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INTRODUCTION |
The v-Crk oncogene has been isolated as the transforming gene of
the avian sarcoma retrovirus CT10 (1). The v-Crk gene product is an
adaptor protein containing a viral gag portion fused with a SH2 and SH3
domain. The corresponding cellular proto-oncogene was shown to exist in
two splice variants, Crk I (28 kDa) and Crk II (42 kDa), of which Crk
II encodes an additional N-terminal SH3 domain (2). In addition,
another closely related gene has been identified, CRKL, which is
similar to Crk II (3). Both c-Crk II and CRKL are ubiquitously
expressed (4), suggesting an important cellular function.
How does v-Crk induce oncogenicity? As an adaptor protein the v-Crk
protein does not possess catalytic activity. Nevertheless the
v-Crk-transformed cells contain enhanced levels of proteins phosphorylated on tyrosine residues, indicating the activation of a
tyrosine kinase or the inactivation of a phosphotyrosine phosphatase
(for review, see Ref. 5). Since tyrosine kinase activities often have
been implicated in signal transduction and oncogenesis, this is likely
to be involved. In v-Crk-transformed cells the most prominently
phosphorylated proteins are Paxillin and p130cas (6-8),
both adapter proteins described as have signaling functions in focal
adhesions (9). Focal adhesions are the sites where cells are bound to
the extracellular matrix through integrin receptors that are connected
to the intracellular actin cytoskeleton. In addition to their
structural function, focal adhesions also serve to transduce
extracellular signals via multiprotein complexes. Focal adhesion
signaling has important functions in the regulation of cell morphology,
cell cycle progression, and cell survival (10, 11). v-Crk not only
induced the phosphorylation of Paxillin and p130cas but also
bound stably to their phosphorylated tyrosine residues through its SH2
domain (6-8). In accordance, v-Crk was shown to localize in focal
adhesions (12). Through its SH3 domain, v-Crk can bind to several
downstream signaling molecules like DOCK180, Sos, and C3G and the
kinases Abl, KHS, and HPK1 (for review, see Ref. 5). These data
suggest that v-Crk enhances focal complex signaling, but the mechanism
resulting in oncogenicity is not known.
v-Crk not only induces oncogenicity in fibroblast model systems;
various data also suggest the involvement of Crk in human tumors.
Particularly, CRKL is a major substrate protein of leukemogenic BCR-ABL
proteins (13). These are the gene products of the chimeric BCR/ABL oncogenes that are generated by the well
known Philadelphia chromosome translocation and which cause chronic
myelogenous leukemia. CRKL- and p130cas-mediated signaling is
thought to play an important role in this disease (for review, see Ref.
14). Furthermore, recently BCAR1, a protein showing 91% amino acid
identity with mouse and rat p130cas, has been identified by a
random retroviral mutagenesis approach as being involved in breast
cancer (15). Particularly, high levels of BCAR1 were associated with
poor survival of patients (16). Since p130cas contains many
binding sites for Crk (8), Crk may be the main downstream effector. In
both cases the mechanism of tumorigenesis is not fully clear. Various
aspects of focal adhesion signaling are shown to be stimulated by Crk,
which might contribute to the oncogenic properties. Cell morphology and
regulation of the actin cytoskeleton are affected by Crk-mediated
activation of Rho-like GTPases, particularly Rac (17, 18). In neuronal
cells, v-Crk was shown to delay apoptosis (19), whereas in experiments
using Xenopus lysates, Crk appeared to have an apoptosis
stimulatory role (20). Furthermore, Crk may play a role in cell cycle
progression (21). In addition, however, various data indicate a role
for Crk in growth factor-stimulation (e.g. Refs. 22 and
23).
In this study we set out to investigate which mechanism could be
involved in the v-Crk-induced oncogenesis by analyzing stably transformed NIH 3T3 fibroblast cell lines expressing v-Crk or v-Crk
mutants that were previously described (12). We and others showed that
v-Crk induces in fibroblasts the capacity to grow in soft agar. This
property is highly correlated with tumorigenicity and is considered to
reflect anchorage independence of growth. In soft agar growth assays,
v-Crk is less oncogenic than e.g. oncogenic Ras variants or
v-Src. Similarly v-Crk induces an intermediary phenotype with respect
to induction of a highly refractile spindle-shaped morphology in
fibroblast cells (12). In this study we compared the oncogenic
properties of v-Crk-transformed NIH 3T3 cells with those of
RasL61-transformed cells and found that v-Crk does not stimulate
serum-independent growth. In contrast, our data indicate that the major
v-Crk-induced effect is the protection of the fibroblast cells against
cell death and particularly against apoptosis, which is also
observed in the RasL61-transformed cells. Analysis of the pathways
involved in the protection by v-Crk indicates the activation of the
PKB1/Akt survival pathway as
one of the mechanisms.
