1 Department 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
2 National Institute for Occupational Safety and Health, Pathology and
Physiology Research Branch, Health Effects Laboratory Division, Morgantown, WV
26505, USA
Author for correspondence (e-mail:
dflynn{at}hsc.wvu.edu)
Accepted 7 March 2003
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Summary |
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Key words: Yes, Src, Actin filaments, SH4 domain, Unique domain
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Introduction |
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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 the exception of
reduced transcytosis of the polyimmunoglobulin (pIg) receptor, the
c-yes-/- mice 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 to
detergent-resistant 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 Fc
receptor and T-cell receptor signaling
(Kabouridis et al., 1997
).
Hoey et al. recently demonstrated that replacement of the Src527F
N-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
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 Src527F with 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).
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Materials and Methods |
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Cells
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 40x total magnification (Plan 2
filter) and photographed with a Nikon N 2000 camera using Kodak TMax 400 black
and white film.
Antibodies
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-Y416 was 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.
Immunofluorescence
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 (63x 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
).
Immunoprecipitation
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.
Cell fractionation
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 [
-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 1x 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, 150 mM NaCl, 10 mM
MgCl2, 10 µg/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
4x105 cells/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% CO2
at 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 10x 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 4x105 cells/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% CO2 for 14
hours. Migrated cells were fixed, stained with 0.1% crystal violet. Washing
five times with 1x 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 assay
Invasion assays were performed according to manufacturer's protocol (BD
Biosciences). The cells were serum-starved overnight. Cells (0.5 ml of
1.0x105 cells/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.
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Results |
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The Y4U32527F and Y4U527F chimeras do not
induce morphological changes or rearrangement of cytoskeletal actin
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
Y4U32527F and Y4U527F chimeras 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
Y3527F and 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
YU527F chimeras 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 Y4527F and YU527F
displayed more evidence for disorganization in monolayer than CEFs. Expression
levels of the Y4527F and YU527F chimeras 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
Y4U32527F to 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 Src527F occurs 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 Src527F to 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 Src527F were 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, Y4527F and YU527F. However, Y4U527F and Y4U32527F appeared 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 cell morphology.
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
Y4U32527F and Y4U527F to 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
Src527F and all Src527F/c-Yes chimeras were associated
predominantly with the membrane fraction (data not shown). Thus the inability
of Y4U32527F to 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 Y4U527F were 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 Src527F and
Y4U32527F remained 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 Y4U32527F to 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 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 Src527F activation 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 Src527F is 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 reprobed 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 Y4U32527F or Y4U527F displayed 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 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,
Y4U32527F or Y4U527F were 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 were strongly immunoreactive
with the anti-phospho-Raf antibody (Fig.
5B, top panel). Y4U32527F lysates 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 Y4U32527F lysates
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, Y4U32527F or Y4U527F using an anti-phospho-Erk antibody. As seen in Fig. 5C, Src527F, Y4U32527F and Y4U527F each 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 Y4U32527F was 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 Y4U32527F can 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 Src527F and 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 Src527F and 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 Src527F to induce
activation of Akt, presumably through PI3K. Interestingly, both
Y4U527F and 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,
Y4U32527F did 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 Y4U32527F is 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.
|
Y4U527F is 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 Y4U527F and Y4U32527F to
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 higher levels of RhoA from CEF cells, as well as
Y4U32527F and Y4U527F cells, 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 integrity.
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, Y4U527F and
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, Y4U527F and
Y4U32527F were unable to increase cell migration efficiency.
|
An invasion assay was also performed using the Matrigel 24-well invasion chamber (Fig. 7C). Under conditions of equal cell numbers, CEF cells expressing Src527F or Y32527F were three-times more invasive than untransfected CEF cells, whereas Y4U527F or Y4U32527F demonstrated 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.
![]() |
Discussion |
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---|
The experiments described in this report were undertaken in an effort to gain an understanding of the roles of the c-Yes functional domains in the apparent inability of c-Yes to compensate for c-Src signals, especially those known to regulate actin cytoskeletal rearrangements. In these 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 of Src527F to 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 morphological and cytoskeletal
changes, the obvious question is why does this occur? One possible mechanism
that may prevent Y4U527F or Y4U32527F from 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 Cys3 in 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 Y4527F was 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 Src527F with 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
Y4U32527F and Y4U527F to 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 Y4U32527F and
Y4U527F is 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 Src527F to
induce Shc/Grb2 complex formation was not ablated by the presence of the c-Yes
N-terminus. Although Y4U32527F did not induce the robust activation
of the MAPK pathway that Src527F did, differences mediated by
Src527F and Y4U527F were less pronounced. The reduced
levels of MAPK pathway activation induced by Y4U32527F may 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, Y4U32527F failed 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 Y4U32527F may 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 Y4U32527F was 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 Y4U32527F is unable to
efficiently induce activation of Akt, as demonstrated by western blot analysis
with the anti-phospho-Akt antibody. However, Y4U527F and
Y32527F were able to induce 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 Y4U32527F to 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 Src527F that 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 Y4U527F and Y5U32527F to
downregulate RhoA. Our data indicate that Src527F/c-Yes N-terminal
chimeras fail to inactivate RhoA, which may explain why the
Y4U32527F and Y4U527F chimeras 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 Y4U32527F unable 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,
Y32527F appears 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
Y4U32527F to 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 Y4U32527F is currently under investigation.
PI3K/Akt activation may not be sufficient to induce disruption of the actin stress fiber network, as Y4U527F was 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.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: Department of Cancer Biology, University of Texas MD
Anderson Cancer Center, PO Box 79, 1515 Holcombe Drive, Houston, TX 77030,
USA
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