErbB-mediated phosphoprotein complex formation. Of particular
interest, Rho activation is required for the tyrosine phosphorylation
of several components of this signaling complex including caldesmon and
p21-activated kinase (data not shown). Careful observation also reveals
a decrease in Shc tyrosine phosphorylation; Shc tyrosine phosphorylation is characteristically elevated in S3v-ErbB-transformed fibroblasts. These data, therefore, suggest the involvement of at least
one tyrosine kinase downstream of Rho activation.
In this regard, several known tyrosine kinases have been
implicated downstream of Rho activation, as well as downstream of cytoskeletal changes within the cell, particularly in transformed cells. One such tyrosine kinase, Src, has been shown to function downstream of Rho via several mechanisms; Src-phosphorylated proteins have been shown to be tyrosine-phosphorylated downstream of Rho, and
Src and Rho share a common mediator linking them in this signaling pathway (16, 17). Specifically, Src has been shown to phosphorylate the
cytoskeletal proteins focal adhesion kinase (Fak) and
p130cas (16, 18-20). More significantly, recent
investigations have linked Rho and Src activation through the Rho
effector mDia (17). The Diaphanous-related formins, namely mDia1 and
mDia2, have been shown to act as effectors for Rho activation and to
link the tyrosine kinase activity of Src to Rho activation by acting as
scaffolding or bridging proteins (17). Together, these results suggest
that Src may be the critical kinase involved in tyrosine
phosphorylation of components of our transformation-associated
phosphotyrosine protein complex, and future studies will be directed
toward testing this hypothesis.
Fak is another tyrosine kinase that has been shown to be required for
the cytoskeletal changes observed as a consequence of Rho activation.
Specifically, Fak has been shown to be a downstream Rho effector.
Stimuli that activate Rho, e.g. sphingolipids, also result
in the tyrosine phosphorylation of Fak (21-23). More directly, Fak has
been shown to be autophosphorylated as well as phosphorylated by other
kinases, e.g. by Src family kinases, in a
Rho-dependent fashion (24, 25). Of additional interest
relevant to this study, EGF can indirectly stimulate Rho activation,
leading to increased activity of Src, as well as to the tyrosine
phosphorylation of Fak. These observations suggest that Fak also might
be an excellent candidate for the kinase responsible for
phosphorylation of our Shc-based phosphoprotein complex (26).
Evidence for yet a third candidate tyrosine kinase,
i.e. Abl, although less compelling, is still worth a
mention. Abl has been shown to be critical for several of the
actin-based cytoskeletal changes correlated with transformation. For
example, in Bcr-Abl-transformed cells, it is the kinase activity of Abl
that is critical for the cytoskeletal changes resulting in
morphological transformation (27). The specific cytoskeletal
alterations correlated with v-Abl transformation of fibroblasts require
Abl-binding partners and substrates, such as ArgB2 and c-Cbl (28, 29).
Moreover, Abl itself contains an F-actin-binding domain that competes
with gelsolin and is critical for F-actin bundling (30). These
observations suggest that Abl also may be a tyrosine kinase downstream
of Rho activation, providing yet another testable candidate for the
kinase responsible for the tyrosine phosphorylation of the
phosphoprotein complex observed in S3v-ErbB-transformed fibroblasts.
This study also demonstrates that Rho is constitutively activated in
S3v-ErbB-transformed fibroblasts, suggesting that a constitutive signal
from S3v-ErbB to Rho may be critical for expression of the phenotypes
observed. In contrast, E1v-ErbB does not constitutively activate Rho,
suggesting that more than just a constitutively active tyrosine kinase
leads to Rho activation. These observations also demonstrate that
although many pathways may be activated by constitutively active
S3v-ErbB, only the pathway to Rho has escaped normal regulation by
governing proteins, such as the GTPase-activating proteins. In this
regard, studying aspects of regulating Rho activity in
v-ErbB-transformed cells may be informative. Recently, the guanine
nucleotide exchange factor Vav has been implicated in Rho activation
via EGF stimulation of the EGFR (31). Specifically, tyrosine
phosphorylation of Vav3 by the EGFR in response to EGF stimulation
activates Rho (32). In contrast, other activators of Rho,
i.e. Ost, Lbc, and Trio have not been shown to be
regulated by EGF. Additional studies will be needed to determine
whether Vav family members are the missing link between S3v-ErbB
oncogenic signaling and Rho activation.
