(Received for publication, October 31, 1994; and in revised form, January 20, 1995)
From the
Addition of guanosine 5` - 3 - O - (thio) triphosphate
(GTPS) to streptolysin O-permeabilized Swiss 3T3 cells
induced tyrosine phosphorylation of M
110,000-130,000 and 70,000-80,000 bands. Specifically,
GTP
S stimulated tyrosine phosphorylation of both focal adhesion
kinase (p125
) and paxillin. GTP
S induced tyrosine
phosphorylation was dose-dependent (EC
of 2.5
µM) and reached maximum levels after 1.5 min for the M
110,000-130,000 band and 2 min for the M
70,000-80,000 paxillin band. Guanosine
5`-O-(2-thiodiphosphate) inhibited GTP
S-induced tyrosine
phosphorylation with an IC
of 100 µM. Protein
kinase C did not mediate GTP
S-induced tyrosine phosphorylation.
Varying the Ca
concentration from 0 to 6 µM did not increase tyrosine phosphorylation above basal levels and
did not affect the ability of GTP
S to induce tyrosine
phosphorylation. GTP
S was able to stimulate tyrosine
phosphorylation in the presence of nanomolar concentrations of
Mg
. Furthermore, 30 µM AlF
only weakly induced tyrosine
phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates
p21
, markedly reduced the ability of GTP
S
to stimulate tyrosine phosphorylation of M
110,000-130,000 and 70,000-80,000 bands including
p125
and paxillin in permeabilized Swiss 3T3 cells.
Furthermore, a peptide of p21
(p21
) inhibited
GTP
S-induced tyrosine phosphorylation in a dose-dependent manner
(IC
1 µM). This peptide also inhibited
tyrosine phosphorylation of p125
and paxillin. In
contrast, 20 µM p21
peptide failed to inhibit GTP
S-induced tyrosine
phosphorylation. Using permeabilized cells, our findings demonstrate
that GTP
S stimulates tyrosine phosphorylation of p125
and paxillin and that a functional p21
is
implicated in this process.
Tyrosine phosphorylation has recently been implicated in the
intracellular signaling of neuropeptides that act as potent cellular
growth factors through receptors with seven transmembrane helices (1, 2, 3, 4, 5, 6, 7, 8) .
Bombesin and other mitogenic neuropeptides stimulate tyrosine
phosphorylation of multiple proteins in Swiss 3T3 cells, a useful model
system for the elucidation of signal transduction pathways leading to
cell proliferation(9) . The tyrosine-phosphorylated proteins
include broad bands of M 110,000-130,000 and
70,000-80,000(2, 4, 10) . The focal
adhesion associated protein p125
, (
)a novel
cytosolic tyrosine kinase which lacks Src homology domains 2 and
3(11, 12) , has been identified as a prominent
tyrosine-phosphorylated protein migrating within the M
110,000-130,000 band stimulated by neuropeptides in Swiss
3T3 cells(6, 13) . Paxillin, another focal adhesion
associated protein (14, 15) has been shown to comprise
the M
70,000-80,000 tyrosine-phosphorylated
protein band stimulated by neuropeptides in these cells(16) .
The rapidity of neuropeptide-stimulated tyrosine phosphorylation is
consistent with p125
and paxillin functioning in a
neuropeptide-activated tyrosine kinase pathway.
Recent evidence
demonstrates that a variety of other agents that modulate cell growth
and differentiation including platelet-derived growth
factor(17) , the bioactive lipid
LPA(18, 19, 20) , sphingosine(21) ,
tumor promoting phorbol esters(13) , extracellular matrix
proteins (22, 23, 24, 25, 26) , and
transforming variants of
pp60(23, 27) , induce
coordinated tyrosine phosphorylation of p125
and
paxillin. In all cases, the induction of tyrosine phosphorylation of
these proteins required the integrity of the actin
cytoskeleton(13, 17, 20, 21, 25) .
Furthermore, both tyrosine phosphorylation and the cytoskeletal changes
induced by LPA and bombesin require functional p21
protein(20, 28, 29, 30) .
These findings support the existence of a tyrosine kinase pathway
involving p125
and paxillin, but the components of this
signal transduction pathway and their upstream and downstream
interactions have not been fully identified.
