From the
Insulin treatment of Chinese hamster ovary cells expressing high
levels of the human insulin receptor resulted in the tyrosine
dephosphorylation of the 125-kDa focal adhesion kinase
(pp125
Focal adhesions are regions of a cell in direct contact with the
extracellular matrix, providing the anchorage site for actin stress
fibers and forming a link between the extracellular matrix and the
actin cytoskeleton (for review, see Refs. 1-3). Several proteins
present in focal adhesion contacts have been shown to undergo tyrosine
phosphorylation (for review, see Refs. 3, 4). One such protein, the
focal adhesion kinase (pp125
Recent studies have
demonstrated that pp125
Previous studies have reported that insulin stimulation
resulted in increased membrane ruffling in both KB and Rat1 fibroblasts
(27, 28) . To investigate the molecular basis for the
insulin-mediated alteration in the actin cytoskeletal structure, we
examined the effect of insulin in CHO/IR cells. In contrast to KB and
Rat1 fibroblasts, acute insulin treatment of CHO/IR cells (100
nM for 15 min) had no effect on membrane ruffling but markedly
reduced the number and length of actin stress fibers as determined by
TRITC-phalloidin staining (Fig. 1, A and B).
Since the cytoskeletal structure of cells may differ depending upon the
extracellular matrix in contact with the cell surface membrane
(1, 30) , we also determined the effect of insulin on
the organization of filamentous actin in cells grown on collagen IV
(Fig. 1, C and D). CHO/IR cells grown on
collagen IV-coated plates displayed increased numbers of actin stress
fibers (Fig. 1 C), which were thicker and shorter than
those observed in cells grown on uncoated plates
(Fig. 1 A). Nevertheless, insulin induced a similar
decrease in actin stress fibers in the CHO/IR cells grown on collagen
IV (Fig. 1 D) compared with cells grown on uncoated
tissue culture plates (Fig. 1 B). This insulin-induced
reduction in stress fibers was also evident in cells grown on collagen
I, laminin, and fibronectin (data not shown).
Although the role of pp125
In contrast to the effect of
PDGF, insulin treatment of CHO/IR cells resulted in the tyrosine
dephosphorylation of pp125
In addition to these divergent responses to insulin and PDGF,
the effect of insulin was highly sensitive and occurred in a typical
linear dose-dependent manner, whereas PDGF displayed a biphasic
dose-response curve. Furthermore, the effect of insulin was transient,
with maximal tyrosine dephosphorylation of pp125
The
physiological role of this complex pattern of filamentous actin
reorganization and pp125
Since all
receptor tyrosine kinases, including the insulin and PDGF receptors,
appear to utilize a similar cadre of effector proteins to elicit
biological responses, the molecular basis for signal specificity has
remained obscure. The divergent, acute effects of insulin and PDGF on
actin stress fibers and pp125
We thank Tom Moninger and Randy Nessler for assistance
with the fluorescent microscopic analysis and Paul Tompach and Dr.
Alice Fulton for helpful advice on the labeling of filamentous actin.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
). The decrease in pp125
tyrosine
phosphorylation paralleled a decrease in the cellular content of actin
stress fibers, and these changes were independent of the extracellular
matrix on which the cells were grown. The reduction in both
pp125
tyrosine phosphorylation and actin stress fibers
occurred in an insulin concentration-dependent manner, with significant
effects at approximately 0.3 nM and a maximal effect at 3
nM. However, in the continuous presence of insulin, the
decreases in the tyrosine phosphorylation state of pp125
and actin stress fiber content were transient. Maximal reduction
of pp125
tyrosine phosphorylation was observed following
15 min of insulin treatment, with a return to unstimulated control
levels by 60 min. Similarly, actin stress fiber content was maximally
reduced by 15 min of insulin treatment and fully recovered by 60 min.
In contrast to insulin, platelet-derived growth factor stimulation
increased actin stress fiber content and enhanced pp125
tyrosine phosphorylation. These data demonstrate a novel
signaling role for insulin in inducing the tyrosine dephosphorylation
of pp125
and a concomitant reorganization of actin stress
fibers, which underlies at least one aspect of signaling divergence
between the insulin and platelet-derived growth factor receptor
tyrosine kinases.