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EXPERIMENTAL PROCEDURES |
Materials--
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) and 4',6'-diamidine-2'-phenylindole dihydochloride
(DAPI) were from Sigma. Polyvinylidene difluoride membranes were from Roche Molecular Biochemicals, and milk proteins (Protifar) were from
Nutricia (Zoetermeer, The Netherlands). ECL (Renaissance) and
[
-32P]ATP were from PerkinElmer Life Sciences
and Amersham Pharmacia Biotech, respectively. p81 phosphocellulose
filters were obtained from Whatman. LY294002 was from Alexis, PD98059
from Biomol, and SB203580 was from Calbiochem. All other reagents were
obtained from Sigma.
Cell Culture--
The NIH 3T3 cells stably transfected with
v-Crk and with v-CrkSH2
and SH3
have previously been
described (12). NIH 3T3 cells stably transfected with RasL61 were
kindly provided by Dr. B. Burgering. COS-1-, NIH 3T3 (ATCC)-, and NIH
3T3-derived cell lines were grown in Dulbecco's modified Eagle's
medium supplemented with 7.5% fetal calf serum (Life Technologies,
Inc.) at 37 °C in a humidified atmosphere with 5%
CO2.
Cell Growth and Viability Assays, Nuclear Condensation, and UV
Treatment--
Relative cell number and viability was determined using
MTT as a substrate (24). In short, cells were cultured in 12-well plates and were incubated with the MTT solution for 60 min at 37 °C.
Cells were lysed, and the formazan salt was solubilized in 0.04 N HCl in isopropanol under continuous shaking. The
absorbance was measured at 570 nm with a microplate reader (Bio-Rad).
The background value without cells (A570 ~ 0.010) was subtracted. For the cell lines used, the MTT signal in the
presented range was linearly correlated with the cell number, as
counted by phase contrast microscopy. The MTT signal per cell was
roughly similar for the cell lines used. For serum starvation survival
experiments cells were incubated in serum-free medium for the indicated
times. For UVC treatment (25) cells were grown in a 12-well dish.
Before UVC treatment cells were incubated for 20 h in Dulbecco's
modified Eagle's medium containing 0.075% fetal calf serum. Medium
was removed, and UV treatment was performed in a Stratalinker
(Stratagene) with the indicated doses (standard: 200 J/m2).
After treatment with UV light, 1 ml of serum-free medium was added, and
cells were incubated at 37 °C. At the indicated time points random
field photographs were taken, and cell death was morphologically scored
as the percentage of cells that are in the process of blebbing, that
have a condensed nucleus, or that have lysed. Also, for visualization
of condensed nuclei, the cells were grown on glass coverslips and
similarly treated. They were fixed in ice-cold methanol (
20 °C)
and subsequently incubated with 1 µg/ml DAPI in methanol for 5 min
(room temperature). Cells were washed once with methanol and once with
phosphate-buffered saline (PBS), air-dried, and mounted in 9 µl of
Mowiol (Hoechst, Frankfurt, FRG) supplemented with 0.1% paraphenylene
diamine. Fluorescent DNA-DAPI complexes were visualized in a
Leica-Aristoplan microscope equipped with epi-illumination.
Alternatively, when phase contrast pictures were taken, cells were
fixed in 4% paraformaldehyde in PBS for 15 min, rinsed with PBS,
permeabilized with ice-cold 0.1% Triton X-100, 0.1% sodium citrate in
PBS for 2 min, rinsed with PBS, stained with 0.4 µg/ml DAPI in PBS
for 1 min, rinsed twice with PBS, and air-dried (26).
Annexin V Plus PI--
For annexin V staining, cells on glass
coverslips were rinsed rapidly in Dulbecco's modified Eagle's medium
(DMEM), 20 mM Hepes, 0.1% bovine serum albumin (BSA) and
incubated in DMEM, 20 mM Hepes, 0.1% BSA containing 0.25 µg of propidium iodine and 1 µl of concentrated annexin V-FITC
(Nexins Research) for 10 min at room temperature in the dark. After
washing in Dulbecco's modified Eagle's medium, 20 mM
Hepes, 0.1% bovine serum albumin cells were mounted in Mowiol/
paraphenylene diamine.
Western Blotting--
Cell lysates and immunoprecipitates were
solubilized in Laemmli sample buffer containing 0.1 M
dithiothreitol and separated by SDS-polyacrylamide gel electrophoresis
(8%). Proteins were blotted onto polyvinylidene difluoride filters
that were blocked with 3% milk proteins (w/v, Protifar) in TBST (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, and 0.1%
Tween 20) for at least 1 h. The filters were probed with primary
antibody in 0.3% protifar in TBST for at least 1 h (or
overnight), washed four times in TBST, incubated with the appropriate
secondary antibody (horseradish peroxidase-coupled goat anti-mouse or
goat anti-rabbit) in 0.3% protifar in TBST buffer for 30 min and
washed again four times with TBST. Proteins were visualized using
enhanced chemiluminescence (Renaissance). For quantification of protein
amounts, a densitometer (Molecular Dynamics) and ImageQuant software
were used. Antibodies against PKB/Akt (1:2000) were from Transduction
Laboratories (Lexington, KY). Where indicated, the phospho-specific
Akt/PKB antibodies against phospho-Ser-473 (1:1500) was obtained from
New England Biolabs (Beverly, MA), whereas in addition,
phospho-Ser-473-PKB antibodies (1:1500) were used from
BIOSOURCE International (Camarillo, CA) since the
Biolabs antibodies gave significantly more background signal with
nonphosphorylated PKB/Akt. The secondary antibodies (1:10000) were from
Jackson ImmunoResearch (Pennsylvania, PA).