Cell permeabilization
has provided a useful approach to study protein phosphorylation and
also to introduce guanine nucleotides to assess the contribution of G
proteins in signal transduction in Swiss 3T3 cells (31, 32, 33) . However, tyrosine
phosphorylation of p125 and paxillin has not been
demonstrated in any permeabilized cell preparation. In the present
study, we show that the nonhydrolyzable GTP analogue, GTP
S,
induced tyrosine phosphorylation of multiple proteins including
p125
and paxillin in SLO permeabilized Swiss 3T3 cells.
Our results suggest that p21
predominantly
mediates this process.
Figure 1:
A, identification of proteins which are
tyrosine phosphorylated by GTPS in permeabilized Swiss 3T3 cells.
Confluent and quiescent cultures of Swiss 3T3 cells in 33-mm dishes
were washed twice in DMEM and then incubated in permeabilization
solution containing 0.4 IU/ml SLO for 1 min. The incubation was
continued for a further 1.5 min in the presence or absence of 5
µM GTP
S as described under ``Experimental
Procedures.'' The cultures were then lysed and the lysates
immunoprecipitated with the anti-Tyr(P) mAb Py72 (left, PY), p125
mAb2A7 (middle, FAK), or anti-paxillin mAb 165 (right, PAX). The
immunoprecipitates were analyzed by immunoblotting with a 1:1 mixture
of 4G-10 and Py20 anti-Tyr(P) mAbs. Autoradiographs shown are
representative of at least two independent experiments. B, time course GTP
S-stimulated tyrosine phosphorylation.
Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM
and permeabilized with 0.4 IU/ml SLO for 1 min prior to addition of 5
µM GTP
S. The cultures were then lysed and
immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by
anti-Tyr(P) immunoblotting. Scanning densitometry of both the M
110,000-130,000 (circles) and
70,000-80,000 (triangles) bands is shown. Each point is
representative of three independent experiments ± S.E. and is
expressed as a percentage of the maximum response. C, dose
response of GTP
S-induced tyrosine phosphorylation. Cultures of
Swiss 3T3 cells were permeabilized for 1 min and incubated in the
presence or absence of increasing concentrations of GTP
S for a
further 1.5 min before lysis. Scanning densitometry of both the M
110,000-130,000 (circles) and
70,000-80,000 (triangles) bands is shown. Each point is
representative of three independent experiments and is expressed as a
percentage of the maximum response. For clarity, only the error bars
(S.E.) for the M
110,000-130,000 band are
shown.
The pattern
of tyrosine phosphorylation induced by GTPS in permeabilized Swiss
3T3 cells was identical to that previously shown to be induced by
bombesin and other agents (see Introduction for details) in intact
cells. These stimuli are known to increase tyrosine phosphorylation of
p125
, which migrates within the M
110,000-130,000, and paxillin which corresponds to the M
70,000-80,000 band. We therefore examined
whether GTP
S also stimulates tyrosine phosphorylation of these
proteins. Accordingly, lysates of permeabilized Swiss 3T3 cells
stimulated with 5 µM GTP
S were immunoprecipitated
with either anti-p125
or paxillin mAbs and the resulting
immunoprecipitates were Western blotted with anti-Tyr(P) mAbs. As shown
in Fig. 1A, GTP
S markedly stimulated tyrosine
phosphorylation of both p125
(5 ± 2-fold) and
paxillin (4 ± 1.2-fold) in permeabilized cells.
Tyrosine
phosphorylation was a rapid consequence of the addition of GTPS to
permeabilized Swiss 3T3 cells. Fig. 1B demonstrates an
increase in tyrosine phosphorylation of the M
110,000-130,000 group of bands after 45 s. Maximum tyrosine
phosphorylation of this band was reached after 1.5 min of incubation
with GTP
S. Tyrosine phosphorylation of the M
70,000-80,000 band, corresponding to paxillin, was delayed
by 30 s, reaching a maximum 2 min after addition of GTP
S (Fig. 1B). GTP
S stimulated tyrosine
phosphorylation of both the M
110,000-130,000 and 70,000-80,000 bands in a
dose-dependent fashion with an identical EC
of 2.5
µM. Maximum tyrosine phosphorylation was achieved at 5
µM GTP
S (Fig. 1C).