),
(
)
is
a 125-kDa protein-tyrosine kinase that is itself tyrosine
phosphorylated in response to a number of stimuli
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
pp125
, in conjunction with another
tyrosine-phosphorylated focal adhesion protein, paxillin, appears to
play an important role in localizing several Src homology 2 (SH2)
domain-containing proteins such as Src, Fyn, phosphatidylinositol
3-kinase and the carboxyl-terminal Src kinase to focal adhesion
contacts
(17, 18, 19, 20, 21, 22) .
In addition to the potential role of pp125
and paxillin
as substrates for the Src-family member kinases and/or docking sites
for the phosphatidylinositol 3-kinase, these proteins may play a role
in promoting the formation of filamentous actin, since the extent of
pp125
and paxillin tyrosine phosphorylation occurs in
direct proportion to the cellular content of actin stress fibers
(11, 16, 23) .
provides an important integration
site for various extracellular signals including integrin receptor
family members, G protein-coupled receptors, and both receptor and
nonreceptor tyrosine kinases (for review, see Refs. 3 and 24). These
signals appear to increase pp125
tyrosine phosphorylation
and enhance the formation of actin stress fibers
(11, 16, 23, 25, 26) . Previous
studies have reported changes in cytoskeletal organization in KB and
Rat1 fibroblasts following insulin treatment
(27, 28) .
To investigate the role of insulin in the regulation of cytoskeletal
structure, we have examined the effect of insulin on pp125
tyrosine phosphorylation and actin stress fiber content. In this
manuscript we demonstrate that in direct contrast to platelet-derived
growth factor (PDGF), insulin stimulation of Chinese hamster ovary
cells expressing the human insulin receptor (CHO/IR) resulted in the
tyrosine-specific dephosphorylation of pp125
with a
concomitant decrease in the length and number of actin stress fibers.
This effect was transient with a complete recovery of pp125
tyrosine phosphorylation and actin stress fiber content within 1
h of continuous insulin stimulation.
Cell Culture
CHO/IR cells expressing 3
10
human insulin receptors/cell were obtained as
described previously
(29) . These cells were maintained in
minimal Eagle's medium containing nucleotides plus 10% fetal
bovine serum. CHO/IR cells were grown to confluency on either uncoated
or collagen IV-coated tissue culture plates (Collaborative Biomedical
Products, Becton-Dickinson Laboratories) as indicated in the figure
legends. To prevent any signaling events induced by the presence of
other growth factors in serum, the cells were cultured in serum-free
medium for 6 h prior to the addition of insulin or PDGF.
Immunoprecipitation of the
pp125
Whole cell extracts were prepared by
detergent solubilization in a lysis buffer (20 mM Hepes, pH
7.4, 1% Triton X-100, 3 mM MgCl, 2 mM
EDTA, 100 mM sodium fluoride, 10 mM sodium
pyrophosphate, 2 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10
µg/ml aprotinin, and 1.5 µM pepstatin) for 1 h at 4
°C. The cell extracts (
5 mg/ml in 150 µl) were then heated
at 100 °C for 5 min in the presence of 1% SDS. The samples were
diluted 10-fold (10 mM Tris, pH 7.4, 1.0% Triton X-100, 0.5%
Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 0.2 mM
sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride) and
immunoprecipitated with 4 µg of a FAK monoclonal antibody
(
FAK, Transduction Laboratories). The immunoprecipitates were
subjected to SDS-polyacrylamide gel electrophoresis and Western
blotting (Enhanced Chemiluminescence detection kit, Amersham Corp.)
using either the 4G10 monoclonal phosphotyrosine antibody (Upstate
Biotechnology Inc.), the PY20-HRP monoclonal phosphotyrosine antibody
conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.)
or the
FAK monoclonal antibody (Transduction Laboratories).
