(Received for publication, May 15, 1995; and in revised form, August 9, 1995)
From the
Sphingosylphosphorylcholine (SPC), a potent mitogen for Swiss
3T3 cells, rapidly induced tyrosine phosphorylation of multiple
substrates including bands of M
110,000-130,000 and M
70,000-80,000 in
Swiss 3T3 cells. Focal adhesion kinase (p125
) and
paxillin were identified as prominent substrates for SPC-stimulated
tyrosine phosphorylation. An increase in tyrosine phosphorylation of
p125
was detected as soon as 30 s after SPC stimulation,
reaching a maximum after 2.5 min. SPC induced tyrosine phosphorylation
of p125
in a concentration-dependent fashion; a
half-maximum effect occurred at 250 nM. Tyrosine
phosphorylation of p125
induced by SPC could be
dissociated from both protein kinase C activation and Ca
mobilization from intracellular stores. SPC induced a unique
pattern of reorganization of the actin cytoskeleton with a rapid
appearance of actin microspikes at the plasma membrane that was
followed by the formation of actin stress fibers. This pattern of
cytoskeletal changes was clearly distinguishable from that induced by
bombesin and 1oleoyl-lysophosphatidic acid. Formation of microspikes
and actin stress fibers were accompanied by striking assembly of focal
adhesion plaques. Cytochalasin D, which disrupts the network of actin
microfilaments, completely prevented SPC-induced tyrosine
phosphorylation of p125
. In addition, tyrosine
phosphorylation of p125
was markedly inhibited in the
presence of platelet-derived growth factor at a concentration (30
ng/ml) that disrupts actin stress fibers. Finally, microinjection of Clostridium botulinum C3 exoenzyme, which inactivates
p21
, prevented SPC-induced formation of actin
stress fibers, focal adhesion assembly, and tyrosine phosphorylation.
Thus, p21
is upstream of both cytoskeletal
reorganization and tyrosine phosphorylation in SPC-treated cells.
Sphingosylphosphorylcholine (SPC) ()acts as a potent
mitogen for Swiss 3T3 cells in the absence of any other exogenously
added growth factor(1, 2) , but the molecular basis
for this effect is largely unknown. The best characterized signaling
event in response to SPC is the release of Ca
from
intracellular stores in a variety of cell
lines(2, 3, 4, 5) . However, it
appeared unlikely that all cellular responses elicited by SPC are
mediated by Ca
mobilization. In the accompanying
paper(6) , we demonstrated that SPC induces transient
activation of MAPK and p90
by a pathway
dependent on the activity of PKC and a pertussis toxin-sensitive
G
protein. In this paper, we identify further cellular and
molecular responses to exogenously added SPC to elucidate the
mechanisms by which lysosphingolipids modulate cell function.
Tyrosine phosphorylation of the cytosolic protein kinase
p125 and of the cytoskeletal associated protein paxillin
has recently been identified as an early event in the action of diverse
signaling molecules that mediate cell growth and differentiation,
including mitogenic neuropeptides(7, 8, 9) ,
growth factors such as PDGF (10) , the bioactive lipid
LPA(11, 12, 13) , sphingosine(14) ,
extracellular matrix
proteins(15, 16, 17, 18, 19) ,
and transforming variants of pp60
(16, 20) . The increases in p125
and paxillin tyrosine phosphorylation are accompanied by profound
alterations in the organization of the actin cytoskeleton and in the
assembly of focal
adhesions(10, 13, 14, 21, 22) ,
the distinct areas of the plasma membrane where p125
and
paxillin are
localized(23, 24, 25, 26) .
Recently, the small G protein p21
, a member of
the Ras superfamily of small GTP binding proteins, has been implicated
in mitogen-stimulated formation of focal adhesions and actin stress
fibers as well as tyrosine phosphorylation of p125
and
paxillin (21, 27, 28, 29) . The
effects, if any, of exogenously added SPC on tyrosine phosphorylation,
the organization of the actin cytoskeleton and focal adhesion assembly,
and the potential involvement of small G proteins such as
p21
in this signaling pathway remain unknown.