Addition of
GDPS inhibited tyrosine phosphorylation of both the M
110,000-130,000 and 70,000-80,000
bands induced by 5 µM GTP
S in a dose-dependent manner
with an IC
of 100 µM (Fig. 2).
Increasing the concentration of GTP
S to 500 µM almost
completely reversed (90%) the inhibitory effect of 250 µM GDP
S, a concentration which reduced tyrosine phosphorylation
stimulated by 5 µM GTP
S by 75% (Fig. 2, inset). These results suggest that a G protein is involved in
tyrosine phosphorylation stimulated by GTP
S in permeabilized Swiss
3T3 cells.
Figure 2:
Effect of GDPS on tyrosine
phosphorylation stimulated by GTP
S. Cultures of Swiss 3T3 cells in
33-mm dishes were washed twice in DMEM and permeabilized with 0.4 IU/ml
SLO in the presence or absence of increasing concentrations of
GDP
S. After 1 min 5 µM GTP
S was added to the
dishes and the incubation continued for a further 1.5 min. The cultures
were then lysed and immunoprecipitates of tyrosine-phosphorylated
proteins were analyzed by anti-Tyr(P) immunoblotting. Scanning
densitometry of both the M
110,000-130,000 (circles) and 70,000-80,000 (triangles) bands
is shown. Each point is representative of three independent experiments
and is expressed as a percentage of the maximum response. For clarity,
only the error bands (S.E.) for the M
110,000-130,000 are shown. Inset, parallel
cultures were permeabilized in the presence (lane 2) or
absence (lane 3) of 250 µM GDP
S for 1 min
prior to addition of 500 µM GTP
S. Lane 1 shows the effect of 250 µM GDP
S alone. An
autoradiograph of a representative experiment is
shown.
Figure 3:
Panel
A, role of PKC in GTPS-stimulated tyrosine phosphorylation.
Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM
and incubated in the absence or presence of 3.5 µM GF109203X for 1 h. The cells were then permeabilized with SLO in
the absence or presence (GF) of 3.5 µM GF109203X
for 1 min and the incubation was continued with (+) or
without(-) addition of 5 µM GTP
S for a further
1.5 min or 200 nM PDBu for a further 4 min. Parallel dishes
were pretreated either in the absence or presence (PDBu) of 800 nM PDBu for 40 h prior to permeabilization and stimulation with
(+) or without(-) 5 µM GTP
S or 200 nM PDBu. The cultures were then lysed and immunoprecipitates of
tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P)
immunoblotting. An autoradiograph of a representative experiment is
shown. In parallel cultures, both pretreatment with 3.5 µM GF109203X and PDBu completely blocked tyrosine phosphorylation
induced by 200 nM PDBu in intact cells (data not shown) in
agreement with results published
previously(13, 16, 20, 21) . Panel B, effect of Ca
concentration on
tyrosine phosphorylation in either resting or GTP
S-stimulated
permeabilized Swiss 3T3 cells. Cultures of Swiss 3T3 cells in 33-mm
dishes were washed twice in DMEM and permeabilized with SLO in the
presence or absence of increasing concentrations of Ca
as described under ``Experimental Procedures.'' After 1
min, 5 µM GTP
S was (solid bars) or was not (open bars) added for a further 1.5 min. The cultures were
then lysed and immunoprecipitates of tyrosine-phosphorylated proteins
were analyzed by anti-Tyr(P) immunoblotting. The bar graph shown represents scanning densitometry from autoradiographs of
tyrosine-phosphorylated proteins migrating with an apparent M
110,000-130,000 and is expressed as a
percentage of the maximum response of three independent experiments
± S.E.
The induction of tyrosine phosphorylation in
some cell systems has been shown to be dependent on Ca oscillations(5, 46) . Permeabilization provides
a convenient procedure to examine directly the effects of increasing
concentrations of Ca
on tyrosine phosphorylation.