Transfections and Immunoprecipitations--
Transient
transfections in COS-1 cells were performed at 40% confluency with 4 µg of DNA/6-cm dish using the Fugene (Roche Molecular Biochemicals)
transfection protocol. For co-transfection 2 µg of each
plasmid/6-cm dish were transfected. Twenty-four hours after
transfection, cells were serum-starved for 16 h. Stimulated and
unstimulated cells were washed once with ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100) with
freshly added 1 mM phenylmethylsulfonyl fluoride, 40 mM
-glycerophosphate, 1 mM sodium vanadate,
50 mM sodium fluoride, and 10 µg/ml aprotinin. Lysates
were incubated on ice for 5 min and centrifuged for 5 min at
15,000 × g, and supernatants were precleared by
incubating with protein A-Sepharose beads (Amersham Pharmacia Biotech)
for 1 h at 4 °C and centrifuging for 5 min at 15,000 × g. HA-PKB was immune-precipitated from aliquots (200 µg of
protein) of the precleared extracts using 2 µg of the monoclonal
anti-HA antibody (12CA5) coupled to a (1:1) mixture of protein
G-agarose beads and protein A-Sepharose beads (Sigma). Immunoprecipitates were washed twice with lysis buffer and twice with
low salt buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2) before Western blot analysis or twice with high salt
buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, and 0.5 M LiCl) and twice with low salt
buffer before activity measurements. One-third of the
immunoprecipitates was used for Western blot analyses. Immune
precipitation of endogenously expressed PKB/Akt from NIH 3T3-derived
cell lines was performed similarly, except that cells were grown in
10-cm dishes and immune precipitation was performed with Immobilized
Akt 1G1 monoclonal antibody (New England Biolabs).
In Vitro Kinase Assays for PKB/Akt--
PKB activity was assayed
with the Crosstide peptide (GRPRTSSFAEG) as a substrate as
previously described (27). Immunoprecipitates were incubated with 45 µl of kinase assay mixture (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 30 µM Crosstide peptide, 1 µM specific
peptide inhibitor of cyclic AMP-dependent protein kinase
(PKI) (Bachem, Bubendorf, Switzerland), 50 µM unlabeled ATP, and 3 µCi of [
-32P]ATP. After incubation for 20 min at 30 °C under continuous shaking, reactions were stopped by the
addition of 5 µl 200 mM EDTA. Proteins were precipitated
by the addition of 12.5 µl 25% trichloroacetic acid and centrifuged
for 1 min at 14,000 rpm. Supernatants containing the phosphorylated
peptide were spotted onto p81 phosphocellulose filters (Whatman),
washed three times with 1% (v/v) orthophosphoric acid, and analyzed by
Cerenkov counting. Under the conditions used, the kinase assays are
linear for at least 60 min.
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RESULTS |
Growth Characteristics of v-Crk-transformed Cells--
To
investigate how v-Crk induces oncogenicity we first analyzed serum
dependence and growth rate of stably transfected NIH 3T3 fibroblast
cell lines expressing v-Crk (3T3v-Crk) or v-Crk mutants, mutated either
in the SH2 (3T3SH2
) or the SH3 (3T3SH3
) domain of v-Crk. These
previously described cell lines (12) were compared with control NIH 3T3
cells (3T3) and RasL61-transformed cells (3T3Ras). Growth of these cell
lines in the presence of 0.5, 2.0, or 7.5% fetal calf serum was
monitored daily using a MTT assay. Fig. 1
shows a representative time point, showing the relative cell densities
after 3 days.

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Fig. 1.
v-Crk has no profound influence on
growth. 7000 cells were seeded in 12-well tissue culture plates
supplemented with medium containing the indicated amount of serum, and
growth was monitored daily using an MTT assay. The
A570 value at day 0 was between 0.05 and 0.065 and is subtracted in the data. The graph indicates the relative cell
densities after 3 days. Shown are the means ± S.E. of three
independent experiments. Cell lines: 3T3, NIH 3T3;
v-Crk, 3T3v-Crk; SH3 , 3T3SH3 ;
SH2 , 3T3SH2 ; Ras, 3T3RasL61. FCS, fetal calf
serum.
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Clearly no profound differences in growth were observed between
3T3v-Crk and NIH 3T3 control cells that could explain oncogenicity. It
appeared that their doubling time is similar and their growth is
similarly correlated with the serum concentration. At 0.5% serum, the
cells complete their cell cycle, but in contrast to RasL61-transformed
cells, none of the other cell lines continued to grow, showing a strict
dependence on serum. The only difference observed is that 3T3v-Crk
cells grow to higher saturation density than the control cells (12), as
was apparent with the MTT assay after 4 days of growth (not shown) and
was confirmed by phase contrast microscopy. For comparison, highly
oncogenic 3T3Ras-transformed cells require less serum, grow more
rapidly, and grow to even higher saturation densities (Fig. 1). They
grow even in serum-free conditions (see next paragraph). The
3T3SH3
cell line that expresses the SH3
mutant v-Crk protein has
growth characteristics similar to NIH 3T3. In contrast, the 3T3SH2
cells grow more slowly, reach lower cell densities, and die in the
presence of 0.5% fetal calf serum. Apparently the SH2 mutant of v-Crk
has a negative effect, both with respect to cell growth and survival.
