The Src family of non-receptor tyrosine kinases is composed
of nine members: c-Src, c-Yes, Fyn, Lyn, Lck, Hck, Blk, Fgr,
and Yrk. These proteins participate in a variety of cellular
signal transduction pathways, governing such diverse
processes as cell division, differentiation, survival, motility,
and vesicular trafficking (Thomas and Brugge, 1997).
Members of the Src family are defined by their amino acid
homology and functional domain architecture. All Src family
members are comprised of an N-terminal membrane-
localization domain (Src Homology 4 or SH4 domain), a
poorly conserved Unique domain, an SH3 domain, an SH2
domain, a tyrosine kinase or SH1 domain, and a short carboxy-
terminal regulatory sequence (Thomas and Brugge, 1997). c-
Src and c-Yes are two of the most ubiquitously expressed and
highly homologous members of the Src family (Sudol and
Hanafusa, 1986; Zhao et al., 1990; Sugawara et al., 1991),
sharing over 80% homology outside of their Unique domains
(Kitamura et al., 1982).
Given the high homology between these two kinases and
their widely overlapping tissue distributions, it is of little
surprise that they are capable of performing redundant
functions. c-Src and c-Yes are both activated downstream of a
multitude of cell surface receptors, including receptor tyrosine
kinases, G-protein-coupled receptors, and cytokine receptors
(Kypta et al., 1990; Landgren et al., 1995; Fuhrer and Yang,
1996a). Additionally, both kinases are activated during the cell
cycle transition from G2 to M phase. Roche et al., provided
strong evidence for functional overlap between c-Src, c-Yes,
and Fyn, in demonstrating that inhibition of all three Src family
kinases blocked cell cycle progression at the G2/M transition
(Roche et al., 1995). Inhibition of c-Src alone did not block
cell cycle progression unless c-Src was the only Src family
member expressed (Roche et al., 1995). Further evidence for
functional redundancy between c-Src and c-Yes has been
derived from the c-src and c-yes knockout mice. While the loss
of neither gene individually is embryonic lethal, mice lacking
both genes fail to survive after birth (Stein et al., 1994).
Despite the evidence for functional overlap, several
studies have also indicated specificity between c-Src and c-
Yes (reviewed by Summy et al., 2003). The two kinases
differ in their sub-cellular localization (Sargiacomo et al.,
1993), intermolecular binding partners (Fuhrer and Yang,
1996b), activation in response to cellular stimulation
(Mukhopadhyay et al., 1995), and ability to mediate
downstream signaling (Schieffer et al., 1996). The
inactivation of the c-src and c-yes genes has provided the
most compelling evidence for functional specificity. With
c-Src and c-Yes are highly homologous members of the Src
family of non-receptor tyrosine kinases. The overall
sequence similarity between c-Src and c-Yes allows them
to perform many overlapping functions. However, the
phenotypes of the c-src and c-yes knockout mice, and
cells derived from them, are quite different, indicating
functional specificity between the two proteins. Specifically,
c-src–/–cells are deficient in several processes that require
dynamic regulation of the actin cytoskeleton. In order to
begin to understand why c-Yes is unable to compensate for
c-Src signaling, we used a series of Src/Yes chimeras in
which the non-catalytic functional domains of Src527F
were replaced by those of c-Yes. Using chicken embryo
fibroblasts as a model system, our results indicate that the
c-Yes N-terminal SH4-Unique domains are sufficient to
inhibit the ability of Src527Fto alter cell morphology, induce
actin filament rearrangements or stimulate motility or
invasive potential. The data also indicate that the SH4-
Unique-SH3-SH2 domains of c-Yes work cooperatively
and prevent activation of signaling proteins associated
transformation, including activation of
phosphatidylinositol 3-kinase, phosphorylation of c-Raf
and Akt and downregulation of RhoA-GTP. These data
indicate that c-Yes may not modulate signals associated
with c-Src-induced changes in actin filament integrity and
may explain why c-Yes fails to compensate for c-Src
signaling in src–/–cells.
Key words: Yes, Src, Actin filaments, SH4 domain, Unique domain
The SH4-Unique-SH3-SH2 domains dictate specificity
in signaling that differentiate c-Yes from c-Src
Justin M. Summy1,*,‡, Yong Qian2,*, Bing-Hua Jiang1, Anne Guappone-Koay1, Amanda Gatesman1,
Xianglin Shi2and Daniel C. Flynn1,§
1Department of Microbiology, Immunology, and Cell Biology, and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine,
PO Box 9300, Morgantown, WV 26506, USA
2National Institute for Occupational Safety and Health, Pathology and Physiology Research Branch, Health Effects Laboratory Division,
Morgantown, WV 26505, USA
*These authors contributed equally to this work
‡Present address: Department of Cancer Biology, University of Texas MD Anderson Cancer Center, PO Box 79, 1515 Holcombe Drive, Houston, TX 77030, USA
§Author for correspondence (e-mail: email@example.com)
Accepted 7 March 2003
Journal of Cell Science 116, 2585-2598 © 2003 The Company of Biologists Ltd
JCS ePress online publication date 6 May 2003
polyimmunoglobulin (pIg) receptor, the c-yes–/–
display no overt phenotype (Luton et al., 1999). However,
mice lacking the c-src gene develop osteopetrosis due to a
perturbation of osteoclast function that prevents bone
resorption (Soriano et al., 1991). The defective osteoclasts
are additionally unable to form membrane ruffles and actin
ring structures (Boyce et al., 1992). Osteoclasts, however,
are not the only cells that are affected by the loss of the
c-src gene. Additional defects in c-src–/–cells include
inefficient motility (Hall et al., 1996), decreased rates of
fibroblast spreading (Kaplan et al., 1995), and neurite
extension (Ignelzi, Jr et al., 1994). It is of interest to note
that all of these processes are dependent on the dynamic
regulation of the actin cytoskeleton. As cytoskeletal
rearrangements are a hallmark of cell transformation, it is
likely that c-Src and c-Yes may play divergent roles in the
onset or progression of the transformed phenotype. In
support of this notion, it has been observed previously that
induction of mammary tumors by middle T antigen is
impaired in c-src–/–cells, whereas tumor formation occurs
normally in the absence of a functional c-yes gene (Guy et
al., 1994). We have hypothesized that functional domain
differences prevent c-Yes from compensating for c-Src in
signaling pathways that regulate actin cytoskeletal dynamics
(Summy et al., 2003).
Given the significant amino acid sequence homology
between c-Src and c-Yes, it is likely that functional domain
specificity may result from subtle differences in amino acid
composition, and thus each functional domain may contribute
to signaling specificity. Previous studies have indicated minor
differences in the ligand specificity of the c-Src and c-Yes SH3
domains in vitro (Rickles et al., 1995). Data obtained in our
laboratory indicate that the c-Yes SH3 domain is incapable of
efficiently binding several c-Src SH3 domain binding partners,
including the actin filament associated protein AFAP-110
(Summy et al., 2000). In contrast to the SH3 domain, little data
exists to suggest specificity between c-Src and c-Yes, or other
Src family members, at the level of the SH2 domain. However,
we recently demonstrated co-immunoprecipitation of an 87
kDa tyrosine-phosphorylated protein (pp87) with Src527F/c-
Yes chimeras containing the c-Yes SH2 domain, indicating that
specificity may also be derived from SH2 domain differences
(Summy et al., 2000).
Several recent studies have pointed to the role of the Src
family N-terminus in dictating signaling specificity between
Src family kinases. All Src family members with the exception
of c-Src and Blk contain one or more cysteine residues
downstream of the myristoylated glycine residue at amino acid
position two (Resh, 1994). These cysteine residues are sites of
palmitoylation and incorporation of one or more palmitate
residues facilitates localization
membrane fractions, also known as lipid rafts (Resh, 1994;
Robbins et al., 1995). Localization to lipid rafts is important
for Src family kinase participation in Fcε receptor and T-cell
receptor signaling (Kabouridis et al., 1997). Hoey et al.
recently demonstrated that replacement of the Src527FN-
terminus (including the SH4 and Unique domains) with that of
c-Yes prevented upregulation of heme oxygenase 1 (HO-1)
message and protein (Hoey et al., 2000). Thus it is evident that
multiple functional domains may contribute to specificity in
exception of reduced transcytosis of the
signaling, and hence function, between c-Src and c-Yes. In the
present study, we have sought to gain a better understanding
of signaling specificity between c-Yes and c-Src. In order to
accomplish this, we have replaced the non-catalytic functional
domains of Src527Fwith those of c-Yes and assessed the ability
of the resulting chimeras to induce differential cellular signals,
morphological and cytoskeletal changes associated with
overexpression of constitutively active c-Src in chicken embryo
fibroblast cells (CEF).
Materials and Methods
cDNA constructs encoding LA29 temperature-sensitive v-Src, c-Src,
Src527F, Y3527F, Y2527F, Y32527F, Y4U32527F, Y4U527F, Y4527Fand
YU527Fwere generated within the Rous Sarcoma Virus (RSV) as
described previously (Felice et al., 1990; Summy et al., 2000; Hoey
et al., 2000).
Chicken embryo fibroblasts (CEFs) were prepared as described
previously from day 10 eggs (Spafas) (Flynn et al., 1993). Cells were
passed 1:4 every 48 hours in Falcon 100 mm tissue culture dishes or
Falcon 250 ml vented-cap tissue culture flasks. CEFs were transfected
at one half confluence with 15 µg of plasmid DNA using the Clontech
Calphos kit, as per the protocol. Confluent cells were viewed through
a Nikon Phase Contrast II microscope at 40× total magnification (Plan
2 filter) and photographed with a Nikon N 2000 camera using Kodak
TMax 400 black and white film.