In this paper, we demonstrate that SPC rapidly stimulates tyrosine
phosphorylation of multiple proteins including p125 and
paxillin in Swiss 3T3 cells and induces a unique pattern of actin
organization, which was accompanied by the formation of focal contacts.
Microinjection of C.botulinum C3 exoenzyme, which
ADP-ribosylates and inactivates p21
, blocked the
changes in the organization of the actin cytoskeleton, the assembly of
focal contacts, and tyrosine phosphorylation in response to SPC.
Figure 8:
SPC-induced changes in actin cytoskeleton,
focal contacts, and tyrosine phosphorylation require the small G
protein p21. Confluent and quiescent Swiss 3T3
were microinjected with C. botulinum C3 exoenzyme at 100
µg/ml and rabbit IgG at 0.5 mg/ml. 40 min after microinjection,
cells were stimulated for 20 min with 5 µM SPC, fixed,
permeabilized, and stained using triple labeling as described under
``Experimental Procedures.'' Cells were stained with TRITC
phalloidin (A and D) to reveal actin filaments, and
the same cells were stained with anti-vinculin mAb (B) to
localize focal contacts or anti-Tyr(P) mAb 4G10 (E). Rabbit
IgG was detected with FITC-conjugated anti-rabbit IgG to localize the
microinjected cells (C and F). For comparison, a
sector of the dish was chosen where C3 exoenzyme-injected cells were
adjacent to non-injected cells. The cytoplasmic staining visible in Fig. 8, B and E, was due to unspecific
labeling by the secondary Cy5-conjugated anti-mouse IgG antibody as
shown in experiments where the cells were microinjected with C3
exoenzyme, treated with SPC as described above, and stained with
Cy5-conjugated anti-mouse IgG without prior incubation with either
anti-vinculin mAb or anti-Tyr(P) mAb (data not
shown).
Figure 1:
Dose response and time
course of SPC-stimulated tyrosine phosphorylation. Left upperpanel, lysates of quiescent Swiss 3T3 cells treated with
5 µM SPC or 10 nM bombesin for 10 min were
immunoprecipitated with anti-Tyr(P) mAb and further analyzed by
anti-Tyr(P) Western blotting followed by autoradiography. Rightupperpanel, quiescent Swiss 3T3 cells were
treated with various concentrations of SPC for 5 min, and cell lysates
were further analyzed as described above. Lower panel,
quiescent Swiss 3T3 cells were treated with 5 µM SPC for
various times, and cell lysates were further analyzed as described
above. The increase in tyrosine phosphorylation of the M 110,000-130,000 band (closedcircles) and
the M
70,000-80,000 band (opencircles) was quantified by scanning densitometry. Values
shown are the mean ± S.E. of at least five independent
experiments and are expressed as percentages of the maximum increase in
tyrosine phosphorylation above unstimulated control values in each
experiment. The dose response of SPC-induced tyrosine phosphorylation
of the M
70,000-80,000 band was virtually
superimposable to that of the M
110,000-130,000 band, which is shown in the upperrightpanel. Where an errorbar is not shown, it lies within the dimensions of the
symbol.
The cytosolic tyrosine kinase
p125(23, 24) and the
cytoskeleton-associated protein paxillin (25, 26) have
been identified as prominent tyrosine-phosphorylated proteins in
bombesin-treated Swiss 3T3 cells(7, 8, 9) .