As compared with RasL61, v-Crk clearly induces more restricted
oncogenic properties.
3T3v-Crk Cells Survive in Serum Starvation Conditions--
To
investigate a possible role of v-Crk in cell survival, the effect of
serum deprivation was further investigated. Serum deprivation results
in rapid cell death of control NIH 3T3 cells; within 48 h, ~95%
of the cells die (Fig. 2). The 3T3v-Crk
cells on the other hand are more resistant against serum withdrawal; ~40-50% of the cells survive for even longer than a week, as is also seen by phase contrast microscopy. RasL61-transformed cells continue growing in serum-free conditions. The surviving
v-Crk-transformed cells were fully capable of growing when serum was
added again (not shown). To determine the role of the SH2 and SH3
domains of v-Crk in mediating protection against cell death, the mutant cell lines were assayed. The 3T3SH3
cell line dies with similar kinetics to NIH 3T3, whereas the 3T3SH2
cell line is significantly more sensitive to serum starvation-induced cell death (Fig. 2). Thus,
v-Crk protects against cell death, and both the SH2 and SH3 domain of
v-Crk are required for this protection. In addition, the v-Crk mutant
protein mutated in the SH2 domain (v-CrkSH2
) enhances cell death,
having a dominant negative function with respect to survival.

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Fig. 2.
v-Crk protects against cell death.
70,000 cells were seeded in 12-well plates and incubated in serum-free
medium, and the relative cell number was assayed daily using an MTT
assay. The cell number is expressed as the percent of cells seeded.
Shown are the means ± S.E. of three independent
experiments.
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v-Crk Protects against Apoptosis--
Figs. 1 and 2 indicate that
the main growth difference between NIH 3T3- and v-Crk-transformed NIH
3T3 cells is the ability of 3T3v-Crk to survive in serum-free
conditions. Inhibition of apoptosis by v-Crk would provide a
physiological selective advantage in vivo for
v-Crk-transformed cells. The MTT assay (Fig. 2) measures the number of
surviving cells but does not distinguish between death by necrosis or
apoptosis. Actually, an important characteristic of apoptosis is that
during the programmed cell death the cell membrane remains impermeable,
and the cell content is not released. Using phase contrast microscopy
we noticed that blebbing apoptotic cells are still capable of forming
dark blue formazan crystals in the MTT assay. A decrease in MTT signal
due to apoptosis is therefore only observed when (apoptotic) cells
detach from the substratum or when they have lysed due to secondary
necrosis. Using nuclear DAPI staining, we found that cell death upon
serum starvation (48 h) results in nuclear condensation, characteristic of apoptosis (Fig. 3A). To
further prove that cell death occurred due to apoptosis, annexin V
binding assays were performed. Annexin V binds to phosphatidylserine, a
phospholipid that normally is present at the inner leaflet of the cell
membrane but during apoptosis is exposed at the outer leaflet. In the
presence of serum, neither NIH 3T3 cells nor 3T3v-Crk cells bound
annexin V. However, after 24 h of serum depletion the NIH 3T3
cells heavily stained with annexin V-FITC, and 3T3v-Crk cultures
stained significantly less (Fig. 3B, panels B and
E). Annexin V-FITC staining of starved NIH 3T3 cells
revealed that many cell fragments (presumably derived from blebs: see
next paragraph) are shedded off. In addition, some NIH 3T3 cells
are in necrosis, as is revealed by their plasma membrane permeability
for propidium iodine (Fig. 3B, panel C, red
arrow). In conclusion, both DAPI and annexin V staining
demonstrate the protective effects of v-Crk expression against
apoptosis induced by serum withdrawal.

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Fig. 3.
A, nuclear condensation upon
starvation in NIH 3T3 cells. NIH 3T3 cells and 3T3v-Crk cells were
cultured on coverslips and directly stained with DAPI
(control) or incubated in serum-free medium for 48 h,
after which nuclei were stained with DAPI. B, annexin V
binds apoptotic NIH 3T3 cells. NIH 3T3 cells (A-C) and
3T3v-Crk cells (D-F) were cultured on coverslips and
incubated in serum-free medium for 28 h. Subsequently they were
stained with annexin V and propidium iodine for 10 min (see
"Experimental Procedures"). A and D, combined
annexin V FITC fluorescence and phase-contrast micrograph. B
and E, annexin V-FITC staining. C and
F, propidium iodine staining. Upon serum starvation for
28 h, NIH 3T3 cells heavily stained with annexin V-FITC
(B, arrows); the cells that in addition are
stained with propidium iodine (C, red arrow) are
permeable and therefore necrotic.