The rabbit polyclonal anti-Src antibody was raised against an epitope
in the Src C-terminus as described previously (Summy et al., 2000).
This antibody was used at a 1:1000 dilution for western blot analysis.
The anti-phosphotyrosine and anti-pp85 cortactin antibodies were
obtained from BD Transduction and were used at a 1:1000 dilution
for western blot analysis. The rabbit anti-phospho-Y416was obtained
from Upstate Biotechnology and used at a dilution of 1:1000. The
anti-Shc antibody is a rabbit polyclonal antibody commercially
available from BD Transduction; it was used at a 1:1000 dilution for
western blot analysis and a 1:200 dilution for immunoprecipitation.
The anti-Grb2 antibody was obtained from BD Transduction and used
at a 1:5000 dilution for western blot analysis. The anti-phospho-c-Raf
antibody was obtained from Biosource International; it recognizes c-
Raf phosphorylated on tyrosines 340 and 341, and was used at a
1:1000 dilution for western blot analysis. The rabbit anti-c-Raf-1
antibody was obtained from BD Transduction; it was used at a 1:1000
dilution for western blot analysis. The monoclonal anti-phospho-MAP
kinase antibody was obtained from Upstate Biotechnology. It is
immunoreactive against Erk 1 and Erk 2 phosphorylated at the pT-E-
pY motif and was used at a dilution of 1:1000 for western blot
analysis. The monoclonal anti-MAP kinase antibody was obtained
from Upstate Biotechnology. This antibody recognizes Erk 1 and 2
and was used at a 1:1000 dilution for western blot analysis. The rabbit
anti-phospho-Akt antibody was obtained from Cell Signaling. This
antibody recognizes Akt-1 phosphorylated at serine 473 and was used
at a 1:1000 dilution for western blot analysis. The monoclonal anti-
Akt antibody was obtained from BD Transduction and used at a 1:500
dilution for western blot analysis. The rabbit anti-rat PI3K p85
antibody was obtained from Upstate Biotechnology and used at a
dilution of 1:1000 for western blot analysis and 1:50 for
immunoprecipitation. Anti-rabbit and anti-mouse horseradish
peroxidase-conjugated secondary antibodies were obtained from
Amersham Life Science and used at a dilution of 1:1000.
Journal of Cell Science 116 (12)
2587 Specificity in signaling by c-Yes
Immunofluorescence was carried out as described previously (Qian et
al., 1998). Briefly, CEF were split onto coverslips and fixed in 3.7%
formaldehyde at 50% confluence. The cells were washed three times
in PBS, permeablized in 0.4% Triton X-100, washed, and stained with
rhodamine-conjugated phalloidin (2 µg/µl in 5% BSA/PBS). Cells
were stained for 20 minutes, washed three times in PBS, and mounted
on coverslips using Flouromount G (Southern Biotechnology
Associates). Cells were visualized using a Zeiss LSM 510 confocal
microscope (63× objective).
Western blot analysis
Cells were lysed at confluence in RIPA buffer as described previously
(Hoey et al., 2000). Cell lysates were quantitated for total protein
content using the Pierce BCA assay as per the protocol. Thirty or fifty
µg of cell lysates were boiled in Laemmli’s sample buffer (LSB) and
resolved by 8% SDS-PAGE. The proteins were transferred to PVDF
membrane and washed as described previously (Hoey et al., 2000).
The membranes were blocked overnight in 1% BSA formulated in
Tris buffered saline with 1% Tween 20 (TBS-T) at 4°C or 5% nonfat
milk in TBS-T for 30 minutes at room temperature. The membranes
were probed with primary antibody (diluted in TBS-T) for 1 hour at
room temperature or overnight in 5% nonfat milk/TBS-T at 4°C.
Secondary antibodies were applied for 45 minutes in TBS-T. Bound
antibodies were visualized by incubation with ECL reagents
(Amersham Pharmacia), followed by X-ray film (Kodak) exposure
(Hoey et al., 2000).
Five hundred µg of RIPA lysates were incubated with antibody for
1.5 hours at 4°C with rotating. Twenty µl of Protein A/G agarose
(Santa Cruz) was added for an additional 1.5 hours at 4°C with
rotation. The beads were centrifuged for 30 seconds at room
temperature, followed by two washes in RIPA and two washes in TBS.
Bound proteins were eluted by boiling in LSB and resolved by SDS-
PAGE. Western blot analysis was carried out as described above.
Cells were separated into Triton X-100 soluble (C) and insoluble (R)
fractions as described previously (Hamaguchi and Hanafusa, 1987).
The insoluble (R) fraction contains the cytoskeletal associated cellular
proteins. Briefly, cells were washed twice with cold Tris-buffered
saline. Cells were then incubated with 1 ml of cold CSK buffer (10
mM PIPES, pH 6.8, 100 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2, 0.3
M sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1%
Triton X-100) on ice with gentle rocking for 1, 4 or 10 minutes. The
soluble material was removed, and the remaining material was
solubilized in 1 ml of RIPA buffer as described previously. Lysates
were clarified by 5 minutes centrifugation at 13,100 g and 4°C in
a Hermle Z 360 K bench-top centrifuge, and supernatants were
quantitated for total protein content. Samples were prepared for
western blot analysis as described above. Triton-soluble fractions
were equilibrated to RIPA by addition of 1% sodium deoxycholate
and 10 mM Tris-HCl, pH 8.0, prior to protein quantitation.
Phosphatidylinositol 3-kinase and RhoA-GTP binding assays
The phosphatidylinositol 3-kinase (PI3K) assay was performed as
described elsewhere (Jiang et al., 1998). Briefly, CEF were scraped
from tissue culture flasks in cold PBS and pelleted at 1000 rpm and
4°C for 5 minutes in a Sorvall RT6000B bench-top centrifuge. Cells
were then lysed and total protein concentration was determined. Four
hundred µg of lysate were pre-cleared with 20 µl of Protein A/G
agarose for 1 hour at 4°C. Lysates were immunoprecipitated with 2 µl
of anti-PI3K antibody for 1 hour at 4°C. Twenty-five µl of protein A/G
agarose beads were added and incubated with rotation for an additional
1 hour at 4°C. The immunoprecipitations were washed with TNE
buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA), centrifuged,
and the pellet was then resuspended in 50 µl of PI3K assay buffer (20
mM HEPES pH 7.5, 10 mM MgCl2, 0.2 mg/ml phosphoinositol, 60
µM ATP, 2 µCi [γ-32P] ATP). The reactions were incubated at room
temperature for 15 minutes. Following the reaction, the products
were extracted by addition of 80 µl 1 M HCl and 160 µl
chloroform/methanol. The upper phase was removed, and the lower
phase, containing the lipid products, was dried. The pellets were then
resuspended in chloroform and spotted on thin layer chromatography
(TLC) plates. Once the buffer front had migrated to the top of the plate,
the plates were removed, dried, and exposed via phosphorimaging.
Rho activation assays were conducted as described (Edlund et al.,
2002). Briefly, the cells were washed with 1× PBS supplemented with
1 mM MgCl2. After the washing, the cells were lysed immediately
with lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2,
10 µg/ml aprotinin and 1 mM PMSF). The lysates were centrifuged
at 18,400 g for 15 minutes. The supernatants were added to GST-
rhotekin in GST beads to pull-down Rho proteins, followed by
incubation at 4°C for 20 minutes. After the incubation, the beads were
washed twice with cold wash buffer (50 mM Tris-HCl pH 7.5, 1%
Triton X-100, 150mM NaCl, 10mM MgCl2, 10µg/ml aprotinin, and
0.1 mM PMSF). The Rho protein was eluted with sample buffer
and subjected to 15% SDS-PAGE. The western blot analysis was
performed using anti-Rho polyclonal antibody. Both ECL cell
attachment matrix and anti-Rho antibody are from Upstate Bio. GST-
rhotekin is a kind gift from Pontus Aspenström (Ludwig Institute for
Cancer Research, Uppsala, Sweden).
Transwell migration assay
Transwell migration assays were conducted using modifications of the
method described by the manufacturer (BD Biosciences). Briefly, the
cells were serum-starved overnight. The transwells were coated with
E-C-L cell attachment matrix (Upstate Biotechnology) at 20 µg/ml
and incubated for one hour at 37°C. The top chambers of the transwell
were loaded with 4×105cells/ml in 0.5% serum DMEM media and
the bottom chambers were filled with 5% FCS DMEM media. The
5% FCS served as an attractant for the cells. The transwells were
incubated in 0.5% CO2at 37°C for 16-18 hours. After the incubation,
the cells that had migrated were fixed with 10% formalin, stained with
Harris Modified Fisher Hematoxylin (Fisher Co.), and mounted on
slides. Cells were also preincubated for 2 hours with 20 µM
LY294002 prior to the migration assay, to determine the effects of
inhibiting PI3K upon migration. The images were taken using an
Olympus inverted microscope, and represent the typical fields per
each sample. Cell numbers were counted, or estimated, depending
upon the degree of cell clumping, within 10× fields of magnification
in order to determine the numbers of cells that migrate. Treatment
with the PI3K inhibitor, LY294002, was performed using a 20 µM
concentration for 22 hours prior to analysis of cells for motility or
invasion. After 22 hours of treatment, cells are routinely analyzed for
survival after removal of the drug. Cell survival was noted with little
evidence of cell death. Cell migration assays were conducted and
quantified as described (Huack et al., 2002). Briefly, the cells were
serum-starved overnight, the transwells were coated with ECL cell
attachment matrix (Upstate Biotechnology) at 20 µg/ml. The top
chamber of transwell was loaded with 0.2 ml of 4×105cells/ml in
serum-free media and the bottom chamber with 0.6 ml of DMEM
medium containing 0.5% FCS. The cells were incubated in the
transwells at 37°C in 5% CO2for 14 hours. Migrated cells were fixed,
stained with 0.1% crystal violet. Washing five times with 1× PBS
removes extraneous, unbound crystal violet, leaving bound crystal
violet associated with only migrated cells. Following washing of the
migrated cells, elution of bound crystal violet with 10% acetic acid
enables quantification of the relative levels of cell-associated dye by
spectrophotometry. The microplate reader was used to measure the
OD of the eluted solutions to determine the migration values. The
mean values were obtained from three individual experiments and
were subjected to a t-test (P<0.01, n=3).
