From the Department of Medicine, Division of
Endocrinology and Metabolism, Whittier Diabetes Program, University of
California, San Diego, La Jolla, California 92093 and Veterans
Administration Medical Center, Medical Research Service, San Diego,
California 92161 and the ¶ Department of Signal Transduction,
Parke-Davis Pharmaceutical Research, Division of Warner Lambert
Company, Ann Arbor, Michigan 48105
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ABSTRACT |
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Crk is a member of a family of adapter proteins
predominantly composed of Src homology 2 and 3 domains, whose role in
signaling pathways is presently unclear. Using an in situ
electroporation system which permits the introduction of glutathione
S-transferase (GST) fusion proteins into cells, we found
that c-CrkII bound to p130cas, but not to paxillin in
serum-starved rat-1 fibroblasts overexpressing the human insulin
receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130cas,
whereas 13 nM insulin-like growth factor-I, 16 nM epidermal growth factor (EGF), and 10% serum each
showed little or no effect. We found that stress fiber formation is
consistent with a change in the p130cas·c-CrkII interactions
before and after growth factor stimulation. Microinjection of either
GST-Crk-SH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited
stress fiber formation before and after insulin-like growth factor-I,
EGF, and serum stimulation. Insulin stimulation by itself caused stress
fiber breakdown and there was no additive effect of microinjection.
Microinjection of anti-p130cas antibody also blocked stress
fiber formation in quiescent cells. Microinjection of the
Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like
growth factor-I, EGF, and serum stimulation, but not after insulin.
These data suggest that the complex containing p130cas·c-CrkII may play a crucial role in actin cytoskeleton
organization and in anchorage-dependent DNA synthesis.
Crk, a member of a family of adapter proteins, was originally
reported from an avian retrovirus encoding the oncogene product v-Crk
(1). The mammalian cellular homologues of v-Crk have been subsequently
identified as c-CrkI and c-CrkII, which are alternatively spliced forms
of a single gene (2, 3). In addition, another closely related gene
product, c-Crk-L has been isolated (4).
A potential role for Crk in the regulation of the mammalian actin
cytoskeleton has been suggested. In v-Crk-expressing cells, the level
of tyrosine phosphorylation of a limited number of specific cellular
proteins is increased, despite the lack of any tyrosine kinase
catalytic domain in v-Crk itself (5). Among these
tyrosine-phosphorylated proteins, components of focal adhesions,
p130cas and paxillin, known to associate with actin stress
fiber formation (6, 7), have been shown to associate with the
SH21 domain of v-Crk (5, 6,
8). v-Crk was shown to activate the Rho GTPase signaling pathway,
thought to regulate actin cytoskeleton organization, and served as a
scaffolding protein during the assembly of focal adhesions (7).
DOCK180, which was cloned as an SH3 domain-binding partner of c-Crk,
alters cell morphology upon translocation to the cell membrane (9).
Thus, c-Crk has been implicated in actin cytoskeleton signaling
pathways, but the detailed mechanisms of its actions are still unclear.
In this study, to examine the mechanism of c-Crk function in growth
factor-induced actin cytoskeleton organization and mitogenic pathways,
we have examined Crk interactions with several signal transduction
molecules in rat-1 fibroblasts overexpressing human insulin receptor
(HIRc cells). The functional involvement of c-Crk in these cellular
functions was directly assessed by single cell microinjection analysis
in the presence and absence of several growth factors in HIRc cells.
Cell Culture and Materials--
Rat-1 fibroblasts expressing
1 × 106 human insulin receptors per cell (HIRc cells)
were cultured as described previously (10). These cells were found to
express c-CrkII but not c-CrkI by Western blot analysis (data not
shown). Porcine insulin and IGF-I were kindly provided by Lilly, and
EGF was purchased from Life Technologies, Inc. (Grand Island, NY).
Bromodeoxyuridine (BrdUrd), a monoclonal rat anti-BrdUrd antibody, and
enhanced chemiluminescence reagents were purchased from Amersham Corp.