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To test if v-Crk also protects against apoptosis induced by other
methods, UVC treatment was applied. After overnight serum starvation,
cells were treated with various doses of UVC light and analyzed for
apoptosis by morphological criteria and by analyzing nuclear
condensation by DAPI staining. After treatment with 200 J/m2 UV light for 5 h, more than 80% of the NIH 3T3
cells are apoptotic or in (secondary) necrosis. Fig.
4A illustrates that UVC
irradiation induces apoptosis, as shown both by nuclear condensation
and cell membrane blebbing. A higher magnification of a typical
blebbing cell shows many cell protrusions that are connected to the
cell body only through a thin stalk, as is nicely illustrated in cells expressing green fluorescent protein (Fig. 4B). Later in the
apoptotic process the protrusions are pinched off while still
containing green fluorescent protein, implying that the plasma membrane
is still intact, which is characteristic of apoptosis (results not shown). The effect of 200 J/m2 UVC on survival of the
different cell lines is represented in Fig. 4C. It shows
that the v-Crk-transformed cells are protected against UV-induced
apoptosis, as compared with NIH 3T3 cells. Similar conclusions were
reached using other UV doses (50, 100, 400, 600 J/m2) and
at different time points after UV treatment (results not shown). Again,
survival of the v-CrkSH3 mutant-expressing cells is similar to the NIH
3T3 cells. The SH2 mutant-expressing cells are slightly more sensitive
to UV-induced apoptosis. RasL61-transformed cells do not undergo
apoptosis at all in these conditions. Other apoptotic stimuli including
H2O2 and osmotic stress were also tested (not
shown). In all cases tested, v-Crk provided protection against
apoptosis.

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Fig. 4.
v-Crk protects against apoptosis.
A, UV induces chromatin condensation and membrane blebbing
in NIH 3T3 cells. DAPI staining shows condensed nuclei. Cells were
cultured on glass coverslips, serum-starved 20 h before UV
treatment, and treated with 200 J/m2 UVC. They were
analyzed by DAPI staining and phase contrast microscopy. B,
illustration of blebbing of a cell expressing green fluorescent protein
(GFP). C, percentage survival 3 h after 200 J/m2 UVC treatment (cells were serum-starved 20 h
before UV treatment). The percentages of apoptotic and dead cells were
quantified by counting blebbing cells and condensed nuclei after DAPI
staining (see Fig. 4A), and the percent viable cells is
indicated. Shown are the means ± S.E. of three independent
experiments.
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Which Signaling Pathways Are Involved in Crk-mediated Protection
against Apoptosis?--
As a first approach in determining which
signaling pathways might be activated by v-Crk to inhibit apoptosis, we
used pharmacological inhibitors. The best-described anti-apoptotic
pathway is the PI 3-kinase-PKB/Akt pathway, which can be inhibited by
the PI 3-kinase inhibitors LY924002 and wortmannin. Because v-Crk has
been implicated in activation of the Ras-Raf-MEK-Erk pathway (28, 29),
which plays a protective role in various systems (30, 31), the
inhibitor PD98059 was used to inhibit activation of MEK. Another MAPK
implicated in apoptosis is the p38 MAPK, which is specifically
inhibited by SB202190 (32). Fig.
5A shows that upon serum
starvation, the v-Crk-induced protection against apoptosis is strongly
decreased by LY294002 (10 µM). The same was found when PI
3-kinase was inhibited with wortmannin (100 nM; not shown),
indicating that v-Crk-induced protection may be mediated by the PI
3-kinase/PKB pathway. Survival of the 3T3Ras cells was not affected by
LY294002, whereas PD98059 (10 µM) largely abrogated
survival. Inhibition of MEK modestly decreased protection of 3T3v-Crk
cells, implying that activation of the MAPK pathway, although to a
lesser extent, is also required for full protection. In contrast,
inhibition of the p38 MAPK did not influence survival. Induction of
apoptosis by UV-induced cell damage yielded a slightly different
result. Again p38 MAPK does not play a role of importance. However,
Fig. 5B shows that both PI 3-kinase- and MEK-mediated
survival contribute only weakly to v-Crk-induced protection. Both in
serum starvation-induced apoptosis and UV-induced apoptosis,
inhibition of both pathways simultaneously hardly showed an additive
effect. Even in the presence of both inhibitors 3T3v-Crk was more
resistant than NIH 3T3, indicating that v-Crk induces yet additional
protective effects, the nature of which still has to be determined.

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Fig. 5.
Signaling pathways involved in protection
against apoptosis. A, percent survival, determined by
MTT assay after a 48-h serum-free incubation without (SF) or
with indicated inhibitors. B, percent survival 3.5 h
after UV treatment (200 J/m2), determined as in Fig.
4C. LY, LY294002 (10 µM);
PD, PD98059 (10 µM); SB, SB202190
(20 µM). Shown are the means ± S.E. of three
independent experiments.