Invasion assays were performed according to manufacturer’s protocol
(BD Biosciences). The cells were serum-starved overnight. Cells (0.5
ml of 1.0×105cells/ml) were loaded on pre-coated matrigel 24-well
invasion chamber (BD Biosciences). 0.5 ml of 5% FCS DMEM media
was added to the wells of the BD Falcon TC Companion plate to serve
as the chemoattractant for the cells. The matrigel invasion chambers
were incubated in 0.5% CO2 at 37°C for 22 hours. Cells were also
preincubated for 2 hours with 20 µM LY294002 prior to the migration
assay, to determine the effects of inhibiting PI3K upon migration.
After the incubation, the invading cells were fixed with 10% formalin,
stained with Harris Modified Fisher Hematoxylin (Fisher Co.), and
mounted onto slides. The invading cells were counted and analyzed
according to manufacturer’s instruction.
Expression of Src527F/c-Yes chimeric constructs in CEF
Chicken embryo fibroblasts were transfected at 50%
confluence with RSV constructs expressing Src527F, or
Src527F/c-Yes chimeric constructs, where Src527Fcontained the
c-Yes SH4-Unique-SH3-SH2 domains (Y4U32527F), the SH4-
Unique domains (Y4U527F), the c-Yes SH3 and SH2 domains
(Y32527F), SH4 domain (Y4527F), Unique domain (YU527F),
SH3 domain (Y3527F) or the c-Yes SH2 domain (Y2527F) (Fig.
1). The temperature-sensitive v-Src variant, LA29 was used as
an additional positive control, and is well known to affect
upregulation of tyrosine phosphorylation and changes in the
actin cytoskeleton at permissive temperature, 35°C (van der
Valk et al., 1987; Felice et al., 1990). After 12 days, cells were
lysed in RIPA, 30 µg of cell lysates were resolved by SDS-
PAGE, and expression of tyrosine phosphorylated proteins
verified by western blot analysis using an anti-phosphotyrosine
antibody. As seen in Fig. 2A, all constructs generated an overall
increase in cellular tyrosine phosphorylation. A light exposure
is shown in order to analyze the cells for changes in the profile
and content of phosphotyrosine-containing proteins that might
correlate with the functional domains present in each chimera
(Fig. 2A). Although most protein bands revealed equivalent
steady-state levels of phosphotyrosine content when
comparing all constructs (e.g. at 75 kDa), there was some
evidence for reduced tyrosine phosphorylation in the
Y4U32527F-expressing cells. These include a reduction in
phosphorylation of 190 kDa, 180 kDa and 65 kDa proteins,
while increased phosphorylation was seen in a 47 kDa protein.
To ensure equal protein loading, cell lysates were probed with
anti-pp85 cortactin, which revealed equivalent levels of pp85
cortactin among each sample (Fig. 2B). Identical results were
obtained by western blot analysis of cell lysates for pp130cas
or pp125FAK (data not shown). Analysis of Src construct
expression levels indicate some minor differences in
expression levels (Fig. 2C). Western blot analysis of Src levels
indicated that Src527F, Y3527Fand Y4U32527Fwere expressed
at comparable levels. Y2527Fand Y32527Fwere expressed at
relatively higher levels than Src527F, while Y4U527Fwas
expressed at relatively lower levels. The temperature-sensitive
vSrc variant, LA29, is always expressed at very low levels
(J.M.S. and D.C.F., unpublished) and was used as a control to
correlate expression with changes in cellular phosphotyrosine
content. Western blot analysis with anti-phosphoY416 revealed
that each of the chimeric constructs, in addition to Src527F,
demonstrated increased phosphorylation at Tyr416, a signature
for activation (Fig. 2C). Src527F, Y3527Fand Y2527Fdisplay
similar levels of autophosphorylation, while Y32527F, Y4U527F
and Y4U32527Fappear to have higher steady state levels of
autophosphorylation. Thus, each of these constructs can be
expressed in CEF cells and are activated based on increased
autophosphorylation and increased
phosphorylation. These data also indicate that although there
may be minor differences in expression levels, each of the
chimeric constructs direct upregulation of cellular tyrosine
phosphorylation levels to equivalent levels.
The Y4U32527Fand Y4U527Fchimeras do not induce
morphological changes or rearrangement of cytoskeletal
We hypothesized that differences in one or more of the
functional domains were responsible for the inability of c-Yes
to compensate for c-Src in regulation of cellular pathways
controlling actin cytoskeletal dynamics. We chose chicken
embryo fibroblast cells (CEFs) as a model system for these
studies, as CEFs have a well-defined system of actin filament
stress fibers that undergo a characteristic rearrangement upon
overexpression of constitutively active Src (Reynolds et al.,
1989). Transfected cells were evaluated for the Src-
transformed phenotype, the hallmarks of which include a
rounded morphology, the lack of a well-organized monolayer,
and an absence of contact-dependent inhibition of cell growth.
As seen in Fig. 3A, the Y4U32527Fand Y4U527F chimeras
Journal of Cell Science 116 (12)
Fig. 1. Src527F/c-Yes chimeric constructs. Src527F/c-Yes chimeras are
depicted subdivided into the following functional domains: SH4
domain; Unique domain; SH3 domain; SH2 domain; protein tyrosine
kinase (PTK) domain; and regulatory sequence (Reg). Gray boxes
represent domains from c-Yes; white boxes represent domains from
Src527F. The generation of the Y3527F, Y2527F, Y32527F, Y4U32527F,
Y4U527F, Y4527Fand YU527Fchimeric constructs has been described
previously (Summy et al., 2000). All proteins were expressed in CEF
via the Rous Sarcoma Virus (RSV) vector.
2589 Specificity in signaling by c-Yes
failed to induce changes in cell morphology, as the morphology
of these cells was difficult to distinguish from that of mock-
transfected cells. Y32527F, however, induced morphological
changes similar to those induced by Src527F(Fig. 3A), as did
Y3527Fand Y2527F(data not shown). As the inability to induce
radical changes in cell morphology were associated primarily
with the c-Yes N-terminal SH4 and Unique domains, Y4527F
and YU527Fchimeras were generated in an attempt to
determine whether these affects could be attributed primarily
to either the c-Yes SH4 or Unique domains individually. Upon
expression of these constructs in cultured fibroblasts, it was
observed that Y4527Fand YU527Fdisplayed more evidence for
disorganization in monolayer than CEFs. Expression levels of
the Y4527Fand YU527Fchimeras were equivalent to those of
Src527F(data not shown). These data indicate that the c-Yes
SH4-Unique-SH3-SH2 domain, collectively, do not permit
Y4U32527Fto induce changes in cell morphology or to affect
the organization of the monolayer. Further, the SH4-Unique
domains alone appear to play a major role in preventing these
changes, and these domains appear to all work cooperatively.