As SPC elicited a pattern of tyrosine-phosphorylated bands similar to
that induced by bombesin (Fig. 1, upperpanel, left), we determined whether p125
and paxillin
were also substrates for SPC-mediated tyrosine phosphorylation. Lysates
of quiescent Swiss 3T3 cells incubated with 5 µM SPC for 5
min were immunoprecipitated with mAb 2A7 directed against p125
or mAb 165 directed against paxillin and further analyzed by
immunoblotting with a mixture of anti-Tyr(P) mAbs. Fig. 2, toppanel, left and right, shows
that SPC markedly induced tyrosine phosphorylation of p125
and paxillin, respectively. Thus, p125
is a
component of the broad M
110,000-130,000
band, and paxillin is a component of the diffuse M
70,000-80,000 band of tyrosine-phosphorylated proteins in
SPC-treated Swiss 3T3 cells. The degree of tyrosine phosphorylation of
these specific substrates in response to 5 µM SPC was
comparable to that induced by 10 nM bombesin in parallel
cultures (Fig. 2, toppanel, left and right). As shown in Fig. 2, middlepanel, SPC induced tyrosine phosphorylation of
p125
in a concentration-dependent manner; half-maximum
and maximum effects were achieved at 0.25 and 1 µM SPC,
respectively. The kinetics of SPC-induced tyrosine phosphorylation of
p125
is shown in Fig. 2, lowerpanel. An increase in tyrosine phosphorylation of
p125
was first detectable after 0.5 min and reached its
maximum after 2.5 min of incubation in the presence of SPC.
Figure 2:
SPC
induces tyrosine phosphorylation of p125 and paxillin in
Swiss 3T3 cells. Upper panel, quiescent Swiss 3T3 cells were
treated with 5 µM SPC or 10 nM bombesin for 5 min
and subsequently lysed. Tyrosine phosphorylation was analyzed by
immunoprecipitation using mAb 2A7 directed against p125
(leftpanel) or mAb 165 directed against
paxillin (rightpanel) followed by Western blotting
with anti-Tyr(P) mAbs. The positions of p125
and paxillin
are indicated by arrows. Middle panel, quiescent
Swiss 3T3 cells were treated with various concentrations of SPC for 5
min and lysed, and the lysates were immunoprecipitated with anti-Tyr(P)
mAb followed by Western blotting with anti-p125
mAb. Lower panel, quiescent Swiss 3T3 cells were treated with 5
µM SPC for various times and lysed, and the lysates were
immunoprecipitated with mAb 2A7 directed against p125
followed by Western blotting with anti-Tyr(P) mAb. Experiments
shown are representative of at least two independent
experiments.
Figure 3:
Role of Ca and PKC
activity in SPC-induced p125
tyrosine phosphorylation. Upperpanel, quiescent Swiss 3T3 cells were incubated
with 30 nM thapsigargin (Thapsigargin, +) or 3 mM EGTA (EGTA, +) for 30 min as indicated. Control cells
received an equivalent amount of solvent(-). Cells were
subsequently stimulated with 5 µM SPC for 5 min and lysed,
and the lysates were immunoprecipitated with anti-Tyr(P) mAb followed
by Western blotting with anti-p125
mAb. Shown is a
representative experiment of three independent experiments. Middle
panel, quiescent Swiss 3T3 cells were pretreated for 48 h with 800
nM PDB (PDB pre, +), incubated for 1 h with GF 109203X
(GF, +), or received an equivalent amount of solvent(-) and
then were stimulated with 200 nM PDB or 5 µM SPC.