Cells were permeabilized for 1 min in permeabilization solution which
contained increasing concentrations of Ca
and then
incubated in the presence or absence of 5 µM GTP
S for
1.5 min prior to lysis. Fig. 3B shows that increasing
Ca
concentrations did not induce any significant
tyrosine phosphorylation in the absence of GTP
S. This was true
even at 6 µM Ca
, a concentration known
to directly activate PIP
-PLC in permeabilized Swiss 3T3
cells(32) . Furthermore, raising the Ca
concentration up to 600 nM had no effect on the ability
of GTP
S to stimulate tyrosine phosphorylation of the M
110,000-130,000 band in permeabilized
Swiss 3T3 cells. Interestingly, at 6 µM Ca
, some inhibition of GTP
S-stimulated
tyrosine phosphorylation was seen. Thus, increasing Ca
concentrations do not directly stimulate tyrosine phosphorylation
or affect GTP
S-induced tyrosine phosphorylation in permeabilized
Swiss 3T3 cells.
Figure 4:
Role of p21 as a
mediator of GTP
S-stimulated tyrosine phosphorylation: effect of C3
exoenzyme pretreatment. Swiss 3T3 cells in 33-mm dishes were incubated
in DMEM supplemented with 10% fetal bovine serum in the presence
(+) or absence(-) of C3 exoenzyme for 48 h at 7.5 µg/ml
as described under ``Experimental Procedures.'' The cells
were then washed twice with DMEM and treated for a further 24 h with 15
µg/ml C3 exoenzyme in DMEM:Waymouth's (1:1, v/v) prior to
permeabilization with SLO for 1 min and then incubation for another 1.5
min in the presence (+) or absence(-) of 5 µM GTP
S as indicated. The cultures were then lysed and the
lysates immunoprecipitated with anti-Tyr(P) mAb Py72 (upper),
anti-p125
mAb2A7 (middle), or anti-paxillin
mAb165 (lower). The immunoprecipitates were analyzed by
immunoblotting with anti-Tyr(P) mAbs. An autoradiograph of a
representative experiment is shown.
To obtain further evidence that p21 was involved in tyrosine phosphorylation stimulated by GTP
S,
we synthesized a peptide corresponding to the effector domain of
p21
(p21
). This
approach was based on previous work demonstrating that amino acid
residues within p21
are necessary
for actin reorganization (53) and that an effector domain
peptide of p21
(p21
)
blocks the interaction of p21
with
p74
(54) . Cells were permeabilized in the
presence or absence of 20 µM p21
peptide for 1 min and then
exposed to 5 µM GTP
S for a further 1.5 min. Fig. 5A (top) shows that tyrosine phosphorylation of
both the M
110,000-130,000 and
70,000-80,000 bands induced by GTP
S was completely inhibited
by p21
. In contrast, addition of
p21
at 20 µM did not
affect GTP
S-stimulated tyrosine phosphorylation. Importantly, Fig. 5, top, also demonstrates that 20 µM p21
could specifically inhibit
p125
(middle panel) and paxillin (lower
panel) tyrosine phosphorylation induced by GTP
S. However,
addition of an identical concentration of
p21
failed to inhibit p125
and paxillin tyrosine phosphorylation stimulated by GTP
S.
Both p21
and
p21
at concentrations up to 20
µM did not affect tyrosine phosphorylation in the absence
of GTP
S. The inhibitory effect of
p21
was dose dependent with an
IC
of 1 µM (Fig. 5, bottom).
These results provide additional evidence that p21
predominantly mediates the stimulatory effect of GTP
S on
tyrosine phosphorylation.
Figure 5:
Effect of p21 and
p21
effector peptides on GTP
S-stimulated
tyrosine phosphorylation. Upper panel, identification of
tyrosine-phosphorylated proteins which are inhibited by the
p21
effector peptide. Cultures of Swiss 3T3
cells in 33-mm dishes were washed twice in DMEM and permeabilized with
SLO in the presence or absence of 20 µM of either the
p21
(Rho) or the
p21
(Ras)
effector peptides for 1 min prior to incubation for a further 1.5 min
in the presence or absence of 5 µM GTP
S. The cells
were then lysed and the lysates immunoprecipitated with anti-Tyr(P) mAb
Py72 (upper), anti-p125
mAb2A7 (middle), or anti-paxillin mAb165 (lower). The
immunoprecipitates were immunoblotted with anti-Tyr(P) mAbs.