The change in cell morphology induced by constitutively
active Src527Foccurs concomitantly with rearrangements of the
actin cytoskeletal structure. Src527F-transformed cells display
a loss of actin stress fibers and focal adhesions, with the actin
repositioning into rosettes, lamellipodia and filopodia. In order
to determine the effects of the c-Yes functional domains on the
ability of Src527Fto exert its influence on the actin-based
cytoskeleton, cells were fixed on coverslips, stained with
rhodamine-conjugated phalloidin, and visualized via confocal
laser microscopy. As can be seen in Fig. 3B, cells expressing
Src527Fwere morphologically distinct, and actin staining was
detected in punctate rosette structures along the cell periphery
or in actin-based motility structures such as lamellipodia or
filopodia. Similar actin staining was also detected in cells
expressing Y32527F, Y4527Fand YU527F. However, Y4U527F
and Y4U32527Fappeared to have contiguous, well-formed
actin filaments (Fig. 3B). These data indicate that the cYes
SH4-Unique-SH3-SH2 domains do not support changes in the
actin based cytoskeleton associated with the activated tyrosine
kinase and that the presence of the c-Yes SH4-Unique domains
is largely responsible for preventing these changes in actin
filament organization, which correlates well with changes in
Src527F/c-Yes N-terminal chimeras are associated with
the Triton X-100 insoluble cytoskeletal fraction
To investigate the mechanisms associated with the inability of
Y4U32527Fand Y4U527Fto alter actin filament integrity, we
next examined the subcellular distribution of these chimeric
proteins. One of the hallmarks of transformation-competent
variants of Src is that they are associated with cellular
membranes via their SH4 domains. Fractionation of cells into
membrane and cytosolic preparations revealed that Src527Fand
Fig. 2. Effects of Src527F/c-Yes chimeras on cellular phosphotyrosine. cell morphology, and the actin cytoskeleton. (A) 30 µg of day 12 RIPA
lysates from mock-transfected CEF or cells expressing Src527F, LA29, or the chimeric constructs were separated by 8% SDS-PAGE. Lysates
were transferred to PVDF membrane, and probed with a rabbit anti-phosphotyrosine antibody Molecular weight markers are shown on the left
side of the figure. Protein bands of note are highlighted by their Mr, on the right side of the figure. (B) 50 µg of cell lysate (as used in Fig. 2A)
was resolved by 8% SDS-PAGE, followed by western transfer and probed with the anti-pp85 cortactin antibody. (C) By using the same lysates
as in Fig. 2A, 30 µg of lysate was resolved by 8% SDS-PAGE and western blot analysis performed with rabbit anti-src antibodies to quantify
the steady state levels of Src and the chimeric constructs (top panel), or with anti-phosphoY416 to detect the activation state of the Src or
all Src527F/c-Yes chimeras were associated predominantly with
the membrane fraction (data not shown). Thus the inability of
Y4U32527Fto induce cytoskeletal rearrangements was not due
to an inability to localize with cellular membranes. Another
feature of transformation-competent variants of Src is that they
associate predominantly with the Triton X-100 insoluble
cytoskeletal fraction, whereas endogenous c-Src is primarily
Triton-soluble (Hamaguchi and Hanafusa, 1987). Thus, in
order to determine whether Src527F/c-Yes N-terminal chimeras
were able to associate with the Triton-insoluble fraction, mock-
transfected CEF or cells expressing c-Src, Src527F, Y4U32527F,
or Y4U527Fwere separated into Triton-soluble and Triton-
insoluble fractions. Both endogenous c-Src from mock-
transfected cells and overexpressed c-Src displayed a shift in
solubility from the Triton insoluble fraction to the Triton
soluble fraction after four minutes of incubation in CSK buffer,
whereas Src527Fand Y4U32527Fremained predominantly
associated with the Triton-insoluble fraction, even after 10
minutes of incubation (Fig. 4). Similar results were obtained
with Y4U527F(data not shown). These results indicate that the
inability of Y4U32527Fto effect actin redistribution does not
correlate with an inability of the protein to partition into the
Triton-insoluble cytoskeletal fraction.
The c-Yes N-terminal region does not prevent activation
of the MAP kinase pathway
The inability of Src527F/c-Yes N-terminal chimeras to induce
rearrangement of the actin cytoskeleton may be attributed to
failure to activate one or more downstream pathways that are
Journal of Cell Science 116 (12)
Fig. 3. Expression of Src527F/c-Yes N-terminal chimeras in CEF and their effects on cell morphology and the actin cytoskeleton. (A) The SH4-
Unique domains of c-Yes prevent Src527Ffrom affecting cell morphology. Confluent CEF that were uninfected, or expressing Y4U32527F,
Y32527FY4U527F, Y4527For YU527Fwere photographed at day 12 post-transfection in 100 mm tissue culture plates (Falcon). Cells were
photographed at 40× total magnification using a Plan 2 filter. (B) The SH4-Unique domains of c-Yes prevent Src527Ffrom affecting actin
filament integrity. CEF expressing Y4U32527F, Y32527FY4U527F, Y4527For YU527Fwere fixed at 50% confluence on coverslips in 3.7%
formaldehyde, permeablized in 0.4% Triton X-100, and stained with rhodamine-phalloidin (2 µg/ml). Cells were visualized via Zeiss LSM 510
confocal microscopy (63× objective). Bars, 10 µm.
2591 Specificity in signaling by c-Yes
normally upregulated in response to oncogenic Src527F. Thus,
we next investigated the activation status of signaling proteins
that function downstream of Src527F. The proteins chosen in
this study can broadly be classified into two distinct yet
overlapping categories: those that are involved in the mitogenic
response to Src527Factivation and those that are associated
with the effects of Src on the actin-based cytoskeleton.
The MAP kinase pathway is one of the most well-
characterized pathways activated in response to Src and is
involved in the induction of a mitogenic response (Gupta et al.,
1992). One of the first steps in the activation of the MAP kinase
pathway downstream of Src527Fis the formation of stable
complex between Shc and Grb2 (Rozakis-Adcock et al., 1992).
Thus in order to assess the effects of the c-Yes N-terminus on
the ability of Src to induce activation of the MAP kinase
pathway,we first assayed complex formation between Shc and
Grb2. CEF lysates were immunoprecipitated with a rabbit anti-
Shc antibody and the immunoprecipitates were resolved by
SDS-PAGE. Western blot analysis was performed using an
anti-Grb2 monoclonal antibody. Blots were stripped and re-
probed with the anti-Shc antibody to demonstrate that equal
amounts of Shc were immunoprecipitated (Fig. 5A, bottom
panel). Minimal complex formation was detected between Shc
and Grb2 in lysates from mock-transfected cells. Robust
complex formation was detected between Shc and Grb2 in
Src527F-transformed cells (Fig. 5A, top panel). Cells expressing
Y4U32527For Y4U527Fdisplayed increased Shc/Grb2 complex
formation over mock-transfected CEF (approximately 2.5-
fold); however, levels were not quite as high as in Src527F-
expressing cells. Similar results were obtained when
Fig. 4. Y4U32527Fis associated with the Triton
X-100 insoluble cytoskeletal fraction. Mock-
transfected CEF or cells expressing c-Src,
Src527For Y4U32527Fwere grown to
confluence in 100 mm dishes. Cells were
incubated in 1 ml of CSK buffer for 1, 4 or 10
minutes. The Triton-soluble fractions were
collected, and the Triton-insoluble material at
each time-point was solublized in RIPA buffer.
50 µg of cell lysates from each fraction were
separated by 8% SDS-PAGE, transferred to
PVDF membrane, blocked with 5% nonfat
milk/TBS-T, and probed with rabbit anti-Src.
Results are shown for mock-transfected CEF,
c-Src, Src527Fand Y4U32527F. C, CSK buffer;
R, RIPA buffer. Cytoskeletal-associated
proteins are defined in the R fraction.
Fig. 5. Activation of the MAP kinase pathway by Src527Fand Src527F/c-Yes chimeras. (A) 500 µg of RIPA lysates from mock-transfected CEF
or cells expressing Src527F, Y4U32527For Y4U527Fwere immunoprecipitated with an anti-Shc antibody and separated by 10% SDS PAGE.
Immunoprecipitated proteins were transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-Grb2 antibody
(top panel). The blots were stripped and re-probed with the anti-Shc antibody, immunoreactive against the 46 and 52 kDa isoforms of Shc
(bottom panel). (B) 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src527F, Y4U32527F, or Y4U527Fwere resolved by
8% SDS-PAGE, transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-phospho-Raf antibody (top panel).
Western blot analysis was also performed with an anti-c-Raf antibody to determine relative protein levels (bottom panel). (C) 50 µg of RIPA
lysates from mock-transfected CEF or cells expressing Src527F, Y4U32527For Y4U527Fwere processed for western blot analysis as described
above. Membranes were probed with an anti-phospho-Erk antibody (top panel) or an anti-p42/44 MAPK antibody to determine relative protein
levels (bottom panel). (A-C) Lane 1, CEF; lane 2, Src527F; lane 3, Y4U32527F; lane 4, Y4U527F.
immunoprecipitating with the anti-Grb2 antibody and probing
with the anti-Shc antibody (data not shown). These data
indicate that the c-Yes N-terminus alone is not sufficient to
abrogate Src-induced Grb2/Shc complex formation.
We next looked further downstream at c-Raf activation. c-
Raf signaling was assessed by western blot analysis using an
antibody against the phosphorylated form of c-Raf, which
recognizes a phosphorylation site (Y340/341) that appears to be
regulated in a Src-dependent manner (Diaz et al., 1997). CEF
lysates from mock-transfected cells, or cells expressing
Src527F, Y4U32527For Y4U527Fwere separated by SDS-PAGE,
and western blots were probed with the anti-phospho-Raf
antibody. As seen in Fig. 5B, no phosphorylation of Y340/341
on c-Raf was detected in mock-transfected cells; however,
lysates from cells expressing Src527F
immunoreactive with the anti-phospho-Raf antibody (Fig. 5B,
top panel). Y4U32527Flysates were weakly immunoreactive
with the anti-phospho-Raf antibody in comparison to Src527F;
however, Y4U527F-induced c-Raf phosphorylation more
closely approximated levels induced by Src527F(Fig. 5B, top
panel). Blots were additionally probed with an anti-c-Raf
antibody in order to determine relative levels of c-Raf present
in the cell lysates (Fig. 5B, bottom panel). The markedly lower
levels of phospho-c-Raf present in Y4U32527Flysates were
somewhat surprising and indicate that the SH3-SH2 domains,
or a combination of the SH4-Unique-SH3-SH2 domains of
cYes do not support activation of c-Raf.
We next assessed the effects of the c-Yes N-terminus on the
ability of Src to induce MAP kinase (MAPK) activation. In
order to evaluate MAPK activation, western blot analysis was
performed on lysates from mock-transfected CEF or cells
expressing Src527F, Y4U32527For Y4U527Fusing an anti-
phospho-Erk antibody. As seen in Fig. 5C, Src527F, Y4U32527F
and Y4U527Feach induced significant Erk activation above
levels detected in mock-transfected cells (Fig. 5C, top panel).