Cells were lysed, and lysates were immunoprecipitated with mAb 2A7
directed against p125
and further analyzed by Western
blotting with anti-Tyr(P) mAbs. Shown is a representative experiment of
three independent experiments. The position of p125
is
indicated by an arrow. The increase in tyrosine
phosphorylation of p125
was quantified by scanning
densitometry. Values shown are the mean ± S.E. of three
independent experiments and are expressed as percentages of the maximum
increase in tyrosine phosphorylation of p125
above
unstimulated control values. PDB or GF 109203X pretreatment of Swiss
3T3 cells reduced tyrosine phosphorylation of p125
in
response to PDB to the same level obtained in PDB or GF 109203X
pretreated but unstimulated cells. These controls were therefore
omitted for clarity. Lower panel, quiescent Swiss 3T3 cells
were pretreated for 1 h with of 3.5 µM GF 109203X (GF,
+) or received an equivalent amount of solvent(-). Cells
were then treated for 30 min with 5 µM SPC, 10 nM
bombesin (Bom) or 12.5 µM sphingosine (Sph) and were subsequently lysed; the lysates were
immunoprecipitated with mAb 165 directed against paxillin and further
analyzed by Western blotting using anti-Tyr(P) mAbs. Shown is a
representative experiment of two independent
experiments.
SPC induces
activation of MAPK by a pertussis toxin-sensitive mechanism, suggesting
the involvement of a G protein of the G/G
subfamily in the signaling pathway(6) . To examine
whether SPC stimulates p125
tyrosine phosphorylation via
G
, quiescent Swiss 3T3 cells were pretreated with various
concentrations of pertussis toxin for 3 h and subsequently challenged
with 5 µM SPC for 5 min. Pertussis toxin at concentrations
between 0.1 and 100 ng/ml did not affect tyrosine phosphorylation of
p125
in response to SPC (data not shown).
In the
accompanying paper(6) , we show that SPC stimulates activation
of PKC in Swiss 3T3 cells. As activation of PKC is also a potential
pathway leading to tyrosine phosphorylation of p125 and
paxillin(8) , we examined the role of PKC in SPC-induced
p125
and paxillin tyrosine phosphorylation. PKC was
either selectively inhibited by pretreatment of quiescent Swiss 3T3
cells for 1 h with the bisindolylmaleimide GF 109203X at 3.5 µM(33) or down-regulated by prolonged exposure (48 h) to 800
nM PDB(34) . As shown in Fig. 3, middlepanel, both treatments completely blocked stimulation of
p125
tyrosine phosphorylation by 200 nM PDB. In
contrast, inhibition or down-regulation of PKC only slightly reduced
p125
tyrosine phosphorylation in response to 5
µM SPC (Fig. 3, middlepanel).
Thus, SPC induces p125
tyrosine phosphorylation largely
through a PKC-independent pathway.
Paxillin contains several potential target sites for phosphorylation by PKC(35) . Tyrosine phosphorylation of paxillin in response to bombesin, LPA, or phorbol esters is accompanied by a mobility shift that results in the appearance of slower migrating forms of this protein, a process mediated by PKC(9) . SPC at 5 µM also induced this mobility shift of paxillin (Fig. 3, lowerpanel). Treatment of Swiss 3T3 cells with GF 109203X did not affect the degree of tyrosine phosphorylation of paxillin induced by SPC but prevented the appearance of the slower migrating forms of paxillin. Interestingly, the sphingolipid breakdown product sphingosine at 12.5 µM induced tyrosine phosphorylation of paxillin to a lesser degree than SPC, and no mobility shift of paxillin could be detected (Fig. 3, lowerpanel). This is probably due to the fact that sphingosine, in contrast to SPC, does not strongly activate PKC(14) .
In conclusion, the results
depicted in Fig. 3indicate that SPC induces tyrosine
phosphorylation of p125 and paxillin through a pathway
largely independent of Ca
mobilization, a G
protein, or PKC activation.
Figure 4: Effect of SPC on the actin cytoskeleton. Swiss 3T3 cells were washed twice with DMEM and incubated with 5 µM SPC for various times as indicated (panelA) or with 5 µM SPC, 10 nM bombesin (BOM), or 2 µM LPA for 3 min (panelB). Cells were subsequently fixed in 4% paraformaldehyde and stained for actin using TRITC-conjugated phalloidin. Changes in the actin cytoskeleton were visualized using a Zeiss Axiophot immunofluorescence microscope.