A mouse monoclonal anti-Crk antibody, a mouse monoclonal
anti-phosphotyrosine (anti-pY) antibody, a mouse monoclonal
anti-p130cas antibody (against carboxyl terminus), a mouse
monoclonal anti-Sos antibody, and a mouse monoclonal anti-paxillin
antibody were from Transduction laboratories (Lexington, KY). A rabbit
polyclonal anti-CrkII antibody, a rabbit polyclonal anti-c-cbl
antibody, a rabbit polyclonal anti-C3G antibody, a rabbit polyclonal
anti-c-abl antibody, a rabbit polyclonal anti-p130cas antibody
(against carboxyl terminus) were from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). A rabbit polyclonal anti-GST antibody was from
Upstate Biotechnology (Lake Placid, NY). Mouse IgG and fluorescent isothiocyanate or rhodamine-conjugated anti-mouse and anti-rat IgG
antibodies were from Jackson Laboratories (West Grove, NY). Electrophoresis reagents were from Bio-Rad. TRITC-phalloidin and all
other reagents were purchased from Sigma.
Glutathione S-Transferase (GST)-Crk-SH2 and GST-Crk-(N) SH3
Domain Fusion Proteins Preparation--
The GST fusion proteins
containing SH2 domain or (N)SH3 domain of c-Crk were a generous gift of
R. B. Birge and H. Hanafusa (8, 11). The fusion proteins were
expressed in Escherichia coli and purified by chromatography
on glutathione-Sepharose 4B. The purified proteins were concentrated
using a Centricon-30 (Amicon Inc., Beverly, MA), and the buffer was
exchanged to 5 mM sodium phosphate (pH 7.4) and 100 mM KCl for microinjection or to phosphate-buffered saline
(PBS) for electroporation.
Western Blotting Studies--
Cell monolayers were starved for
36 h in serum-free Dulbecco's modified Eagle's medium (DMEM)
containing 0.1% BSA. The cells were then treated with 17 nM insulin, 13 nM IGF-I, 16 nM EGF, or 10% fetal bovine serum for the indicated times at 37 °C. Cells were lysed in a buffer containing 20 mM Hepes, 3 mM MgCl2, 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate,
1% Triton X-100, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin (pH
7.4). The cell lysates were centrifuged to remove insoluble materials. The supernatants were used for immunoprecipitation with the indicated antibodies or precipitation with glutathione-Sepharose 4B for 3 h
at 4 °C. The precipitates were separated by SDS-polyacrylamide gel
electrophoresis and transferred to Protran (Schleicher & Schuell) using
a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer
containing 50 mM Tris, 150 mM NaCl, 0.1% Tween
20, 4% BSA (pH 7.5) for 2 h at room temperature. The membranes
were then probed with specified antibodies for 2 h at room
temperature. After washing the membranes in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20 (pH
7.5), blots were incubated with horseradish peroxidase-linked
second antibody followed by enhanced chemiluminescence detection using
the ECL reagent according to the manufacture's instructions (Amersham
Corp.).
Cellular Electroporation--
HIRc cells were plated on glass
slides coated with conductive indium tin oxide and grown within windows
(7 × 15 mm) framed by nonconducting adhesive. Cells approaching
confluence were starved for 24 h in serum-free Dulbecco's
modified Eagle's medium containing 0.1% BSA. Electroporation was
performed with an Epizap pulse generator and electrode manufactured by
Ask Science (12). Plated cells were subjected to six consecutive
electrical pulses, each at 25 V to a capacitance of 16 microfarads.
During pulsations, the cells were submerged in PBS containing 7 mM of the indicated GST fusion protein. The cells were
washed once with serum-free Dulbecco's modified Eagle's medium
containing 0.1% BSA and incubated in the same medium for 12 h at
37 °C. The cells were lysed as described above. The cell lysates
from 10 slides (400 µg of protein) were used for immunoprecipitation
with anti-CrkII antibody and for precipitation with
glutathione-Sepharose 4B (Amersham Corp.).
Microinjection--
Cells were grown on glass coverslips and
rendered quiescent by starvation for 36 h in serum-free
Dulbecco's modified Eagle's medium containing 0.1% BSA. Anti-Crk
antibody and GST fusion proteins were solubilized in microinjection
buffer consisting of 5 mM sodium phosphate and 100 mM KCl (pH 7.4), and then microinjected using glass
capillary needles. Immunofluorescent staining as described below of the
injected cells indicated that about 80% of the cells were successfully
microinjected. In all injection experiments, results represent the mean
of at least three identical experiments in which at least 250 cells
were injected.
Actin Localization--
One hour after microinjection, cells
were stimulated with 17 nM insulin, 13 nM
IGF-I, 16 nM EGF, or 10% fetal bovine serum for 5 min and
fixed with 3.7% formaldehyde in PBS for 10 min at room temperature.