Western blots were additionally probed with an anti-MAPK
antibody, specific for Erk 1/2, to ensure that equal levels of
protein were present in the lysates (Fig. 5C, bottom panel).
These results indicate that the presence of the c-Yes N-terminus
is insufficient to abrogate Erk activation downstream of
Src527F. These data indicate that the SH3-SH2 domains of c-
Yes can enable formation of the Shc/Grb2 complex and
activation of Erk1/2. However, specificity in signaling by the
SH3-SH2 domains is evident in that Y4U32527Fwas unable to
direct increased phosphorylation of c-Raf at Tyr340/341. Thus
the inability of chimeric proteins with the c-Yes N-terminus to
induce rearrangement of the actin cytoskeleton and changes in
cellular morphology do not correlate with an inability to
activate the MAPK pathway. Further, these data indicate that
either Y4U32527Fcan activate Map kinase signaling in a c-Raf-
independent manner or that only low levels of c-Raf activation
are required to achieve subsequent MAPK activation.
Differential signaling to the PI3K/Akt and RhoA pathway
While many proteins are known to be important for Src-
mediated actin filament rearrangement, the precise pathway
that regulates this process has not been defined.
Phosphatidylinositol 3-kinase (PI3K), a lipid and protein
kinase named for its ability to phosphorylate the 3′ hydroxyl
group of inositol phospholipids, may mediate some of the
effects of Src on the actin cytoskeleton, as it functions
downstream of Src and is known to be involved in actin
filament rearrangements (Penuel and Martin, 1999). We first
assessed the activation of PI3 kinase indirectly, using the
activation state of Akt as an indicator of PI3 kinase activity.
Akt is a Ser/Thr kinase that is activated downstream of PI3K
and an important mediator of cellular survival signals
(Krasilnikov, 2000). In order to evaluate Akt activation,
western blot analysis was performed, using an antibody against
the phosphorylated and active form of Akt, on lysates from
cells that were mock-transfected or expressing Src527Fand
the chimeric constructs. As shown in Fig. 6A, no Akt
phosphorylation was detected in lysates of mock-transfected
CEF; however, significant anti-phospho-Akt immunoreactivity
was detected in cell lysates expressing Src527Fand all the
chimeric constructs, with the exception of Y4U32527F(Fig. 6A,
top panel). These results indicate that the presence of the c-Yes
SH4-Unique-SH3-SH2 domains were sufficient to ablate the
ability of Src527Fto induce activation of Akt, presumably
through PI3K. Interestingly, both Y4U527Fand Y32527F
induced activation of Akt phosphorylation, indicating that the
combination of the c-Yes SH4-Unique-SH3-SH2 domains may
coordinately play a role in directing activation of Akt. In order
to assess directly the ability of these proteins to induce
PI3K activity, cell lysates were immunoprecipitated with an
antibody against the 85 kDa subunit of PI3K, and the
immunoprecipitated proteins were subjected to a PI3K assay.
The radio-labeled kinase assay products were then spotted on
thin layer chromatography plates for separation. The results
were visualized using phosphorimager analysis. Little PI3K
activity was detected in lysates from mock-transfected cells,
while PI3K activity was readily detected in Src527F-transfected
cells (Fig. 6B, top panel). However, Y4U32527Fdid not induce
PI3K activity above background levels (Fig. 6B, top panel).
Western blot analysis of PI3K immunoprecipitates using an
anti-PI3K p85 antibody revealed that equivalent amounts of
PI3K were present (Fig. 6B, bottom panel). These results
confirm that Y4U32527Fis unable to induce activation of PI3K
and indicate that the c-Yes SH4-Unique-SH3-SH2 domains
may function interdependently to prevent PI3K activation,
while the same domains from c-Src enable PI3K activation.
Y4U527Fis able to stimulate phosphorylation of Akt, as well
as PI3K activity (data not shown), but is unable to induce
changes in actin filament integrity. These data indicate that
activation of the PI3K pathway is not linked with the failure of
Y4U527Fand Y4U32527Fto alter actin filament integrity. The
actin cytoskeleton is dynamically regulated and re-organized
in transformed cells and during cell motility. The family of
small GTPases of the Rho family, in particular Rac1, RhoA
and Cdc42, regulate the organization of the actin cytoskeleton
and provides the force for cell motility (Wittmann and
Waterman-Storer, 2001; Ridley, 2001). RhoA activation is
associated with the formation of stress filaments, and its
activity has been reported to be downregulated in Src-
transformed cells, which may be important for changes in actin
filament integrity (Fincham et al., 1999). CEF cells expressing
the chimeric constructs were assessed for endogenous RhoA-
GTP activity by affinity absorption from cell lysates with the
GST-Crib fusion protein generated from rhotekin, which has
higher affinity for RhoA-GTP over RhoA-GDP. Western blot
analysis indicates that GST-Crib was able to affinity absorb
Journal of Cell Science 116 (12)
2593Specificity in signaling by c-Yes
higher levels of RhoA from CEF cells, as well as Y4U32527F
and Y4U527Fcells, relative to the Src527F-transformed cells or
the Y32527F-transformed cells (Fig. 6C). These data indicate
that the presence of the SH4-Unique domains of c-Yes is
associated with a failure to downregulate RhoA activity, which
can be linked with a failure to induce changes in actin filament
The SH4-Unique domains of c-Yes do not support
increased motility or invasive potential
As the SH4-Unique domains of c-Yes do not permit significant
changes in actin filaments, it was predicted that chimeric
constructs that contain the c-Yes SH4-Unique domains may be
less motile or invasive, compared with Src527F. To test this,
CEF cells were infected with retrovirus encoding Src527F,
Y32527F, Y4U527Fand Y4U32527F, the cells achieved confluent
expression of these constructs within 12 days and were
subjected to a transwell migration assay. Fig. 7A qualitatively
demonstrates that upon loading of equal cell numbers, Src527F-
and Y32527F-expressing cells migrated across the transwells
more efficiently than normal CEF cells. Y4U527F- and
Y4U32527F-expressing cells did not exhibit increased
migration potential relative to CEF. To quantify these changes
in migration, the migrated cells were processed for analysis by
spectrophotometry to measure migration relative to each group
of CEF cells expressing the chimeras (Fig. 7B, see Materials
and Methods for details). The assay indicates that Y32527F- and
Src527F-expressing cells were more efficient in migrating
across the transwells than the CEF cells. In addition, the
Y4U527F- and Y4U32527F-expressing cells were no more
efficient in migration than CEF cells. These data indicate that,
consistent with an inability to affect changes in actin filament
integrity, Y4U527Fand Y4U32527Fwere unable to increase cell
An invasion assay was also performed using the Matrigel
24-well invasion chamber (Fig. 7C). Under conditions of
equal cell numbers, CEF cells expressing Src527For Y32527F
were three-times more invasive than untransfected CEF
cells, whereas Y4U527For Y4U32527Fdemonstrated invasive
potential equivalent to untransfected CEF cells. These data
indicate that the SH4-Unique domains do not support increased
motility or invasion, which correlates well with an inability to
support changes in cell morphology or dynamic changes in
actin filament integrity.
c-Yes and c-Src are two of the most highly homologous
members of the Src family of non-receptor tyrosine kinases,
yet despite their significant similarity, specificity in signaling
exists between the two proteins. The c-src and c-yes gene
knockout mice and cells derived from them have proved useful
in uncovering cellular processes and pathways in which these
proteins function distinctly from one another. As mentioned
above, several studies carried out in cells derived from the c-
src–/–mice have indicated deficiencies in cellular processes that
are dependent on dynamic regulation of the actin cytoskeleton.
c-Yes, despite normal expression in these cells, is unable to
compensate for the loss of c-Src in these processes.
The experiments described in this report were undertaken in
an effort to gain an understanding of the roles of the c-Yes
Fig. 6. Differential
signaling to the PI3K/Akt
and RhoA pathway. (A) Akt
phosphorylation. 50 µg of
RIPA lysates from mock-
transfected CEF or cells
expressing Src527For the
chimeric constructs were
resolved by 8% SDS-
PAGE, transferred to PVDF
membrane, blocked with
5% nonfat milk/TBS-T, and
probed with an anti-
phospho-Akt antibody (top
panel) or an anti-Akt
antibody to determine
relative protein levels
(bottom panel). (B) PI3K
assay. 400 µg of lysates
from mock-transfected CEF
or cells expressing Src527F
immunoprecipitated with an
anti-PI3K antibody and
subjected to PI3K assay as
described in Materials and Methods. Kinase assay products were resolved by thin layer chromatography and visualized by Phosphorimager
analysis (top panel). Anti-PI3K immunoprecipitates were also resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked in 5%
nonfat milk/TBS-T, and probed with an anti-PI3K p85 antibody to verify that there were equal amounts of PI3K in the lysates (bottom panel).
(C) RhoA-GTP assay. CEF cells expressing the chimeric constructs or Src527Fwere lysed and 500 µg of cell lysate processed for affinity
absorption with GST-Crib, expressing the Crib domain of rhotekin.
functional domains in the apparent inability of c-Yes to
compensate for c-Src signals, especially those known to
regulate actin cytoskeletal
experiments, the cytoskeletal and morphological changes that
occur concomitantly with Src527F-induced transformation of
primary and mortal CEF cells were used as a model system.