Both the
neuropeptide bombesin and the bioactive lipid LPA stimulate the
reorganization of actin with a time course comparable to that of SPC.
However, these compounds primarily induce organization of actin into
stress fibers visible as early as 1 min after addition of the factors
but not into microspikes(21) . ()Therefore, the
pattern of the changes in actin organization induced by SPC seemed to
be unique. To further substantiate this observation, we compared the
early effects of 5 µM SPC, 10 nM bombesin, and 2
µM LPA on the actin cytoskeleton in parallel cultures. As
shown in Fig. 4B, SPC induced the typical microspike
pattern of actin arrangement. In contrast, bombesin induced actin
stress fiber formation and membrane ruffling, whereas LPA predominantly
induced actin stress fiber formation in accordance with previous
observations(13, 21) . Therefore, the pattern of actin
rearrangements in response to SPC showing first microspikes and later
stress fibers is clearly distinguishable from that induced by other
factors that cause rapid changes in the organization of the actin
cytoskeleton in Swiss 3T3 cells.
Figure 5: Effect of SPC on focal contact assembly and relation to SPC-induced microspike formation. Panel A, Swiss 3T3 cells were washed with DMEM and incubated with 5 µM SPC for various times, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with anti-vinculin mAb and FITC-linked anti-mouse IgG antibody. Panel B, Swiss 3T3 cells were incubated with 5 µM SPC for 5 min, and double labeling was used to compare the concomitant changes in the organization of the actin cytoskeleton (stained with TRITC-conjugated phalloidin) with those of vinculin (stained with anti-vinculin mAb) in focal adhesions (left and rightpictures, respectively). Confocal imaging was performed as described under ``Experimental Procedures.''
To corroborate these findings and clarify the spatial relationship between microspikes and focal contacts in response to SPC, we used double labeling to visualize actin and vinculin in the same cells. Addition of 5 µM SPC to quiescent Swiss 3T3 cells for 5 min led to recruitment of actin into microspikes at the plasma membrane, giving the cells a ``hedgehog''-like appearance (Fig. 5B, leftpicture). Vinculin staining of focal contacts was clearly visible at the ends of these microspikes (Fig. 5B, rightpicture). Thus, in SPC-stimulated cells, focal adhesion assembly with localization of vinculin to focal contacts clearly occurred before formation of actin stress fibers and was also related to a different form of actin organization, namely the formation of actin microspikes.
Figure 6:
Cytochalasin D inhibits SPC-induced
tyrosine phosphorylation of p125. Panel A,
quiescent Swiss 3T3 cells were treated for 2 h in the presence of
various concentrations of cytochalasin D as indicated and then
incubated with 5 µM SPC for a further 5 min. Cells were
then lysed, and lysates were further analyzed by immunoprecipitation
using mAb 2A7 directed against p125
followed by
anti-Tyr(P) Western blotting. SPC-induced tyrosine phosphorylation of
p125
obtained after pretreatment with various
concentrations of cytochalasin D was quantified by scanning
densitometry. The values shown are expressed as the percentage of the
maximum increase in p125
tyrosine phosphorylation above
control, unstimulated values and are the mean ± S.E. of three
independent experiments. The maximum increase in p125
tyrosine phosphorylation was induced by 5 µM SPC in
cells that were not pretreated. The inset shows a
representative experiment of three independent experiments. The
position of p125
is indicated by an arrow. Panel B, effect of cytochalasin D on SPC-induced intracellular
Ca
release. Quiescent cultures of Swiss 3T3 cells
were incubated with fura-2-tetraacetoxymethyl ester and transferred to
a cuvette by gentle scraping.
[Ca
]
was measured as
described under ``Experimental Procedures.'' The basal
[Ca
]
was monitored for
1 min before treating the cells with 5 µM SPC (A)
or 2 µM cytochalasin D 2 min before adding 5 µM SPC (B). In C, cells were pretreated for 2 h
with 2 µM cytochalasin D prior to loading with the
Ca
indicator and then challenged with 5 µM SPC. The arrows indicate addition of SPC to the cells.