Cells were permeabilized in 0.2% Triton X-100 for 5 min, washed in
PBS, and incubated at room temperature for 45 min with TRITC-phalloidin
(0.5 µg/ml) in PBS. Fluorescent isothiocyanate-labeled donkey
anti-mouse IgG antibody (1:100) was added to the incubation mixture for
coverslips containing injected cells.
BrdUrd Incorporation--
One hour after microinjection, cells
were incubated with BrdUrd plus vehicle, 17 nM insulin, 13 nM IGF-I, 16 nM EGF, or 10% fetal bovine serum
for 16 h at 37 °C. The cells were fixed with 3.7% formaldehyde
in PBS for 20 min at room temperature. The fixed cells were
permeabilized and blocked with a solution containing 5% BSA and 0.5%
Nonidet P-40 in PBS. The cells were incubated with rat polyclonal
anti-BrdUrd antibody in a buffer containing 10 mM
MgCl2 and DNase I for 1 h at room temperature. The
cells were then stained by incubation with rhodamine-labeled donkey anti-rat IgG antibody, fluorescent isothiocyanate-labeled donkey anti-mouse IgG antibody, and Heuchst (Sigma), which stains all nuclei,
for 1 h at room temperature.
After staining, coverslips were washed successively in PBS and
deionized H2O for 5 min and mounted in PBS containing 15%
polyvinyl alcohol, 33% glycerol, and sodium azide. After the
coverslips were mounted, the cells were analyzed with a Axioskop
fluorescence microscope (Zeiss, Germany). For actin localization,
individual cells displaying parallel actin fibers that colocalized with
the nucleus were then scored as positive for stress fibers. Cells that
showed actin staining at the periphery were scored as positive for
membrane ruffles. The percentage of total counted cells displaying each
phenotype is represented by the stress fiber and ruffling indexes, respectively.
c-CrkII Association with Tyrosine-phosphorylated
p130cas--
We first evaluated tyrosine-phosphorylated
proteins which are co-immunoprecipitated with c-Crk in the basal state
in HIRc cells. Immunoprecipitation by anti-CrkII antibody followed by Western blotting with anti-pY antibody showed a tyrosine-phosphorylated 130-kDa band (pp130 in Fig. 1). This band
was also immunoprecipitated by another anti-Crk antibody which
recognizes a distinct epitops (Fig. 1). The pp130 was identified as
p130cas by subsequent anti-p130cas blotting of the
anti-Crk antibodies immunoprecipitants (Fig. 1, middle
panel). c-CrkII was faintly tyrosine phosphorylated (pp40) in the
basal state (Fig. 1). c-CrkI was not detected by Western blotting with
anti-Crk antibody in HIRc cell lysates (data not shown). A quantitative
analysis of p130cas·c-CrkII binding using Western blotting
and densitometry showed that an estimated 43% of total c-CrkII binds
to p130cas, and 21% of total p130cas binds to c-CrkII
in quiescent and serum-starved HIRc cells.
In Vivo Assessment of CrkII Associated Molecules by Electroporation
of GST Fusion Proteins--
To determine which signaling molecules
bind to which domain(s) of c-CrkII in vivo, we
electroporated GST fusion proteins containing either the Crk SH2 domain
or the Crk-(N)SH3 domain. Cell lysates were then immunoprecipitated
with anti-CrkII antibody (Fig. 2, lanes 1-3) and the supernatants from this procedure were
precipitated with glutathione-Sepharose 4B (Fig. 2, lanes
4-6). The final supernatants are seen in Fig. 2, lanes
7-9. The CrkII antibody precipitates >95% of endogenous CrkII
(Fig. 2, panel A), and since this antibody is directed
against the COOH-terminal CrkII SH3, as predicted, it does not
precipitate either the GST-Crk-SH2 or GST-Crk-(N)SH3 (Fig. 2,
panel B, lanes 1-3). The GST fusion proteins were
efficiently electroporated into the cells and were quantitatively
precipitated by glutathione-Sepharose 4B, as seen in Fig. 2,
panel B, lanes 4-6. Control studies using immunostaining of
cells and Western blotting with GST antibody showed that >90% of the
cells incorporate GST fusion proteins at an average level of 10 pmol of
GST protein/mg of total cellular protein under our electroporation
conditions (data not shown). As seen in Fig. 2, panels C and
D, when the electroporated cells are precipitated with CrkII
antibody, which does not recognize the GST fusion proteins,
incorporating GST-Crk-SH2 into the cells completely disrupts the
association between endogenous CrkII and a tyrosine-phosphorylated
p130cas (lane 2), whereas GST-Crk-(N)SH3 is without
effect (lane 3). In addition, precipitation of the GST
fusion proteins from the c-CrkII immunodepleted supernatants revealed
that p130cas was now associated with GST-Crk-SH2, but not
GST-Crk-(N)SH3 (Fig. 2, C and D, lanes 4-6).