We discovered that replacement of the Src SH4, Unique, SH3
and SH2 domains with the corresponding c-Yes domains
resulted in a loss of actin repositioning from stress fibers
to punctate rosettes and actin-based membranous motility
structures such as lamellipodia and filopodia. Replacement of
the c-Src N-terminus with that of c-Yes eliminated the ability
rearrangements. In these
of Src527Fto induce the phenotypic changes that are typically
observed upon overexpression of the protein. The inability to
induce morphological and cytoskeletal changes was associated
primarily with the c-Yes N-terminus (SH4 and Unique
domains). Differences in signaling were also associated with a
combination of the SH4-Unique-SH3-SH2 domains of c-Yes.
These data indicate that the ability to induce morphological
changes may be largely due to the SH4-Unique domains and
that signaling specificity may be interdependent upon each of
these functional domains.
Having identified the c-Yes N-terminus as the region
responsible for the failure of these chimeric proteins to induce
Journal of Cell Science 116 (12)
CEF Y4U527F Y4U32527F Y32527F Src527F
Invaded cells (%)
Fig. 7. Y4U32527Fis unable to induce motility or invasion of CEF cells. (A) Equal numbers of mock transfected CEF cells, or CEF cells
expressing Src527F, Y32527F, Y4U527F, Y4U32527Fsubjected to (A) a transwell migration assay where cells migrated across the transwell and
were visualized by staining. (B) The transwell migration assay was repeated and the total number of cells that migrated was isolated and
quantified spectrophotometrically, to determine the relative numbers of cells that migrated. The experiment was done in triplicate and error bars
indicate standard deviation. (C) A Matrigel invasion assay was also performed under the same conditions with the same cells, where the number
of cells capable of invading through the Matrigel were counted. The experiment was carried out twice.
2595Specificity in signaling by c-Yes
morphological and cytoskeletal changes, the obvious question
is why does this occur? One possible mechanism that may
prevent Y4U527For Y4U32527Ffrom altering actin filament
integrity is altered subcellular localization. Although we found
no evidence for gross changes in cellular localization to
membranes or the cytoskeleton, it is possible that changes in
subcellular regions may not be detected by these methods, but
may be highly relevant to stimulating specific signaling
cascades. One obvious difference between the SH4 domains of
c-Yes and c-Src is palmitoylation of Cys3in c-Yes, which could
direct it to lipid rafts. Raft localization may not be the full
explanation, however, as the palmitoylation site is sufficient for
localization of Src family kinases to lipid rafts (Shenoy-Scaria
et al., 1994), yet Y4527Fwas predicted to be palmitoylated and
yet was able to induce actin filament rearrangement and
morphological changes. It is possible that the Unique domain
also contributes to membrane compartmentalization, as occurs
with Lck (Bijlmakers et al., 1997), or targets the kinase to
proteins with which it would not normally interact. Conversely,
the c-Yes Unique domain may prevent the interaction of
Src527Fwith substrates or binding partners that are necessary
for repositioning of cellular actin and induction of
morphological changes. However, the ability of the YU527F
chimera to induce actin filament rearrangement and
morphological changes suggests that this is also not the
complete story and that both the SH4 and Unique domains may
work cooperatively to contribute to the inability of Src527F/c-
Yes N-terminal chimeras to induce morphological changes and
actin filament rearrangement. The SH4 and Unique domains
may act synergistically, both through sequestration to lipid raft
fractions and through altered protein/protein and/or protein/
substrate interactions. The function of the c-Yes Unique
domain has not been previously explored.
If altered sub-cellular localization is responsible for the
inability of Y4U32527Fand Y4U527Fto induce actin filament
rearrangements and morphological changes consistent with
cell transformation, it must result in failure to activate the
appropriate signaling pathways necessary to effect these
transformation-associated changes. Unfortunately, determining
which signaling pathway or pathways that are normally
activated by transforming Src variants but not activated by
Y4U32527Fand Y4U527Fis a particularly vexing issue, as there
are many proteins that are involved in Src-mediated actin
filament rearrangement, and they do not necessarily function
in a linear pathway. In fact, it is likely that the effectors of Src
transformation act through multiple pathways, both parallel
and overlapping. One pathway that is activated downstream of
Src and is essential for cell transformation by oncogenic Src
is the MAPK pathway (Cowley et al., 1994). MAPKs are
activated downstream of Src through a pathway that can be
initiated through Shc/Grb2 interaction (Rozakis-Adcock et al.,
1992). Shc/Grb2 complex formation allows the Grb2/SOS
complex to activate Ras, which is followed by activation of
Raf, MEK1/2, and finally the MAP kinases themselves (Klein
and Schneider, 1997). In these studies, the ability of Src527Fto
induce Shc/Grb2 complex formation was not ablated by the
presence of the c-Yes N-terminus. Although Y4U32527Fdid not
induce the robust activation of the MAPK pathway that Src527F
did, differences mediated by Src527Fand Y4U527Fwere less
pronounced. The reduced levels of MAPK pathway activation
induced by Y4U32527Fmay be due in part to the presence of
the c-Yes SH3 and SH2 domains, which, as noted above, have
been previously demonstrated to differ in their ligand-binding,
and hence signaling, capacities (Sparks et al., 1996).
Interestingly, Y4U32527Ffailed to induce phosphorylation of
c-Raf on Tyr340/341, unlike Y4U527F. This phosphorylation of
c-Raf is associated with c-Src activity. Thus the reduced c-Raf
phosphorylation associated with Y4U32527Fmay be due to
either differences in SH3-SH2 mediated signaling, or to
interdependent signaling by the combination of the c-Yes SH4-
Unique-SH3-SH2 domains. The inability of c-Yes to
phosphorylate and possibly activate c-Raf may have functional
meaning. c-Yes will associate with adherens junctions (Tsukita
et al., 1991). Nusrat et al. demonstrated that the tight-junction-
associated protein, occludins, uniquely associate with c-Yes
and not c-Src (Nusrat et al., 2000). Occludins are
transmembrane proteins that regulate extracellular interactions
in tight junctions. Activation of Raf-1 is associated with
downregulation of occludin expression (Li and Mrsny, 2000).
These data are consistent with a role for activated c-Yes as a
binding signaling partner for occludins, while activation of c-
Src might be predicted to direct phosphorylation and activation
of Raf-1 and downregulation of occludins. Thus, it is possible
that activated c-Yes may play a role in participating in the
maintenance of tight junction interactions, whereas activation
of c-Src is known to cause their dissociation. Thus, it may not
be functionally advantageous for c-Yes to activate c-Raf, if c-
Yes plays a role in regulating occludin function.
While activation of the MAP kinase pathway is important
for the mitogenic response to Src, the role that MAP kinase
activation plays in rearrangement of the actin cytoskeleton
downstream of Src is unclear. Fincham and colleagues
demonstrated mitogeneis-independent inactivation of Rho, a
key modulator of the actin cytoskeleton, downstream of Src,
indicating that separate pathways may be involved in the
induction of cytoskeletal and mitogenic responses to Src
activation (Fincham et al., 1999). Mek has also been shown to
play a role in regulating actin filament dynamics in some cells
(Pawlak and Helfman, 2002); however, in our CEF system,
Mek inhibitors were unable to block changes in actin filament
integrity (data not shown). Although Y4U32527Fwas able
to induce activation of Erk1/2, it was unable to induce
phosphorylation of c-Raf. Interestingly, it has been
demonstrated that c-Raf-knockout cells (raf–/–) or raf–/–cells
that are engineered to express c-RafY340/341F(c-raf-FF) are able
to grow, and Erk activation in these cells is normal (Huser et
al., 2001). These data indicate that Erk1/2 activation may
proceed in a Raf-independent manner.
PI3K is another important downstream mediator of Src,
which has been implicated in directing actin cytoskeleton
rearrangements. It has been reported previously that PI3K may
function in parallel with the MAP kinase pathway (Penuel and
Martin, 1999). The 85 kDa subunit of PI3K is both a substrate
and SH3 domain binding partner of Src, and the ability of Src
to associate with PI3K correlates with its ability to induce cell
transformation (Hamaguchi et al., 1993). Fincham et al.
recently demonstrated that binding of the v-Src SH3 domain
to PI3K is important in targeting Src to focal adhesion
structures (Fincham et al., 2000). The exact mechanism by
which PI3K exerts its influence on the actin cytoskeleton
remains unclear; however, it may be involved in the regulation
of Rho family members, such as Rac-1, Cdc42 and RhoA (Reif
et al., 1996). One of the major effector proteins downstream of
PI3K is Akt (Krasilnikov, 2000). Akt activation contributes to
cell survival and may also play a role in cell transformation
(Krasilnikov, 2000). Our data indicate that Y4U32527Fis
unable to efficiently induce activation of Akt, as demonstrated
by western blot analysis with the anti-phospho-Akt antibody.
phosphorylation of Akt, indicating that interdependent
signaling between each of these functional domains may divert
c-Yes from being able to activate Akt. This conclusion
correlates with the inability of Y4U32527Fto induce activation
of PI3K. PI3K has been implicated in modulating changes in
actin filament integrity, cell motility and invasion and,
consistent with these observations, CEF cells expressing
chimeric constructs of Src527Fthat contained the c-Yes SH4-
Unique-SH3-SH2 domains were less motile and invasive
compared with Src527F. Interestingly, Y4U527F-expressing cells
were also less motile and invasive, which correlates well with
the morphology of cells expressing this construct, although this
chimera was able to direct phosphorylation of Akt, indicating
that PI3K activation may not be sufficient to induce changes
in cell morphology in CEF cells. The reason for this may be
attributed to an inability of Y4U527Fand Y5U32527Fto
downregulate RhoA. Our data indicate that Src527F/c-Yes N-
terminal chimeras fail to inactivate RhoA, which may explain
why the Y4U32527Fand Y4U527Fchimeras failed to affect
changes in actin filament integrity.