Autoradiographs shown are representative of at least two independent
experiments. Lower panel, dose response of
rhop21
effector peptide on GTP
S-stimulated
tyrosine phosphorylation. Identical cultures were permeabilized in the
presence of increasing concentrations of the
p21
effector peptide (closed symbols) or 20 µM p21
effector peptide (open symbols) for 1 min prior to incubation for another 1.5
min in the presence or absence of 5 µM GTP
S. The
cells were then lysed and immunoprecipitates of tyrosine-phosphorylated
proteins were analyzed by anti-Tyr(P) immunoblotting. Scanning
densitometry of both the M
110,000-130,000 (circles) and 70,000-80,000 (triangles) bands
is shown. Each point is representative of two independent experiments
and is expressed as a percentage of the maximum
response.
We verified that the effector peptides
were not inhibiting bombesin-stimulated tyrosine phosphorylation in a
cell-free kinase assay. Confluent and quiescent cultures of Swiss 3T3
cells were incubated in the presence or absence of 10 nM bombesin for 10 min, a concentration and time known to induce
maximum tyrosine phosphorylation(10) . The cells were then
lysed and the lysates immunoprecipitated with anti-mouse IgG
agarose-linked mAbs directed against Py72 for 4 h. A cell-free in
vitro kinase assay was performed in the presence or absence of
either 20 µM p21 or
p21
peptides. Neither peptide
affected basal or bombesin-stimulated phosphorylation of proteins in
this kinase assay (data not shown).
We demonstrate for the first time that GTPS can rapidly
stimulate tyrosine phosphorylation of M
110,000-130,000 and 70,000-80,000 bands in
permeabilized cells. The pattern of GTP
S-induced tyrosine
phosphorylation is similar to that recently reported for neuropeptides (2, 6, 13, 16) and LPA (20) in intact cells. Indeed, GTP
S specifically induces
tyrosine phosphorylation of the focal adhesion associated proteins,
p125
, and paxillin. Interestingly, tyrosine
phosphorylation of the M
70,000-80,000
paxillin band lagged behind the M
110,000-130,000 band by 30 s which would be consistent with
previous in vitro evidence suggesting that paxillin is a
substrate of p125
(55) .
GTPS-stimulated
tyrosine phosphorylation could be inhibited by GDP
S in a
dose-dependent fashion. This indicates that GTP
S-stimulated
tyrosine phosphorylation is mediated by a G protein. We have previously
shown that GTP
S can activate PIP
-PLC in permeabilized
Swiss 3T3 cells(31, 32, 33) .
PIP
-PLC catalyzes the hydrolysis of inositol phospholipids
into diacylglycerol and inositol 1,4,5-trisphosphate which activate PKC
and mobilize Ca
, respectively(56) .
Activation of PKC by phorbol esters or membrane permeable
diacylglycerols has been shown to induce tyrosine phosphorylation of
p125
and paxillin in intact Swiss 3T3
cells(13, 16) . It was therefore possible that
GTP
S stimulates tyrosine phosphorylation via heterotrimeric G
protein mediated activation of PKC. In this respect, integrin-induced
stimulation of p125
tyrosine phosphorylation appears to
be mediated by PKC(26) . In contrast, our results show that
GTP
S stimulates tyrosine phosphorylation of both the M
110,000-130,000 band and
70,000-80,000 paxillin band through a PKC-independent pathway as
judged by the fact that neither down-regulation nor inhibition of PKC
prevented GTP
S-mediated tyrosine phosphorylation.
In liver
epithelial cells, angiotensin II increases tyrosine phosphorylation of
cellular components, including a M 125,000 band,
apparently through a Ca
-dependent
pathway(5) . It has also been reported that the induction of
Ca
oscillations are required for GPIIb-IIIa-induced
tyrosine phosphorylation of a M
125,000 protein in
cells expressing this integrin(46) . It is likely but unproven
that these M
125,000 bands are related to
p125
. In view of these findings we tested whether
Ca
could stimulate tyrosine phosphorylation in
permeabilized Swiss 3T3 cells. Our results show that increasing the
Ca
concentration in the absence of GTP
S, to
mimic Ca
mobilization from internal stores, failed to
increase tyrosine phosphorylation over background levels. Furthermore,
the presence or absence of Ca
had little effect on
GTP
S-induced tyrosine phosphorylation. These results strongly
suggest that tyrosine phosphorylation occurs via a Ca
independent pathway in permeabilized Swiss 3T3 cells. This is in
agreement with our previous reports showing that neuropeptides, LPA,
and sphingosine induce tyrosine phosphorylation via a PKC- and
Ca
-independent pathway in intact Swiss 3T3
cells(6, 13, 16, 17, 20, 21) .