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Protection against Apoptosis Is Correlated with Enhanced PKB/Akt
Activation and Enhanced PKB/Akt Phosphorylation on
Ser-473--
The inhibitory effect of PI 3-kinase inhibitors on
v-Crk-induced protection against apoptosis caused us to investigate the activity of PKB/Akt in v-Crk-expressing cells. PKB/Akt was
immune-precipitated and assayed for kinase activity using a specific
peptide (Crosstide) as a substrate (27). Whereas serum-starved
v-Crk-transformed cells and control NIH 3T3 cells contain similar
amounts of PKB/Akt (Fig. 6B),
it appeared that the PKB/Akt activity was at least 3-fold more in
v-Crk-transformed cells than in control NIH 3T3 cells (Fig.
6A). As a positive control, PKB/Akt activation by PDGF was
also assayed. For both, the addition of PDGF (20 ng/ml for 10 min)
resulted in a further induction of PKB/Akt activity. Thus, v-Crk
expression results in a 3-fold activation of PKB/Akt even in the
absence of growth factors and induces an enhancement of PDGF
stimulation of PKB/Akt activity.

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Fig. 6.
PKB/Akt is constitutively activated in
3T3v-Crk cells. A, relative in vitro PKB/Akt
kinase activity (using the Crosstide peptide as a substrate)
measured after immune-precipitation of PKB/Akt from NIH 3T3 and
3T3v-Crk cells. The PKB/Akt activity of serum-starved 3T3v-Crk is
(arbitrarily) taken as 100%. SF, serum starvation for
20 h with 0.075% fetal calf serum; +PDGF, induction
with 20 ng/ml PDGF for 10 min. B, Western blot analysis of
lysates of stably transfected NIH 3T3 cell lines obtained after a 24-h
serum starvation (SF) using -phospho-Ser-473-PKB/Akt
antibody (Biolabs). The control panel, treated with PKB, shows the
total PKB/Akt independent of the phosphorylation state.
+PDGF, induction with 20 ng/ml PDGF for 10 min.
C. Western blot analysis of lysates of NIH 3T3 and 3T3v-Crk,
using -phospho-Ser-473-PKB/Akt antibody
(BIOSOURCE International). Lysates were obtained
after starvation for 24 h (SF) and after starvation
plus, in addition, treatment with LY294002 (10 µM) for 30 min (SF+LY).
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Generally PKB/Akt activity is correlated with phosphorylation of
Ser-473 and Thr-308, both contributing to maximal PKB/Akt activity (for
reviews, see Refs. 33-35). Using phospho-specific antibodies, we found
that upon starvation, the Ser(P)-473 PKB levels in 3T3v-Crk are
increased as compared with NIH 3T3 (Fig. 6B), which
correlates well with the PKB/Akt activity in these cells (as determined
in Fig. 6A). The phosphorylation of PKB/Akt on Ser-473 is
abrogated by inhibition of PI 3-kinase with the chemical inhibitor
LY924002 (Fig. 6C), also indicating the involvement of PI 3-kinase-mediated signaling in v-Crk-induced PKB/Akt activation. Again, both the SH2 and SH3 domains of v-Crk are required; Ser-473 PKB
phosphorylation in the SH3 mutant is the same as in NIH 3T3 cells,
whereas the SH2 mutant displays decreased levels of Ser-473 PKB
phosphorylation. Thus, the PKB/Akt phosphorylation levels in these cell
lines correspond well to their resistance against serum
starvation-induced apoptosis (Fig. 2). The RasL61-transformed cells
display low levels of PKB/Akt phosphorylation, indicating that other
signaling pathways may be involved in protection against apoptosis in
these cells, as was also clear in Fig. 5A. In conclusion, v-Crk expression induces a PI 3-kinase-dependent activation
of PKB/Akt and stimulates the phosphorylation of Ser-473.
v-Crk Also Enhances PKB/Akt Phosphorylation upon Transient
Transfection in COS-1 Cells--
To further investigate the activation
of PKB/Akt by v-Crk, transient transfection experiments were performed.
COS-1 cells were transfected with HA-tagged PKB/Akt or co-transfected
with HA-tagged PKB/Akt and v-Crk together. PKB/Akt was
immune-precipitated and analyzed for activity (Fig.
7A) and for phosphorylation
state using the phosphospecific antibody against Ser-473 (Fig.
7B). Fig. 7A shows that immune-precipitated
PKB/Akt displays a basal level of PKB activity even in serum starvation
conditions (bars 1 and 2), which is stimulated by
PDGF treatment (bar 4) and is PI
3-kinase-dependent (bars 3 and 5).
Co-transfection with v-Crk results in clearly enhanced PKB/Akt
activity, both in serum starvation conditions (bars 7 versus
2) and after PDGF induction (bars 9 versus 4), which is
also mostly PI 3-kinase-dependent (bars 8 and
10). Thus, these results demonstrate that transient expression of v-Crk
also activates a pathway resulting in PKB/Akt activation. Fig.
7B shows the parallel analysis of the PKB/Akt
phosphorylation state. It appears that in this transient assay system,
v-Crk also induces the phosphorylation of Ser-473, which is nicely
correlated with the observed PKB/Akt activity. The phosphorylation of
transiently expressed PKB/Akt as well as the enhanced phosphorylation
due to v-Crk expression is completely PI 3-kinase-dependent
(lanes 3, 5, and 8, 10,
respectively).