Even though we demonstrate here that Rho activity is
required for transformation of fibroblasts by S3v-ErbB, it remains a point of controversy whether expression of a constitutively active Rho
is sufficient for fibroblast transformation. It has been shown, however, that constitutive activation of Rho can transform fibroblasts in the presence of constitutively active Raf (Raf-CAAX) (33). The
authors of this particular study conclude that these two pathways are
required for transformation of fibroblasts via Rho; one pathway is
independent of Raf activity and regulates stress fiber dynamics, and a
second pathway is dependent on Raf activity (33). In addition, Rho and
Rac also have been shown to cooperate in the transformation of
fibroblasts (34). Rho also has been implicated in the regulation of the
EGFR endosomal trafficking, such that the EGFR is delayed from moving
past the late endosome when Rho is constitutively active (35). The
regulation of S3v-ErbB trafficking may perhaps provide an alternate
mechanism for the contribution of Rho in S3v-ErbB-mediated
transformation. Furthermore, constitutive activation of proteins shown
to regulate the activity of Rho, such as the guanine nucleotide
exchange factor Lbc, also have the ability to induce
anchorage-independent cell growth (36). In contrast, several
investigators have demonstrated that the overexpression of a
constitutively activated mutant Rho directly leads to the formation of
tumors in nude mice (3, 37). Additional studies will be required to
determine the basis for these discrepancies and to more precisely
define the relationship between the role of Rho in regulating stress
fiber dynamics versus transformation.
In conclusion, we believe that Rho plays a critical role in
the transformation of fibroblasts by S3v-ErbB. We further hypothesize that Rho is constitutively activated by signals downstream of S3v-ErbB
and that the normal regulatory proteins governing Rho activation are
either outnumbered or disregulated themselves in S3v-ErbB-transformed
fibroblasts. This constitutive activation of Rho leads to the
constitutive activation of a nonreceptor tyrosine kinase, such as Src,
Fak, or Abl, resulting in the tyrosine phosphorylation of a signaling
complex of proteins including Shc, caldesmon, and p21-activated kinase.
These tyrosine phosphorylation events not only result in cytoskeletal
changes such as stress fiber disassembly, but also provide signals that
lead to the transformation of fibroblasts. Together, these observations
support the working hypothesis that ligand-independent oncogenic
signaling by S3v-ErbB is distinct from the ligand-dependent
mitogenic pathway that regulates normal cell division. Further
delineation of this putative oncogenic signaling pathway may reveal new
therapeutic targets unique to transformed cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF
).
Cells were lysed in Rho extraction buffer (50 mM Tris (pH
7.5), 20 mM MgCl2, 150 mM NaCl, 5%
Nonidet P-40, 1 mM aprotinin, 1 mM
phenylmethylsulfonyl fluoride) and immunoprecipitated with an antibody
against Rho (Santa Cruz Biotechnology). GDP and GTP were eluted
from the immunocomplexes in 1 M
KH2PO4 (pH 3.4) at 100 °C for 3 min. Eluants
were separated using thin-layer chromatography developed in 1 M KH2PO4 (pH 4.5), and
chromatograms were exposed to x-ray film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
N19RhoA, N17Rac, or N17Cdc42 coexpression
with E1v-ErbB or S3v-ErbB. Lysates from cells coinfected with RCAS
viruses containing N19RhoA, E1v-ErbB, and S3v-ErbB were resolved by
SDS-PAGE, followed by Western blot analysis with antibodies specific
for v-ErbB (top panel in A, B, and
C) (T2, (13)), Rho (Santa Cruz Biotechnology;
bottom panel in A), Cdc42 (Santa Cruz
Biotechnology; bottom panel in B), and Rac
(Transduction Labs; bottom panel in C).