Cells electroporated with PBS alone showed the same results as the
cells electroporated with GST alone (data not shown). These results
indicate that endogenous CrkII binds to p130cas via its SH2
domain and that GST-Crk-SH2 is able to displace endogenous CrkII from
p130cas in vivo.
Crk-SH2 domain preferentially binds to (p)YXXP motifs (13).
Paxillin possesses (p)YXXP motifs as does p130cas
(14), and paxillin is tyrosine phosphorylated in these cells in the
basal state (data not shown and Ref. 15). v-Crk has been shown to bind
to paxillin in v-Crk transformed cells (8), and GST-Crk-SH2 binds to
paxillin (pp68) in vivo (Fig. 2, C and E, lane 5), however, anti-CrkII antibody failed to
co-immunoprecipitate any detectable paxillin (Fig. 2, C and
E, lanes 1-3). In vitro GST fusion protein
precipitation studies showed that 1 µg of GST-Crk-SH2 precipitated
both p130cas and paxillin but 0.1 µg of GST-Crk-SH2 did not,
while anti-CrkII antibody immunoprecipitated only p130cas but
not paxillin (data not shown). Another anti-Crk antibody also did not
immunoprecipitate the pp68 band (Fig. 1). These data suggest that the
full-length c-CrkII protein binds only to tyrosine-phosphorylated p130cas, and not to tyrosine-phosphorylated paxillin, although
GST-Crk-SH2 can bind to both docking proteins similarly, even in
vivo. In the basal state, c-cbl, which also has been reported to
bind to the Crk-SH2 domain (16, 17), did not bind to c-CrkII or to the
GST fusion proteins (data not shown). The electroporated GST-Crk-(N)SH3 partially blocked the association between c-CrkII and the guanine nucleotide exchange factor C3G, or between c-CrkII and c-abl in vivo (Fig. 2, F and G). Sos, another
exchange factor, was not detected in anti-CrkII antibody
immunoprecipitants, whereas a small amount of Sos was precipitated with
the GST-Crk-(N)SH3 in vivo (Fig. 2H).
Effect of Growth Factor Stimulation on Tyrosine Phosphorylation of
CrkII or on CrkII Association with Signaling Molecules--
As shown
in Fig. 1, there is a low level of tyrosine phosphorylation of c-CrkII
in the basal state and it has been shown that Tyr221 can
undergo phosphorylation in response to growth factors (18, 19). To
assess this and the effect of growth factor stimulation on c-CrkII
interactions, we stimulated HIRc cells with 17 nM insulin, 13 nM IGF-I, 16 nM EGF, or 10% serum followed
by immunoprecipitation with anti-CrkII antibody and Western blotting of
the precipitates with anti-pY antibody, anti-CrkII antibody,
anti-p130cas antibody, anti-paxillin antibody, or anti-c-cbl
antibody. All three growth factors caused a time-dependent
increase in c-CrkII tyrosine phosphorylation, whereas serum was without
effect (Fig. 3A).
As regards the interactions of c-CrkII, as shown in Fig. 3C,
c-CrkII strongly associates with a tyrosine-phosphorylated 130-kDa band
in the basal state, and this association is almost completely disrupted
after 5 min of insulin stimulation. p130cas blotting confirmed
that this tyrosine-phosphorylated 130-kDa molecule is p130cas
(Fig. 3D). A slight effect of IGF-I on dissociation of
c-CrkII·p130cas was also detected, and EGF caused a transient
(1 min only) effect which was coinduced with the transient appearance
of an 120-kDa tyrosine-phosphorylated band. This EGF-induced
tyrosine-phosphorylated band was identified as c-cbl by anti-c-cbl
antibody blotting (Fig. 3E). Serum stimulation had no
significant effect (Fig. 3, C-E). There were no other
detectable tyrosine-phosphorylated bands in the anti-CrkII antibody
immunoprecipitants before or after growth factor stimulation (data not shown).