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Fig. 7.
v-Crk also enhances PKB/Akt phosphorylation
upon transient transfection in COS-1 cells. COS-1 cells were
transfected with HA-PKB/Akt with or without co-transfected v-Crk.
HA-PKB was immune-precipitated, and immune precipitates were split and
subjected to PKB/Akt kinase activity assays (A) and Western
blot analysis (B). A, PKB/Akt activity assays, as
described under "Experimental Procedures." The PKB/Akt activity of
cells transfected with PKB-HA only is (arbitrarily) taken as 100%.
B, Western blot analysis with phosphorylation-specific
antibodies using -phospho-Ser-473-PKB/Akt antibody
(BIOSOURCE International) and control with
antibodies recognizing phosphorylation-independent total PKB/Akt
(PKB). SF, serum depletion for 24 h;
+PDGF, induction with 20 ng/ml PDGF for 10 min;
+LY, pretreatment with LY924002 (10 µM) for 30 min.
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DISCUSSION |
v-Crk Protects against Apoptosis--
The v-Crk oncogene product
induces in fibroblasts the capacity to form tumors (1), which is
correlated with a transformed phenotype and the capacity to grow in
soft agar. Here we further investigated which mechanisms are involved
in the transformation. Compared with Ras-transformed cells, v-Crk
appears mildly transforming. Ras-transformed cells are more aggressive
in soft agar growth and when injected in animals. In correlation with
this, we found that v-Crk does not induce obvious growth advantage as
compared with NIH 3T3. In contrast to the activated Ras mutant RasL61, v-Crk does not induce growth in serum-free conditions, and the serum
requirement and growth rate are similar to NIH 3T3 control cells.
However, similar to RasL61-transformed cells upon serum starvation,
v-Crk-expressing cells were largely protected against cell death. In
view of the physiological relevance of apoptosis, we further
investigated whether the protection against cell death by v-Crk could
be due to the inhibition of apoptosis.
Whereas 3T3v-Crk cells were clearly protected against cell death by
serum starvation, the propensity of fibroblasts to undergo apoptosis is
not very well defined. The hallmark of apoptosis is that upon a death
stimulus, the cell content is largely degraded within the boundary of
the intact cell membranes, and therefore, no leakage of cell content
and degrading enzymes occurs. This is generally accompanied by the
typical apoptotic changes in morphology of cells called blebbing. We
found that the majority of dead fibroblast cells generated by serum
starvation did not meet this criterion since their membranes were
permeable for ethidium bromide and propidium iodine. They did, however,
display condensed nuclei, which is another feature taken as a criterion
for apoptosis. The explanation is that in tissue cultures, apoptosis is
followed by secondary necrosis. Annexin V staining of serum-starved
cells provided further evidence that the NIH 3T3 cells indeed undergo apoptosis before lysis. Annexin V staining showed that many large cell
fragments (most likely derived from blebs) are shedded off from the
dying cells, which made this method less useful for quantitative purposes. Nevertheless, the 3T3v-Crk cells were protected also by this criterion.
Other methods of apoptosis induction were also tested. A very suitable
method appeared to be UVC treatment, which induced clear blebbing of
the cells within hours. During blebbing the fibroblasts displayed
cellular protrusions typically attached to the cell only by a stalk,
which were subsequently pinched off. The blebbing preceded nuclear
condensation, and both were significantly delayed in 3T3v-Crk. Other
apoptosis-inducing agents like hydrogen peroxide and osmotic stress
also showed that v-Crk protects against apoptosis, but these displayed
less the blebbing phenotype. Whereas serum starvation most likely
induces apoptosis by deregulating cellular metabolism, UVC will do so
by radiation damage. v-Crk was shown to protect in both cases.
v-Crk Activates Multiple Protection Pathways--
In search of
signaling pathways involved in protection against apoptosis and
considering that focal adhesion signaling may be stimulated by v-Crk,
we investigated the possible involvement of the well known
anti-apoptotic PI 3-kinase/PKB pathway. Furthermore, among the
signaling pathways that have been reported to be activated by v-Crk (5,
13, 28, 29), the Ras-Erk pathway has been ascribed anti-apoptotic
properties (30, 31) and was, therefore, also investigated. Using
pharmacological inhibitors, we found that v-Crk-mediated protection
against serum starvation-induced apoptosis was largely abrogated by
inhibiting the PI 3-kinase/PKB pathway, whereas the MEK-Erk pathway
played a smaller role. Indeed, v-Crk induced in a PI
3-kinase-dependent manner the phosphorylation and
activation of PKB/Akt, as is outlined below. Remarkably, the protection
against UV-induced apoptosis appears to involve other mechanisms, since
inactivation of PKB/Akt by LY924002 treatment only poorly decreased
resistance against UV-induced apoptosis. This indicates that v-Crk
activates, in addition to the PI 3-kinase/PKB pathway, another
pathway(s) that plays a role in protection against UV-induced
apoptosis. Comparably, the Ras oncogene was recently also reported to
inhibit different apoptotic induction mechanisms independently of
each other, which in that case was attributed to independent activation
of both the Ras/PKB and the Ras/Erk pathway (36). Inhibition of the
Ras-Erk pathway by MEK inhibitor PD98059 showed that in our cells the
MAPK pathway does provide some protection. However, whereas our data
indicate that in 3T3RasL61 cells this is the major protection pathway
upon serum starvation, this is not the case in 3T3v-Crk. For UV-treated
cells, the effect of the MEK inhibitor PD98059 is even stronger in NIH
3T3 cells than in 3T3v-Crk, indicating that v-Crk does not play a role
in the MEK-Erk-mediated protection. In accordance with this, it was recently reported that Erk is not activated by v-Crk (37) and, therefore, cannot be causally correlated with protection against cell
death. Others have shown that the functions of Erk and c-Crk II
represent two distinct pathways involved in protection from apoptosis
in a three-dimensional collagen matrix (38). Our data indicate that yet
another pathway is activated by v-Crk that protects against apoptosis.