Two bands are observed in the DNCdc42- and DNRac-containing lanes
because the dominant negative mutants are both Myc-tagged and,
therefore, migrate slower on SDS-PAGE.
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Fig. 2.
Phosphoprotein complex formation
characteristic of S3v-ErbB-transformed cells is blocked in CEF
expressing N19RhoA. Lysates from cells coexpressing dominant
negative mutants (as indicated) and S3v-ErbB were immunoprecipitated
with anti-Shc antibodies; proteins were resolved by SDS-PAGE, followed
by Western blot analysis with anti-phosphotyrosine antibodies (4G10,
Upstate Biotechnology).
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Fig. 3.
N19RhoA expression inhibits S3v-ErbB-mediated
soft agar colony formation. CEF coexpressing S3v-ErbB and DNRhoA,
DNRac, or DNCdc42 were plated in soft agar for 3 weeks, and colonies
were counted using light microscopy. The average number of colonies for
S3v-ErbB CEF were normalized to 100 for the purpose of comparison. Each
experiment consisted of four replicates, and each experiment was
repeated four times. N19RhoA/S3v-ErbB colony numbers are statistically
significant, with a p value of <0.05. Parallel cultures
were maintained to ensure that cell viability would not be a concern
for the cells in soft agar.
and metabolically labeled with
32Pi. Cell lysates were immunoprecipitated with
anti-Rho antibodies, and the associated nucleotides were separated by
thin-layer chromatography. The resultant chromatogram was analyzed
using computer-aided densitometry. Fig. 4
illustrates a graphical representation of these data. These results
indicate that the level of Rho activity in CEF expressing S3v-ErbB is
comparable with the level of Rho activation seen in
-stimulated
cells. In contrast, cells expressing E1v-ErbB exhibit Rho activity
levels comparable with the basal levels observed in serum-starved
cells. These results suggest that constitutive tyrosine kinase activity
(e.g. of the nontransforming E1v-ErbB mutant) is not
sufficient to stimulate constitutive Rho activity. In addition,
S3v-ErbB-expressing fibroblasts do not exhibit constitutive activation
of Rac (data not shown). Taken together, these data suggest a unique
role for Rho in S3v-ErbB-transformed fibroblasts.
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Fig. 4.
Rho activity is constitutively active in
S3v-ErbB-transformed fibroblasts. CEF expressing E1v-ErbB or
S3v-ErbB were serum-starved, metabolically labeled with
32Pi, and stimulated with TGF . Cell
lysates were immunoprecipitated with anti-Rho antibodies, and
associated guanine nucleotides were eluted with hot acid and separated
by thin-layer chromatography. Radiolabeled GTP or GDP was detected
using x-ray film, and images were analyzed via computer-aided
densitometry. This graph is representative of the results obtained in
two independent experiments. TGF, transforming growth factor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Trace Christensen and Andy Danielsen for their technical assistance. We also thank Anne Ridley, Alan Hall, and Marc Symons for the dominant negative Rac, Rho, and Cdc42 constructs, respectively.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA75238 (to M. J. M.) and CA 79808 (to N. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Submitted in partial fulfillment of doctoral thesis requirements in the Mayo Graduate School. Supported by National Institutes of Health Training Grant CA 75926 (to the Tumor Biology Program of the Mayo Graduate School). Current address: Dept. of Microbiology, University of Virginia Health Science Center, Charlottesville, VA 22908.
Current address: Dept. of Molecular and Experimental Medicine,
Scripps Research Inst., La Jolla, CA 92037.
** To whom correspondence should be addressed: Mayo Clinic, 200 1st St. SW, Rochester, MN 55905. Tel.: 507-284-8121; E-mail: Maihle@mayo.edu.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M003801200
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ABBREVIATIONS |
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The abbreviations used are:
EGFR, epidermal
growth factor (EGF) receptor;
CEF, chicken embryo fibroblast(s);
SDS-PAGE, SDS-polyacrylamide gel electrophoresis;
Fak, focal adhesion
kinase;
TGF, transforming growth factor-
.
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REFERENCES |
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