Fluorescent Labeling of Filamentous
Actin
Confluent cultures of CHO/IR cells following treatments
indicated in the figure legends were washed twice with ice-cold
phosphate-buffered saline (PBS; 6.6 mM
KHPO
, 1.5 mM
KH
PO
, pH 7.4, 2.7 mM KCl, 137
mM NaCl, 1 mM EGTA, and 2 mM
MgCl
). The cells were then fixed in 4% paraformaldehyde (in
PBS) for 20 min at 4 °C followed by quenching for 15 min at 25
°C in a solution containing 0.1 M glycine and 0.2
M Tris-HCl, pH 7.4. The fixed cells were rinsed twice in PBS
and permeabilized by incubation for 5 min at 25 °C in 0.5% Triton
X-100. The cells were then rinsed twice again with PBS and blocked with
a solution containing 0.1% bovine serum albumin in PBS for 15 min at 25
°C. Immunofluorescent labeling of filamentous actin was carried out
by incubation for 30 min at 37 °C with 0.1 mg/ml of fluorescein
isothiocyanate (FITC) or carboxytetramethylrhodamine isothiocyanate
(TRITC)-conjugated phalloidin (Sigma). Following incubation with the
fluorochrome-conjugated phalloidin, the cells were rinsed three
additional times with PBS, and the samples were mounted for viewing on
a Bio-Rad MRC 600 confocal microscope.
Figure 1:
Insulin-induced decrease in actin
stress fiber content in CHO/IR cells. CHO/IR cells were grown to
confluency on uncoated ( A and B) or collagen type
IV-coated ( C and D) tissue culture plates. The cells
were incubated without ( A and C) or with 100
nM insulin ( B and D) for 15 min at 37
°C. The cells were then fixed, detergent permeabilized, and stained
for filamentous actin using TRITC-phalloidin as described under
``Experimental Procedures.''
It has been well
documented that the formation of actin stress fibers parallels focal
adhesion formation and is accompanied by increased tyrosine
phosphorylation of pp125 and paxillin
(11, 16, 23, 26) . We therefore examined
the tyrosine phosphorylation state of pp125
following
insulin treatment of CHO/IR cells (Fig. 2 A). Compared
with unstimulated cells (Fig. 2 A, lane1), or cells treated with 0.1 nM insulin
(Fig. 2 A, lane2), there was a
detectable decrease in pp125
tyrosine phosphorylation
following treatment with 0.3 nM insulin
(Fig. 2 A, lane3). The apparent lower
level of tyrosine-phosphorylated pp125
observed in
lane1 resulted from underloading of this particular
sample as determined by
FAK Western blotting
(Fig. 2 B, lane1). Incubation of the
cells with increasing concentrations of insulin from 1 nM
(Fig. 2 A, lane4) to 3 nM
(Fig. 2 A, lane5) resulted in a
progressive decline in the extent of pp125
tyrosine
phosphorylation. Maximal reduction of tyrosine phosphorylation occurred
at 10 nM insulin (Fig. 2 A, lane6) and was not further affected by treatment with higher
concentrations (30 nM) of insulin (Fig. 2 A,
lane7). This decrease in phosphotyrosine content
occurred with no significant alteration in the amount of pp125
protein immunoprecipitated under these conditions
(Fig. 2 B).
Figure 2:
Concentration dependence of
insulin-stimulated pp125 tyrosine dephosphorylation.
CHO/IR cells were grown to confluency on uncoated tisssue culture
plates and incubated in the absence ( lane1) or
presence of 0.1 ( lane2), 0.3 ( lane3), 1 ( lane4), 3 ( lane5), 10 ( lane6), and 30 nM
( lane7) insulin for 10 min at 37 °C. Cell
extracts were prepared and subjected to immunoprecipitation with a
monoclonal antibody (
FAK) directed against pp125
as described under ``Experimental Procedures.''
A, the
FAK immunoprecipitates were then Western blotted
with an antibody directed against phosphotyrosine ( 4G10).
B, the Western blot transfer membrane that was probed with the
4G10 antibody in A was stripped and probed with the monoclonal
antibody (
FAK) directed against
pp125
.
In agreement with the dose response
relationship of pp125 tyrosine dephosphorylation, the
decrease in TRITC-phalloidin-labeled actin stress fibers was also
observed to be insulin concentration-dependent (Fig. 3), with a
dose response paralleling that observed for tyrosine dephosphorylation
of pp125
. There was a small but significant reduction in
the length and number of actin stress fibers at 0.1 nM insulin
(Fig. 3 C) compared with untreated cells
(Fig. 3 A) or cells treated with 0.03 nM insulin
(Fig. 3 B). A marked decrease in filamentous actin was
observed following incubation with 1 nM insulin, and maximal
reduction occurred at 3 nM insulin (Fig. 3, D and E). Treatment of CHO/IR cells with 10 (data not
shown), 30 (Fig. 3 F), or 100 nM insulin (data
not shown) had no additional effect on the disassembly of actin stress
fibers.