The investigation of one question invariably leads to the
uncovering of another: in this case, why is Y4U32527Funable
to induce activation of PI3K? Traditionally, Src has been
hypothesized to induce PI3K activation through direct binding
and/or tyrosine phosphorylation (Pleiman et al., 1994). Binding
of Src to PI3K occurs through an interaction between the Src
SH3 domain and the 85 kDa subunit of PI3K (Pleiman et al.,
1994). Mutants of v-Src that fail to bind PI3K also fail to
induce cell transformation (Catling et al., 1994). While there
is no previous evidence of direct interaction between c-Yes and
PI3K, there is little reason to believe that the c-Yes SH3 domain
would be unable to bind to p85, as the SH3 domain residues
that have been reported to be essential for p85 binding in other
Src family members are conserved in c-Yes (Mak et al., 1996),
and v-Yes is able to induce elevated PI3K activity (Fukui et al.,
1991). Indeed, Y32527Fappears to co-immunoprecipitate with
PI3K as efficiently as Src527F(data not shown). It is possible
that sequestration to the lipid raft fraction may be in part
responsible for the inability of Src527F/c-Yes N-terminal
chimeras to activate the PI3K pathway and thus induce
morphological and cytoskeletal changes. This explanation
would be in agreement with the ability of v-Yes to induce PI3K
activation and cell transformation, as v-Yes is an N-terminal
fusion of the Yes protein with the retroviral Gag protein and is
not palmitoylated, relying instead on the Gag protein for
membrane targeting (Ghysdael et al., 1981). It should also be
noted that the inability of Y4U32527Fto induce PI3K activation
in CEF may be unrelated to direct interaction between the
chimeric proteins and PI3K. It has been demonstrated that in
Shp-2 knockout cells, the PI3K/Akt pathway was inefficiently
activated by v-Src, and this correlated with reduced complex
formation between c-Cbl and p85 (Hakak et al., 2000). The
reason behind the reduced activation of the PI3K/Akt pathway
by Y4U32527Fis currently under investigation.
were able to induce
PI3K/Akt activation may not be sufficient to induce
disruption of the actin stress fiber network, as Y4U527Fwas
able to activate Akt phosphorylation (a signature for PI3K
activation) but did not exert a significant effect on actin
filament organization. We speculate that the SH4-Unique-SH3-
SH2 domains may work cooperatively to position c-Yes to
activate specific signals that are not associated with actin
filament changes. We speculate that the SH4-Unique domains
may function as one functional domain and position c-Yes
in specific subcellular regions that prevent it from affecting
significant changes in actin filament organization and/or
activation of specific signals associated with actin filament
rearrangements. Together, the SH4-Unique-SH3-SH2 domains
of c-Yes may target this tyrosine kinase for distinct functions,
which may explain why c-Yes is unable to compenstate for
cSrc in affecting dynamic changes in actin filament
organization in src–/–cells.
The authors thank Jenny Zheng for technical assistance. This work
was supported by a grant from the NCI (CA60731) and a grant from
NCRR Cobre program (RR16440) to D.C.F. J.M.S. was supported by
a fellowship from the Arlen G. and Louise Stone Swiger Foundation.
B.H.J. was supported by a grant from the NIH/NCRR Cobre program
(RR16440). A.G. was supported by an NIH training grant
Bijlmakers, M. J., Isobe-Nakamura, M., Ruddock, L. J., Marsh, M. (1997).
Intrinsic signals in the unique domain target p56(lck) to the plasma
membrane independently of CD4. J. Cell Biol. 137, 1029-1040.
Boyce, B. F., Yoneda, T., Lowe, C., Soriano, P. and Mundy, G. R. (1992).
Requirement of pp60c-src expression for osteoclasts to form ruffled borders
and resorb bone in mice. J. Clin. Invest. 90, 1622-1627.
Catling, A. D., Fincham, V. J., Frame, M. C., Haefner, B. and Wyke, J.
A. (1994). Mutations in v-Src SH3 and catalytic domains that jointly
confer temperature-sensitive transformation with minimal temperature-
dependent changes in cellular tyrosine phosphorylation. J. Virol. 68, 4392-
Cowley, S., Paterson, H., Kemp, P. and Marshall, C. J. (1994). Activation
of MAP kinase kinase is necessary and sufficient for PC12 differentiation
and for transformation of NIH 3T3 cells. Cell 77, 841-852.
Diaz, B., Barnard, D., Filson, A., MacDonald, S., King, A. and Marshall,
M. (1997). Phosphorylation of Raf-1 serine 338-serine339 is an essential
regulatory event for Ras-dependent activation and biological signaling. Mol.
Cell. Biol. 17, 4509-4516.
Edlund, S., Landstrom, M., Heldin, C. H. and Aspenstrom, P. (2002).
Transforming growth factor-beta-induced mobilization of actin cytoskeleton
requires signaling by small GTPases Cdc42 and RhoA. Mol. Biol. Cell 13,
Felice, G. R., Eason, P., Nermut, M. V. and Kellie, S. (1990). pp60v-src
association with the cytoskeleton induces actin reorganization without
affecting polymerization status. Eur. J. Cell Biol. 52, 47-59.
Fincham, V. J., Chudleigh, A. and Frame, M. C. (1999). Regulation of p190
Rho-GAP by v-Src is linked to cytoskeletal disruption during
transformation. J. Cell Sci. 112, 947-956.
Fincham, V. J., Brunton, V. G. and Frame, M. C. (2000). The SH3 domain
directs acto-myosin-dependent targeting of v-Src to focal adhesions via
phosphatidylinositol 3-kinase. Mol. Cell Biol. 20, 6518-6536.
Flynn, D. C., Leu, T. H., Reynolds, A. B. and Parsons, J. T.
(1993). Identification and sequence analysis of cDNAs encoding a 110-
kilodalton actin filament-associated pp60src substrate. Mol. Cell Biol. 13,
Fuhrer, D. K. and Yang, Y. C. (1996a). Activation of Src-family protein
tyrosine kinases and phosphatidylinositol 3-kinase in 3T3-L1 mouse
preadipocytes by interleukin-11. Exp. Hematol. 24, 195-203.
Fuhrer, D. K. and Yang, Y. C. (1996b). Complex formation of JAK2 with
PP2A, P13K, and Yes in response to the hematopoietic cytokine interleukin-
11. Biochem. Biophys. Res. Commun. 224, 289-296.
Journal of Cell Science 116 (12)
2597Specificity in signaling by c-Yes
Fukui, Y., Saltiel, A. R. and Hanafusa, H. (1991). Phosphatidylinositol-3
kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo
fibroblasts. Oncogene 6, 407-411.
Ghysdael, J., Neil, J. C. and Vogt, P. K. (1981). A third class of avian sarcoma
viruses, defined by related transformation-specific proteins of Yamaguchi 73
and Esh sarcoma viruses. Proc. Natl. Acad. Sci. USA 78, 2611-2615.
Gupta, S. K., Gallego, C., Johnson, G. L. and Heasley, L. E. (1992). MAP
kinase is constitutively activated in gip2 and src transformed rat 1a
fibroblasts. J. Biol. Chem. 267, 7987-7990.
Guy, C. T., Muthuswamy, S. K., Cardiff, R. D., Soriano, P. and Muller, W.
J. (1994). Activation of the c-Src tyrosine kinase is required for the
induction of mammary tumors in transgenic mice. Genes Dev. 8, 23-32.
Hakak, Y., Hsu, Y. S. and Martin, G. S. (2000). Shp-2 mediates v-Src-
induced morphological changes and activation of the anti-apoptotic protein
kinase Akt. Oncogene 19, 3164-3171.
Hall, C. L., Lange, L. A., Prober, D. A., Zhang, S. and Turley, E. A. (1996).
pp60(c-src) is required for cell locomotion regulated by the
hyaluronanreceptor RHAMM. Oncogene 13, 2213-2224.
Hamaguchi, M. and Hanafusa, H. (1987). Association of p60src with Triton
X-100-resistant cellular structure
transformation. Proc. Natl. Acad. Sci. USA 84, 2312-2316.
Hamaguchi, M., Xiao, H., Uehara, Y., Ohnishi, Y. and Nagai, Y. (1993).
Herbimycin A inhibits the association of p60v-src with the cytoskeletal
structure and with phosphatidylinositol 3′ kinase. Oncogene 8, 559-564.
Hauck, C. R., Hsia, D. A., Puente, X. S., Cheresh, D. A. and Schlaepfer,
D. D. (2002). FRNK blocks v-Src-stimulated invasion and experimental
metastases without effects on cell motility or growth. EMBO J. 21, 6289-
Hoey, J. G., Summy, J. and Flynn, D. C. (2000). Chimeric constructs
containing the SH4/Unique domains of cYes can restrict the ability of
Src(527F) to upregulate heme oxygenase-1 expression efficiently. Cell
Signal. 12, 691-701.