We then evaluated the tyrosine phosphorylation state of c-CrkII binding
molecules by stimulation with growth factors, followed by precipitation
with anti-p130cas and anti-c-cbl antibodies and Western
blotting with anti-pY antibody. p130cas was strongly tyrosine
phosphorylated in the basal state, and insulin led to marked
dephosphorylation of this protein, which was almost complete by 5 min,
while IGF-I had a more modest effect. EGF led to a transient
dephosphorylation of p130cas, which peaked at 1 min, and
returned to basal level by 5 and 20 min (Fig.
4, right panels). Taken
together, our results indicate that c-CrkII binds to p130cas
via the CrkII SH2 domain in the basal state, and this
c-CrkII·p130cas complex can be disrupted by the tyrosine
dephosphorylation of p130cas induced by insulin, weakly by
IGF-I, and transiently by EGF stimulation.
With respect to c-cbl, insulin, IGF-I, and serum stimulation had no
effect on c-cbl tyrosine phosphorylation. EGF had a measurable, but
transient effect which peaked at 1 min (Fig. 4, left
panels), and this event may induce the formation of the
c-CrkII·c-cbl complex seen in Fig. 3, C and
E.
Effects of Microinjection of GST Fusion Proteins and Antibodies on
Cytoskeleton Organization and DNA Synthesis--
As seen in Fig. 2,
C and D, electroporation of GST-Crk-SH2 into the
cells almost completely inhibited the endogenous CrkII SH2 domain
binding to p130cas, whereas GST-Crk-(N)SH3 was less competitive
for the endogenous CrkII-(N)SH3 domain binding to C3G and to c-abl
(Fig. 2, F and G). To see if these interactions
were important for actin cytoskeleton signaling and for mitogenic
signaling, we carried out single cell microinjection studies in which
GST-Crk-SH2, GST-Crk-(N)SH3, and anti-Crk antibody were introduced into
HIRc cells followed by stimulation with insulin, IGF-I, EGF, or serum.
Following stimulation, actin cytoskeletal organization and BrdUrd
incorporation into newly synthesized DNA were monitored by
immunofluorescence staining.
As can be seen in Fig. 5, A
and B, 62% of serum-starved HIRc cells have stress fibers.
The stress fiber response to growth factor stimulation in preimmune
mouse IgG-injected cells (Fig. 5B, white bars) was quite
comparable to the changes in the association between c-CrkII and
p130cas by growth factor stimulation (Fig. 3). Microinjection
of the anti-Crk reagents induced partial stress fiber breakdown in the basal state, and even after IGF-I, EGF, or serum stimulation (Fig. 5,
A and B), while control injections of the same
concentration of GST, GST-Grb2-SH2, or GST-Grb2-(N)SH3 domain had no
effect (Fig. 5C). Insulin stimulation by itself caused
marked stress fiber breakdown, and there was no additive effect, or
inhibition, resulting from microinjection of these reagents.
Microinjection of anti-p130cas antibody also decreased actin
stress fiber formation, indicating that the p130cas·c-Crk-II
pathway is important in maintenance of the cytoskeletal structure (Fig.
5C).
We next evaluated the effect of microinjection of these anti-Crk
reagents on membrane ruffling of HIRc cells. Insulin and IGF-I led to a
membrane ruffling response in 81 and 65% of the cells, respectively
(Fig. 6, A and B),
whereas EGF and serum stimulation were without effect (data not shown).
All of the injected anti-Crk reagents inhibited membrane ruffling
induced by insulin and IGF-I (Fig. 6B). Taken together with
the data in Fig. 5, it seems likely that c-CrkII plays a role in the
signaling pathway leading to stress fiber formation and membrane
ruffling.
As can be seen in Fig. 7, none of these
anti-Crk reagents had any influence on basal rates of DNA synthesis.
Microinjection of the anti-Crk reagents did not inhibit
insulin-stimulated BrdUrd labeling, whereas all three anti-Crk reagents
partially inhibited IGF-I, EGF, and serum stimulated BrdUrd labeling
(Fig. 7, A and B). Thus, ongoing c-CrkII function
as an adapter protein is necessary for IGF-I, EGF, and serum to exert
their mitogenic stimulatory effects, but not for insulin in HIRc cells.