The nature of this pathway still has to be determined.
v-Crk Regulation of PKB--
Further analysis of the activation
state of PKB mediated by v-Crk showed a clear correlation between
enhanced phosphorylation of Ser-473, PKB activity, and survival in
serum starvation conditions in the cells stably transformed with
various v-Crk constructs. Activation of PKB/Akt by v-Crk also occurred
upon transient co-expression of both in COS-1 cells, indicating that it
is not the result of the transformed phenotype of the v-Crk transformed
cells but directly due to v-Crk-induced signaling. Both the
v-Crk-induced PKB/Akt activation in stably transformed cells and in
transient assays are largely PI 3-kinase-dependent.
Furthermore, whereas v-Crk activates PKB/Akt, the cell line stably
expressing the v-Crk SH2 mutant showed a reduced amount of Ser(P)-473
even in serum-containing conditions (not shown). A lack of
phosphorylation correlated with enhanced sensitivity to cell death
inducers. In a recent study, it was shown that soft agar growth of
v-Crk-transformed cells was abrogated by inhibition of PI 3-kinase,
confirming the correlation with oncogenic properties (37). Using the
Ser(P)-473-specific antibody, this study showed that v-Crk also induces
PKB/Akt phosphorylation in chicken fibroblasts (37).
How v-Crk activates PKB/Akt still has to be established. Ser(P)-473
phosphorylation was abrogated by LY924002, indicating PI 3-kinase
dependence. That PI 3-kinase can localize to focal adhesion complexes
(39) suggests that PKB/Akt activation might be due to enhanced PI
3-kinase activity in v-Crk-transformed cells, correlating with enhanced
focal complex signaling. In this respect both focal adhesion kinase and
c-Src and p130cas have been implicated in signaling toward PI
3-kinase (39-41). PI 3-kinase binds with the SH2 domains of its
regulatory subunit, p85, to focal adhesion kinase, Src, and
p130cas. Interestingly, Src has also been shown to stimulate PI
3-kinase, and moreover, Src is activated in v-Crk-transformed cells
(6). Furthermore, both focal adhesion kinase and p130cas are
phosphorylated in v-Crk-transformed cells, creating binding sites for
PI 3-kinase. This might result in activation of this lipid kinase.
The results obtained with the v-CrkSH2
mutant indicate that the
regulation of PKB/Akt by Crk isoforms is not restricted to the viral
v-Crk only. The v-CrkSH2
mutant protein is considered to act as a
dominant negative protein disturbing normal c-Crk function. The
v-CrkSH2
mutant-expressing cell line grew more slowly than control
cells, showed down-regulated phosphorylation of PKB/Akt, and displayed
enhanced sensitivity for apoptosis. This implies that the v-Crk SH2
protein titrates-out proteins that are required for protection against
cell death and suggests that endogenous c-Crk may also have a function
in regulating PKB/Akt and protection against cell death. Further
investigations are required to determine if the decreased growth rate
of the v-Crk SH2 mutant NIH 3T3 cell line is also correlated with
PKB/Akt activity or is due to other dominant negative effects by this
v-Crk mutant (e.g. see Ref. 21). As indicated in the
Introduction, various data suggest that v-Crk and c-Crk play an
important role in focal adhesion signaling. Our results are in
agreement with a model in which v-Crk enhances focal adhesion complex
signaling; previously, it was shown that PKB/Akt can be activated by
focal adhesion signaling (42). Similarly, in general the effect of
dominant negative v-Crk SH2 may be the disturbance of focal adhesion
complex signaling.
In conclusion we show that v-Crk expression in fibroblast cells results
in protection against apoptosis induced by various methods. Protection
is mediated by activation of several protection pathways, among them
the PI 3-kinase/PKB pathway. We show that in fibroblasts, v-Crk
activates PKB/Akt and induces phosphorylation of Ser-473. Furthermore,
our results with dominant negative forms of v-Crk suggest that
endogenously expressed Crk also plays a role in PKB/Akt activation and
regulation of apoptosis. Since, normally, cells that are deregulated
will undergo apoptosis, inhibition of apoptosis is thought to be a very
important requirement in the process of tumorigenesis. Our results
suggest that inhibition of apoptosis is the main mechanism involved in
the oncogenesis induced by v-Crk.