Figure 3:
Insulin dose-dependent decrease of actin
stress fiber content. CHO/IR cells were grown to confluency on uncoated
tissue culture plates and incubated in the absence ( panelA) or in the presence of 0.03 ( panelB), 0.1
( panelC), 1.0 ( panelD), 3
( panelE), and 30 ( panelF)
nM insulin for 15 min at 37 °C. The cells were then fixed,
detergent permeabilized, and TRITC-phalloidin labeled for actin stress
fibers as described under ``Experimental
Procedures.''
To further characterize the tyrosine dephosphorylation of
pp125, we examined the time-dependence of this effect
following insulin treatment (Fig. 4). At saturating insulin
concentrations (100 nM), pp125
tyrosine
dephosphorylation was initially detected by 2 min
(Fig. 4 A, lane2). There was a
progressive decline in pp125
tyrosine phosphorylation
that reached a maximum at 10-15 min of insulin treatment
(Fig. 4 A, lanes4 and 5).
However, the insulin-mediated decrease in pp125
tyrosine
phosphorylation was transient and began to recover 30 min subsequent to
the addition of insulin (Fig. 4 A, lane6). Following 60 min of insulin treatment, the tyrosine
phosphorylation state of pp125
was similar to that
observed for the unstimulated control CHO/IR cells
(Fig. 4 A, compare lanes1 and
7). To insure that the observed changes in phosphotyrosine
blotting of pp125
did not result from alterations in
pp125
expression or differential immunoprecipitation, the
FAK immunoprecipitates were subjected to Western blotting with the
FAK antibody (Fig. 4 B). During this period of
insulin treatment (0-60 min), there was no significant alteration
in the amount of immunoprecipitated pp125
. Thus, the
insulin-mediated decrease and subsequent recovery of pp125
phosphorylation observed in the phosphotyrosine immunoblot was a
direct result of changes in the tyrosine phosphorylation state of
pp125
.
Figure 4:
Time dependence of insulin-stimulated
pp125 tyrosine dephosphorylation. CHO/IR cells were grown
to confluency on uncoated tissue culture plates and incubated in the
absence ( lane1) or presence of 100 nM
insulin for 2 ( lane2), 5 ( lane3),
10 ( lane4), 15 ( lane5), 30
( lane6), and 60 ( lane7) min at 37
°C. Cell extracts were prepared and subjected to
immunoprecipitation with a monoclonal antibody (
FAK)
directed against pp125
. A, the
immunoprecipitates were then Western blotted with an antibody directed
against phosphotyrosine ( 4G10) as described under
``Experimental Procedures.'' B, the Western blot
transfer membrane that was probed with the 4G10 antibody in panelA was stripped and probed with the monoclonal antibody
(
FAK) directed against
pp125
.
Consistent with the time-dependent change in
pp125 tyrosine phosphorylation (Fig. 4), there was
a concomitant decrease in the number and length of actin stress fibers,
which was followed by recovery to normal levels (Fig. 5,
A-F). We consistently observed a small but significant
decline in stress fibers following 3 min of insulin treatment
(Fig. 5 B) compared with unstimulated control cells
(Fig. 5 A). However, there was a marked reduction in
FITC-phalloidin labeling following 5 min of insulin treatment
(Fig. 5 C). A maximal decrease in actin stress fibers was
routinely detected after 15 min of insulin exposure
(Fig. 5 D). In addition, in the continuous presence of
insulin, restoration of the actin stress fiber network was apparent
within 30-60 min (Fig. 5, E and F). These
time-dependent alterations in actin stress fibers closely paralleled
the changes in the tyrosine phosphorylation state of pp125
(Fig. 4). To insure that the transient nature of actin
stress fiber rearrangement and pp125
tyrosine
phosphorylation was not due to depletion of the exogenously added
insulin, we performed radioimmunoassay analyses of the culture media.