The adapter protein p130cas was originally identified as a
prominent tyrosine-phosphorylated protein in v-Crk- and
v-Src-transformed cells (20). p130cas contains an SH3 domain,
two proline-rich regions, and a substrate domain consisting of 15 potential SH2-binding motifs (20). In fact, 9 of these 15 tyrosine
phosphorylation sites conform to the SH2-binding motif for Crk
((p)YXXP), suggesting that Crk is the primary adapter
protein for p130cas. Paxillin also has three (p)YXXP
motifs at tyrosine positions 31, 118, and 181. Tyrosines at positions
31 and 118 of paxillin may be important in binding the v-Crk-SH2 domain
(14). In the present study, c-CrkII is shown to bind to
p130cas, but not to paxillin, although both p130cas and
paxillin are members of focal adhesions and tyrosine phosphorylated in
the basal state. c-CrkL competes with c-CrkII to bind to paxillin at
phosphorylated tyrosine residues 31 and 118 in rat uterus cells, because c-CrkL is more abundant in these cells (21). We could not
detect any association between paxillin and c-CrkII, even after growth
factor stimulation, thus, paxillin does not seem to have an important
function through binding to c-CrkII, in contrast to
p130cas·c-CrkII interaction, in HIRc cells.
Several studies have recently suggested that Crk may regulate the actin
cytoskeleton through activation of the Rho/Rac family of small GTPases
(6, 7, 22-26). In the present study, we directly demonstrated that
c-CrkII plays an important role in the cytoskeletal organization of
stress fiber formation and membrane ruffling. Both the SH2 domain and
(N)SH3 domain of c-CrkII are required for these effects. Although
c-CrkII transiently binds to c-cbl after EGF stimulation, stress fiber
formation was correlated with the association between p130cas
and c-CrkII via the SH2 domain of c-CrkII. Our results are also consistent with a role for p130cas in the maintenance of stress
fibers. Furthermore, a recent report showed that the p130cas
knockout embryo exhibits decreased stress fiber formation (27). Taken
together with the previous reports noted above, c-CrkII is now
implicated in the regulation of the Rho/Rac family of small GTPases via
p130cas and Crk-(N)SH3 interaction.
The introduction of activated Rho into cells induces tyrosine
phosphorylation of focal adhesion kinase (FAK), paxillin, and p130cas, placing one or more tyrosine kinase downstream of Rho
activity (28). Disruption of the actin cytoskeleton with cytochalasin D
inhibits p130cas tyrosine phosphorylation. Thus, Rho activation
followed by stress fiber formation seems to be essential for
p130cas tyrosine phosphorylation (29). In contrast, the
treatment of cells with a tyrosine kinase inhibitor, tyrphostin, blocks
Rho activation specifically at an upstream step (30). Thus, tyrosine kinases seem to be required at signaling steps both upstream and downstream of Rho. Recently, v-Crk was reported to activate Rho GTPase
in PC12 cells (7). Thus, it is possible that c-CrkII binds to
tyrosine-phosphorylated p130cas, and then activates Rho GTPase,
which is important in the production of phosphorylated
4,5-phosphatidylinositol (PIP2) and mediates stress fiber
formation (7). Therefore, Rho activation followed by stress fiber
formation seems to be interdependent with p130cas·c-CrkII association.
The present study strongly suggests that a p130cas·c-CrkII
complex acts in the maintenance of stress fibers. Insulin stimulation of HIRc cells induces stress fiber breakdown and dissociation of
p130cas and c-CrkII. Insulin-induced stress fiber breakdown
appears to involve the conversion from PIP2 to
PIP3 by phosphatidylinositol 3-kinase activation (31). This
conversion decreases the amount of PIP2, necessary for
stress fiber maintenance, in the plasma membrane (32). Insulin
stimulation causes c-Src inactivation through the activation of Csk and
tyrosine dephosphorylation of focal adhesion proteins (33). Because
c-Src activity is important for the tyrosine phosphorylation of
p130cas (34, 35), its inactivation may lead to tyrosine
dephosphorylation of p130cas following insulin stimulation.
Insulin-induced p130cas dephosphorylation followed by
disruption of the p130cas·c-CrkII complex may also contribute
to insulin-induced breakdown of stress fibers.
The membrane ruffling induced by insulin or IGF-I stimulation is
inhibited by microinjection of Crk inhibitory reagents. These data are
consistent with a previous report which showed that anti-Crk antibody
microinjection inhibited insulin-induced membrane ruffling (36). The
breakdown of stress fibers in the basal state induced by microinjection
of anti-Crk reagents suggests that the CrkII complex may regulate
activation of Rho GTPase, which stimulates production of
PIP2 through the activation of PIP-5-kinase (32, 37).