GTPS has been shown to bind and irreversibly activate both
heterotrimeric and small GTP binding proteins. It was therefore
important to assess the contribution of these two classes of G proteins
in mediating GTP
S-induced tyrosine phosphorylation. The fact that
GTP
S could induce tyrosine phosphorylation almost to the same
degree at nanomolar and millimolar concentrations of Mg
suggests that a small GTP binding protein predominantly mediates
GTP
S-induced tyrosine
phosphorylation(37, 47, 48) . This data is
supported by the fact that AlF
, a direct
activator of heterotrimeric G proteins(49) , only weakly
stimulated tyrosine phosphorylation in permeabilized Swiss 3T3 cells.
Tyrosine phosphorylation of p125 and paxillin
stimulated by bombesin, LPA, and sphingosine is closely related to
changes in the organization of actin microfilaments induced by these
ligands in Swiss 3T3 cells. Disruption of the cytoskeleton with
cytochalsin D inhibits the ability of these agents to stimulate
tyrosine
phosphorylation(13, 16, 20, 21) .
Furthermore, both tyrosine phosphorylation and the cytoskeletal changes
induced by LPA and bombesin require functional p21
protein(20, 28, 29) . Consequently, it
was of interest to determine whether p21
mediated
GTP
S-induced tyrosine phosphorylation of p125
and
paxillin in permeabilized cells.
The C. botulinum C3
exoenzyme has been shown to ADP-ribosylate the Asn of
p21
, a residue within the effector domain of this small G
protein and thereby prevents the interaction of p21
with
its downstream targets(41, 51, 52) .
Pretreatment with C3 exoenzyme markedly inhibits GTP
S-induced
tyrosine phosphorylation of multiple substrates including p125
and paxillin. These results suggest that p21
predominantly mediates the effect of GTP
S and consequently,
that p21
lies upstream of tyrosine phosphorylation.
Interestingly, we have recently shown that pretreatment with C3
exoenzyme also inhibits bombesin and endothelin-induced tyrosine
phosphorylation in intact cells(30) .
In a separate approach
to test whether p21 mediates GTP
S-induced tyrosine
phosphorylation we used a synthetic peptide corresponding to the
effector domain of p21
(p21
). This region of
p21
and more recently p21
has been
identified as necessary for actin
reorganization(53, 57) . Furthermore, an effector
domain peptide of p21
(p21
) can block the ability
of p21
to interact with p74
in vitro(54) . We reasoned that a
p21
peptide could interfere with
the interaction between this small G protein and its effector(s). The
peptide p21
inhibited tyrosine
phosphorylation of both the M
110,000-130,000 and 70,000-80,000 bands induced by
GTP
S in a dose-dependent fashion. In contrast,
p21
at identical concentrations did
not affect GTP
S-stimulated tyrosine phosphorylation in
permeabilized Swiss 3T3 cells. Importantly,
p21
but not
p21
could specifically inhibit
p125
and paxillin tyrosine phosphorylation induced by
GTP
S. These results provide additional evidence that p21
predominantly mediates GTP
S-induced tyrosine
phosphorylation. The mechanism(s) linking p21
activation
with tyrosine phosphorylation of p125
and paxillin
warrants further investigation.
In conclusion, using permeabilized
Swiss 3T3 cells we demonstrate for the first time that GTPS can
induce tyrosine phosphorylation of multiple substrates including
p125
and paxillin. As these effects can occur at
nanomolar Mg
concentrations and are blocked by either
C3 exoenzyme or p21
peptide, we
suggest that the small GTP binding protein p21
predominantly mediates GTP
S-induced tyrosine
phosphorylation.