These data demonstrated that insulin levels decreased from 100 to 50
nM following the 1-h incubation time (data not shown). Thus,
the concentration of insulin remaining in the media at 60 min was more
than sufficient to maximally stimulate acute tyrosine dephosphorylation
of pp125
and reduce the cellular content of actin stress
fibers (Figs. 2 and 3).
Figure 5:
Time course of insulin treatment on actin
stress fiber content. CHO/IR cells were grown to confluency on collagen
IV-coated tissue culture plates and incubated in the absence ( panelA) or in the presence of 100 nM insulin for 3
( panelB), 5 ( panelC), 15
( panelD), 30 ( panelE), and 60
( panelF) min at 37 °C. The cells were then
fixed, detergent permeabilized, and FITC-phalloidin-labeled for actin
stress fibers as described under ``Experimental
Procedures.''
Previous studies have demonstrated that
various extracellular signals increase pp125 tyrosine
phosphorylation and enhance the formation of actin stress fibers
(11, 16, 23, 25, 26) . In
particular, low concentrations of PDGF (
0.2 nM) have been
observed to increase the cellular content of actin stress fibers and
pp125
tyrosine phosphorylation. Paradoxically, high
concentrations of PDGF (>1.2 nM) have been reported to have
no effect or to reduce the cellular levels of filamentous actin and
tyrosine-phosphorylated pp125
(16, 31) .
We therefore directly compared the tyrosine phosphorylation state of
pp125
in cells stimulated with insulin and PDGF
(Fig. 6). As previously observed, stimulation of CHO/IR cells
with 100 nM insulin for 15 min decreased in the extent of
tyrosine-phosphorylated pp125
(Fig. 6 A,
lanes1 and 2). In direct contrast,
incubation with 0.2 nM (5 ng/ml) PDGF for 15 min resulted in
an increase in pp125
tyrosine phosphorylation
(Fig. 6 A, lane3), whereas 0.4
nM (10 ng/ml) and 1.2 nM (30 ng/ml) PDGF had little
effect on the extent of tyrosine-phosphorylated pp125
(Fig. 6 A, lanes4 and
5). Although the relative increase in pp125
tyrosine phosphorylation at 0.2 nM (5 ng/ml) PDGF was
small, it was highly reproducible (1.56 ± 0.04, p <
0.01 compared with control, n = 3). To control for the
efficiency of pp125
immunoprecipitation, we also
determined the amount of pp125
by
FAK immunoblot
analyses. Under these conditions, there were no significant differences
in pp125
protein levels (Fig. 6 B,
lanes1-5).
Figure 6:
Comparison between insulin and PDGF
stimulation of the tyrosine phosphorylation state of
pp125. CHO/IR cells were grown to confluency on uncoated
tisssue culture plates and incubated in the absence ( lane1) or presence of 100 nM insulin ( lane2), 0.2 nM PDGF (5 ng/ml, lane3), 0.4 nM PDGF (10 ng/ml, lane4), or 1.2 nM PDGF (30 ng/ml, lane5) for 15 min at 37 °C. Cell extracts were prepared
and subjected to immunoprecipitation with a monoclonal antibody
(
FAK) directed against pp125
as described
under ``Experimental Procedures.'' A, the
FAK
immunoprecipitates were then Western blotted with an antibody directed
against phosphotyrosine ( PY20- HRP). B, the
Western blot transfer membrane that was probed with the PY20-HRP
antibody in panelA was stripped and probed with the
monoclonal antibody (
FAK) directed against
pp125
. Please note that the higher background observed in
panelB compared with Figs. 2 and 4 resulted from use
of the PY20-HRP antibody in the initial immunoblot. Although PY20-HRP
resulted in a lower background for the phosphotyrosine immunoblot
( A), this antibody cannot be completely removed for the second
FAK immunoblot ( B).
We next determined the effect of
PDGF treatment on the cellular content of actin stress fibers in
comparison with that of insulin (Fig. 7). As observed in Figs. 3
and 5, incubation with insulin for 15 min decreased the length and
number of actin stress fibers (Fig. 7, A and
B). In contrast, incubation with 0.2 nM (5 ng/ml)
PDGF enhanced the actin stress fiber content of the CHO/IR cells
(Fig. 7 C). Consistent with the inability of 1.2
nM (30 ng/ml) PDGF to effect pp125 tyrosine
phosphorylation (Fig. 6), this concentration had no significant
effect on the actin cytoskeleton compared with untreated cells
(Fig. 7 D). These data directly demonstrated that the
identical CHO/IR cell population responded to insulin and low
concentrations of PDGF in an opposite manner with respect to
pp125
tyrosine phosphorylation and actin stress fiber
formation.