Membrane ruffling induced by insulin stimulation is dependent upon
phosphatidylinositol 3-kinase (38), which converts PIP2 to
PIP3. Thus, microinjection of Crk inhibitory reagents may
lead to a decrease in PIP2 in the plasma membrane through
Rho GTPase inactivation. This effect would be expected to reduce
PIP3 production, preventing membrane ruffling.
Ishiki et al. (36) reported that c-CrkII·c-cbl complex
formation induced by EGF stimulation may be important for mitogenic signaling. Our results with EGF are consistent with these observations, however, we also demonstrated that c-CrkII·c-cbl complex independent signaling in response to IGF-I or serum is also blocked by
microinjection of anti-Crk reagents (Fig. 7). This suggests that
c-CrkII plays a c-cbl-independent role in mitogenic signaling in
response to IGF-I or serum. The activation of Rho, Rac, and Cdc42,
members of Rho/Rac family of small GTPases, regulates actin
cytoskeleton organization and is required for S phase progression (39).
The present study shows that the formation of a
p130cas·c-CrkII complex, which may localize at focal
adhesions, paralleled both stress fiber formation and DNA synthesis
after growth factor stimulation in HIRc cells. Thus, a c-CrkII
containing complex may mediate signals to actin cytoskeleton
organization and to DNA synthesis through the regulation of Rho/Rac
family small GTPases.
HIRc cells possess a DNA synthesis response to 17 nM
insulin stimulation (Fig. 7), although 17 nM insulin
stimulation causes almost complete breakdown of stress fibers (Fig. 5,
A and B) and tyrosine dephosphorylation of focal
adhesions (40). Insulin receptor overexpression in 184B5 epithelial
cells led to ligand-dependent transformation, and these
cells grow in an anchorage-independent manner (41). Thus,
insulin-stimulated HIRc cells may be deficient in normal
anchorage-dependent growth control (Fig.
8). This hypothesis is consistent with
the ability of HIRc cells to retain high DNA synthesis activity in the
absence of stress fibers, which may be accomplished through the robust
activation of Shc/Grb2/Sos/Ras in this cell type (42). EGF receptor
overexpressing rat-1 fibroblasts also demonstrated growth in the
absence of stress fibers after EGF stimulation (43).
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
Tyrosine-phosphorylated p130cas was
co-immunoprecipitated with c-CrkII using two different anti-Crk
antibodies in quiescent-HIRc cells. 400 µg of HIRc cell lysate
was used for immunoprecipitation with anti-CrkII antibody (2 µg/1
mg/ml cell lysates) or anti-Crk antibody (1 µg/1 mg/ml cell lysate).
40 µg of cell lysate was loaded in the cell lysates lane for
comparison.
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Fig. 2.
Western blotting analysis of in vivo
GST fusion protein binding experiments using an in situ
electroporation system (see "Experimental Procedures").
The samples in lanes 1-3 were immunoprecipitated
(IP) with anti-CrkII antibody 12 h after in
situ electroporation of GST (lanes 1, 4, and
7), GST-Crk-SH2 (lanes 2, 5, and 8),
or GST-Crk-SH3 (lanes 3, 6, and 9). Lanes
4-6 are the precipitants with glutathione-Sepharose 4B using the
supernatants of the first immunoprecipitation with anti-Crk antibody.
40 µg of the final supernatants were then loaded in lanes
7-9. Following electrophoresis and transfer to nitrocellulose,
samples were immunoblotted with anti-CrkII antibody (A),
anti-GST antibody (B), anti-phosphotyrosine (anti-pY)
antibody (C), anti-p130cas antibody (D),
anti-paxillin antibody (E), anti-C3G antibody
(F), anti-c-abl antibody (G), or anti-Sos
antibody (H) and detected by chemiluminescence.
IgH, immunoglobulin heavy chain. Data shown are
representative of results from each of three separate
experiments.
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Fig. 3.
Time course of tyrosine phosphorylation of
c-CrkII (A), or interaction between c-CrkII and tyrosine
phosphorylated molecules (C, D, and E) after
stimulation with 17 nM insulin, 13 nM IGF-I, 16 nM EGF, or 10% fetal bovine serum in HIRc cells. Cell
lysates were immunoprecipitated with anti-CrkII antibody, and then
immunoblotted using anti-phosphotyrosine antibody (anti-pY,
A and C), anti-Crk antibody (B),
anti-p130cas antibody (D), or anti-c-cbl antibody
(E). Data shown are representative of results from each of
two separate experiments.