Huser, M., Luckett, J., Chiloeches, A., Mercer, K., Iwobi, M., Giblett, S.,
Sun, X. M., Brown, J., Marias, R. and Pritchard, C. (2001). MEK kinase
activity is not necessary for Raf-1 function. EMBO J. 20, 1940-1951.
Ignelzi, M. A., Jr, Miller, D. R., Soriano, P. and Maness, P. F. (1994).
Impaired neurite outgrowth of src-minus cerebellar neurons on the cell
adhesion molecule L1. Neuron 12, 873-884.
Jiang, B. H., Zheng, J. Z. and Vogt, P. K. (1998). An essential role of
phosphatidylinositol 3-kinase in myogenic differentiation. Proc. Natl. Acad.
Sci. USA 95, 14179-14183.
Kabouridis, P. S., Magee, A. I. and Ley, S. C. (1997). S-acylation of LCK
protein tyrosine kinase is essential for its signalling function in T
lymphocytes. EMBO J. 16, 4983-4998.
Kaplan, K. B., Swedlow, J. R., Morgan, D. O. and Varmus, H. E. (1995).
c-Src enhances the spreading of src–/– fibroblasts on fibronectin by a kinase-
independent mechanism. Genes Dev. 9, 1505-1517.
Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J. and Parise, L. V.
(1997). Cdc42 and Rac1 induce integrin-mediated cell motility and
invasiveness through PI(3)K. Nature 390, 632-636.
Kitamura, N., Kitamura, A., Toyoshima, K., Hirayama, Y. and Yoshida,
M. (1982). Avian sarcoma virus Y73 genome sequence and structural
similarity of its transforming gene product to that of Rous sarcoma virus.
Nature 297, 205-208.
Klein, N. P. and Schneider, R. J. (1997). Activation of Src family kinases by
hepatitis B virus HBx protein and coupled signaling to Ras. Mol. Cell Biol.
Krasilnikov, M. A. (2000). Phosphatidylinositol-3 kinase dependent
pathways: the role in control of cell growth, survival, and malignant
transformation. Biochemistry Mosc. 65, 59-67.
Kypta, R. M., Goldberg, Y., Ulug, E. T. and Courtneidge, S. A. (1990).
Association between the PDGF receptor and members of the src family of
tyrosine kinases. Cell 62, 481-492.
Landgren, E., Blume-Jensen, P., Courtneidge, S. A. and Claesson-Welsh,
L. (1995). Fibroblast growth factor receptor-1 regulation of Src family
kinases. Oncogene 10, 2027-2035.
Li, D. and Mrsny, R. J. (2000). Oncogenic Raf-1 disrupts epithelial tight
junctions via downregulation of occludin. J. Cell Biol. 148, 791-800.
Luton, F., Verges, M., Vaerman, J. P., Sudol, M. and Mostov, K. E. (1999).
The SRC family protein tyrosine kinase p62yes controls polymeric IgA
transcytosis in vivo. Mol. Cell 4, 627-632.
Mak, P., He, Z. and Kurosaki, T. (1996). Identification of amino acid residues
required for a specific interaction between Src-tyrosine kinase and proline-
rich region of phosphatidylinositol-3′ kinase. FEBS Lett. 397, 183-185.
correlates with morphological
Mukhopadhyay, D., Tsiokas, L., Zhou, X. M., Foster, D., Brugge, J. S. and
Sukhatme, V. P. (1995). Hypoxic induction of human vascular endothelial
growth factor expression through c-Src activation. Nature 375, 577-581.
Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M.,
Quan, C. and Mrsny, R. J. (2000). The coiled-coil domain of occludin can
act to organize structural and functional elements of the epithelial tight
junction. J. Biol. Chem. 275, 29816-29822.
Pawlak, G. and Helfman, D. M. (2002). MEK mediates v-Src-induced
disruption of the actin cytoskeleton via inactivation of the Rho-ROCK-LIM
kinase pathway. J. Biol. Chem. 277, 26927-26933.
Penuel, E. and Martin, G. S. (1999). Transformation by v-Src: Ras-MAPK
and PI3K-mTOR mediate parallel pathways. Mol. Biol. Cell 10, 1693-
Pleiman, C. M., Hertz, W. M. and Cambier, J. C. (1994). Activation of
phosphatidylinositol-3′ kinase by Src-family kinase SH3 binding to the p85
subunit. Science 263, 1609-1612.
Qian, Y., Baisden, J. M., Westin, E. H., Guappone, A. C., Koay, T. C. and
Flynn, D. C. (1998). Src can regulate carboxy terminal interactions with
AFAP-110, which influence self-association, cell localization and actin
filament integrity. Oncogene 16, 2185-2195.
Reif, K., Nobes, C. D., Thomas, G., Hall, A. and Cantrell, D. A. (1996).
Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-
dependent effector pathways. Curr. Biol. 6, 1445-1455.
Resh, M. D. (1994). Myristylation and palmitylation of Src family members:
the fats of the matter. Cell 76, 411-413.
Reynolds, A. B., Roesel, D. J., Kanner, S. B. and Parsons, J. T. (1989).
Transformation-specific tyrosine phosphorylation of a novel cellular protein
in chicken cells expressing oncogenic variants of the avian cellular src gene.
Mol. Cell. Biol. 9, 629-638.
Rickles, R. J., Botfield, M. C., Zhou, X. M., Henry, P. A., Brugge, J. S. and
Zoller, M. J. (1995). Phage display selection of ligand residues important
for Src homology 3 domain binding specificity. Proc. Natl. Acad. Sci. USA
Ridley, A. J. (2001). Rho proteins, PI 3-kinases, and monocyte/macrophage
motility. FEBS Lett. 498, 168-171.
Robbins, S. M., Quintrell, N. A. and Bishop, J. M. (1995). Myristoylation
and differential palmitoylation of the HCK protein- tyrosine kinases govern
their attachment to membranes and association with caveolae. Mol. Cell
Biol. 15, 3507-3515.
Roche, S., Fumagalli, S. and Courtneidge, S. A. (1995). Requirement for
Src family protein tyrosine kinases in G2 for fibroblast cell division. Science
Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li,
W., Batzer, A., Thomas, S., Brugge, J. and Pelicci, P. G. (1992).
Association of the Shc and Grb2/Sem5 SH2-containing proteins is
implicated in activation of the Ras pathway by tyrosine kinases. Nature 360,
Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M. P. (1993). Signal
transducing molecules and glycosyl-phosphatidylinositol-linked proteins
form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol. 122,
Schieffer, B., Paxton, W. G., Chai, Q., Marrero, M. B. and Bernstein, K.
E. (1996). Angiotensin II controls p21ras activity via pp60c-src. J. Biol.
Chem. 271, 10329-10333.
Shenoy-Scaria, A. M., Dietzen, D. J., Kwong, J., Link, D. C. and Lublin,
D. M. (1994). Cysteine3 of Src family protein tyrosine kinase determines
palmitoylation and localization in caveolae. J. Cell Biol. 126, 353-363.
Soriano, P., Montgomery, C., Geske, R. and Bradley, A. (1991). Targeted
disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell
Sparks, A. B., Rider, J. E., Hoffman, N. G., Fowlkes, D. M., Quillam, L.
A. and Kay, B. K. (1996). Distinct ligand preferences of Src homology 3
domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.
Proc. Natl. Acad. Sci. USA 93, 1540-1544.
Stein, P. L., Vogel, H. and Soriano, P. (1994). Combined deficiencies of Src,
Fyn, and Yes tyrosine kinases in mutant mice. Genes Dev. 8, 1999-2007.
Sudol, M. and Hanafusa, H. (1986). Cellular proteins homologous to the viral
yes gene product. Mol. Cell Biol. 6, 2839-2846.
Sugawara, K., Sugawara, I., Sukegawa, J., Akatsuka, T., Yamamoto, T.,
Morita, M., Mori, S. and Toyoshima, K. (1991). Distribution of
c-yes-1 gene product in various cells and tissues. Br. J. Cancer 63, 508-
Summy, J. M., Guappone, A. C., Sudol, M. and Flynn, D. C. (2000). The
SH3 and SH2 domains are capable of directing specificity in protein
2598 Download full-text
interactions between the non-receptor tyrosine kinases cSrc and cYes.
Oncogene 19, 155-160.
Summy, J. M., Sudol, M., Monteiro, A. N., Eck, M. J., Gatesman, A. and
Flynn, D. C. (2003). Specificity in signaling by c-Yes. Front. Biosci. 8,
Thomas, S. M. and Brugge, J. S. (1997). Cellular functions regulated by Src
family kinases. Annu. Rev. Cell Dev. Biol. 13, 513-609.
Tsukita, S., Oishi, K., Akiyama, T., Yamanashi, Y., Yamamoto, T. and
Tsukita, S. (1991). Specific proto-oncogenic tyrosine kinases of src family
are enriched in cell-to-cell adherens junctions where the level of tyrosine
phosphorylation is elevated. J. Cell Biol. 113, 867-879.
van der Valk, J., Verlaan, I., de Laat, S. W. and Moolenaar, W. H. (1987).
Expression of pp60v-src alters the ionic permeability of the plasma
membrane in rat cells. J. Biol. Chem. 262, 2431-2434.
Wittmann, T. and Waterman-Storer, C. M. (2001). Cell motility: can Rho
GTPases and microtubules point the way? J. Cell Sci. 114, 3795-3803.
Zhao, Y. H. and Krueger, J. G. and Sudol, M. (1990). Expression of cellular-
yes protein in mammalian tissues. Oncogene 5, 1629-1635.
Journal of Cell Science 116 (12)