Figure 7:
Effect of insulin and PDGF treatment on
actin stress fiber content. CHO/IR cells were grown to confluency on
uncoated tissue culture plates and incubated in the absence ( panelA) or in the presence of 100 nM insulin
( panelB), 0.2 nM PDGF (5 ng/ml, panelC), or 1.2 nM (30 ng/ml, panelD) for 15 min at 37 °C. The cells were then fixed,
detergent permeabilized, and FITC-phalloidin-labeled for actin stress
fibers as described under ``Experimental
Procedures.''
in growth factor
signaling has not been elucidated to date, it appears to be an
important integration point for the actions of G protein-coupled
receptors, the non-tyrosine kinase integrin receptor family members,
and both receptor and nonreceptor tyrosine kinases
(3, 24) . The enhanced tyrosine phosphorylation of
pp125
induced by these signaling pathways parallels an
increase in the formation of filamentous actin structures
(11, 16, 23, 26) . In particular,
stimulation of Swiss 3T3 fibroblasts with low concentrations of PDGF
(
5 ng/ml) was observed to enhance pp125
tyrosine
phosphorylation and actin stress fiber formation
(16, 31, 32) . Interestingly, the effect of PDGF
appeared to be biphasic with high concentrations of PDGF (>1.2
nM) having either no or an inhibitory effect
(16, 32) . We have confirmed these observations in
CHO/IR cells stimulated with both low and high concentrations of PDGF.
Under our experimental conditions, CHO/IR cells incubated with 0.2
nM (5 ng/ml) PDGF for 15 min stimulated both pp125
tyrosine phosphorylation and actin stress fiber formation,
whereas incubation of cells with 1.2 nM (30 ng/ml) PDGF had no
detectable effect on pp125
tyrosine phosphorylation and
little effect on actin stress fibers.
that correlated with a
decrease in the cellular content of actin stress fibers. Thus,
activation of the insulin or PDGF receptor tyrosine kinases resulted in
diametrically opposite signaling events in terms of pp125
tyrosine phosphorylation and the reorganization of filamentous
actin.
and
reduction in actin stress fibers occurring at 15 min. However,
following 60 min in the continuous presence of insulin, both
pp125
tyrosine phosphorylation and actin stress fibers
recovered to the unstimulated state. Although 0.2 nM (5 ng/ml)
PDGF increased actin stress fibers, we have also observed that this
response occurred in a transient manner with the near complete return
of actin stress fiber content to the basal state following 60 min of
PDGF addition (data not shown). These data demonstrate that the acute
effects of insulin and PDGF were divergent in terms of pp125
tyrosine phosphorylation and assembly of actin stress fibers.
However, in each case the cells ultimately recovered to steady-state
levels that were similar to the unstimulated state.
tyrosine phosphorylation is
completely unknown. In addition to the structural role for the actin
cytoskeleton and its involvement in cellular adhesion, it may also
provide important elements necessary for mediating metabolic and/or
mitogenic actions of various growth factors. In this regard, it has
recently been suggested that the actin cytoskeleton is an essential
component necessary for the insulin-stimulated translocation of GLUT4
protein-containing intracellular vesicles
(33) .
tyrosine phosphorylation is
likely to underlie an important component of receptor tyrosine kinase
signaling specificity. In future studies, analyses of the signaling
pathways that couple to these events will not only elucidate the role
of these events in biological responsiveness, but they will also
provide important information defining the molecular basis of tyrosine
kinase receptor signaling specificity.
, 125-kDa focal adhesion kinase; PDGF,
platelet-derived growth factor; CHO/IR, Chinese Hamster ovary cells
expressing the human insulin receptor; SH2, Src homology 2 domain; PBS,
phosphate-buffered saline; TRITC-phalloidin;
car-boxytetramethylrhodamine isothiocyanate-conjugated phalloidin;
FITC-phalloidin, fluorescein isothiocyanate-conjugated phalloidin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.