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Fig. 4.
Time course of tyrosine phosphorylation of
p130cas or c-cbl following stimulation by 17 nM
insulin, 13 nM IGF-I, 16 nM EGF, or 10% fetal
bovine serum in HIRc cells. Cell lysates were immunoprecipitated
with anti-p130cas antibody (A) or anti-c-cbl
antibody (B), then immunoblotted using anti-phosphotyrosine
antibody (anti-pY). Data shown are representative of results
from each of two separate experiments.
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Fig. 5.
Effect of microinjections of (A
and B) GST alone, GST-Crk-SH2 domain, (B)
GST-Crk-SH3 domain, mouse monoclonal anti-Crk antibody, (C)
GST-Grb2-SH2 domain, GST-Grb2-(N)SH3 domain, and rabbit polyclonal
anti-p130cas in HIRc cells. Serum-starved cells were
microinjected with each protein at a concentration of 5 mg/ml. After
stabilization for 1 h, cells were stimulated with the indicated
concentrations of insulin, IGF-I, EGF, or 10% fetal bovine serum for 5 min at 37 °C. Actin localization in the injected cells was
determined as described under "Experimental Procedures." Results
are expressed as the percent of total cells, and are the mean ± S.E. of three separate experiments.
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Fig. 6.
Effect of microinjections of (A
and B) GST alone, GST-Crk-SH2 domain, (B)
GST-Crk-(N)SH3 domain and anti-Crk antibody on membrane ruffling
induced by 17 nM insulin, or 13 nM IGF-I in
HIRc cells. Serum-starved cells were microinjected with each
protein at a concentration of 5 mg/ml. After stabilization for 1 h, cells were stimulated with the indicated concentrations of insulin
or IGF-I for 5 min at 37 °C. Membrane ruffling in the injected cells
was determined as described under "Experimental Procedures."
Results are expressed as the percent of total cells, and are the
mean ± S.E. of three separate experiments.
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Fig. 7.
Effect of microinjections of (A
and B) GST alone, GST-Crk-SH2 domain, (B)
GST-Crk-(N)SH3 domain and anti-Crk antibody on DNA synthesis in HIRc
cells. Serum-starved cells were microinjected as described in the
legend to Fig. 5. After stabilization for 1 h, cells were
stimulated with indicated concentrations of growth factors for 16 h at 37 °C. BrdUrd incorporation into the injected cells was
determined as described under "Experimental Procedures." Results
are expressed as the percent of total cells, and are the mean ± S.E. of three separate experiments.
DISCUSSION
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Fig. 8.
Model for the regulation of the c-CrkII
complex by growth factors and its function in HIRc cells. This
complex appears to function as a positive regulator of stress fiber
formation and DNA synthesis. Both tyrosine phosphorylation of
c-CrkII-Y221 and p130cas dissociation from c-CrkII are needed
for the interaction of phosphorylated Tyr221 with the SH2
domain, resulting in the masking of the amino-terminal SH3 domain (22).
Because insulin stimulation rapidly disrupts this complex, the
mitogenic pathway of insulin stimulation is considered not to employ
the p130cas·c-CrkII complex in HIRc cells.
We established an in vivo GST fusion protein binding system
using in situ electroporation in this study. Compared with
the transfection of GST fusion protein constructs, the advantages of
this in vivo system are the rapid and homogeneous
introduction of fusion proteins into cells, and the facility of
controlled fusion protein concentration. This system also permits
the subsequent assay of biological functions. Thus, this can be a
powerful and simple method to analyze signal transduction pathways.
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FOOTNOTES |
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* This work was supported in part by the Manpei Suzuki Diabetic Foundation (to N. N.), a grant from the Uehara Memorial Foundation (to K. E.), and National Institutes of Health Research Grant R01 DK36651 (to J. M. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Career Development Award from the American Diabetes Association.
To whom correspondence should be addressed: University of
California, San Diego, Dept. of Medicine (9111-G), 9500 Gilman
Dr., La Jolla, CA 92093. Tel.: 619-534-6651; Fax: 619-534-6653; E-mail: jolefsky{at}ucsd.edu.
The abbreviations used are: SH2, Src homology domain 2; IGF-I, insulin-like growth factor I; EGF, epidermal growth factor; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; PIP2, phosphatidylinositol 3,4-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate.
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REFERENCES |
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