The traces shown are representative of three independent
experiments.
The inhibitory effect of cytochalasin D on
SPC-induced tyrosine phosphorylation was specific, as cytochalasin D at
the concentrations used profoundly disrupted the network of actin
filaments and focal contacts in Swiss 3T3 cells (data not shown) but
did not interfere with SPC-induced Ca mobilization (Fig. 6B) or with SPC-stimulated MAPK activation as
shown in the accompanying paper(6) . This suggests that SPC
induces tyrosine phosphorylation of p125
and MAPK
activation by clearly distinct mechanisms.
Figure 7:
Effect of PDGF on SPC induced
reorganization of the actin cytoskeleton and p125 tyrosine phosphorylation. PanelA, effect of
SPC and PDGF on the actin cytoskeleton. Quiescent Swiss 3T3 cells were
washed and incubated for 10 min in DMEM at 37 °C. The cells were
incubated for a further 20 min following no addition (upperleftpanel) or addition of 5 or 30 ng/ml PDGF (middle and lowerleftpanels,
respectively), 5 µM SPC (upperrightpanel), or 5 µM SPC in the presence of 5 or
30 ng/ml PDGF (middle and lowerrightpanels, respectively). Subsequently, cells were washed
with PBS and fixed in 4% paraformaldehyde; actin was then labeled with
TRITC-conjugated phalloidin (0.25 µg/ml) in PBS for 10 min at room
temperature. Panel B, effects of PDGF on SPC-induced tyrosine
phosphorylation of p125
. Quiescent Swiss 3T3 cells were
incubated for 20 min at 37 °C with 5 or 30 ng/ml PDGF, 5 µM SPC (+), or 5 µM SPC in the presence of 5 or 30
ng/ml PDGF. Control cells received an equivalent amount of
solvent(-). Tyrosine phosphorylation of p125
was
analyzed by immunoprecipitation with mAb 2A7 directed against
p125
and immunoblotting with anti-Tyr(P) mAbs. The
position of p125
is indicated by an arrow.
SPC-induced tyrosine phosphorylation of p125
in the
presence of 5 or 30 ng/ml PDGF was quantified by scanning densitometry.
The values shown are expressed as the percentage of the maximum
increase in p125
tyrosine phosphorylation above control,
unstimulated values and are the mean ± S.E. of three independent
experiments.
The results shown in Fig. 6demonstrated that SPC
stimulated p125 tyrosine phosphorylation by a mechanism
dependent on the integrity of the actin cytoskeleton. In view of the
results depicted in Fig. 7A, we examined whether
SPC-stimulated tyrosine phosphorylation of p125
could
also be affected by high concentrations of PDGF. As shown in Fig. 7B, PDGF at 30 ng/ml markedly reduced tyrosine
phosphorylation of p125
induced by 5 µM SPC
in agreement with its effects on SPC-induced actin reorganization.
To examine whether C3
exoenzyme could also prevent tyrosine phosphorylation in response to
SPC, microinjected cells were stained for actin, Tyr(P), and rabbit IgG
as described under ``Experimental Procedures.'' In control
cells stimulated with 5 µM SPC for 20 min, anti-Tyr(P)
staining was visible at the ends of actin stress fibers in focal
contacts (Fig. 8, D and E). In microinjected
cells, this pattern of staining was not detectable, indicating that C3
exoenzyme inhibited tyrosine phosphorylation in response to SPC (Fig. 8, D-F). Thus, p21 is an
upstream regulator of both cytoskeletal changes and tyrosine
phosphorylation in response to SPC.
The results presented here demonstrate that SPC induces
tyrosine phosphorylation of multiple substrates in Swiss 3T3 cells. We
identified two substrates that were phosphorylated on tyrosine residues
in response to SPC, p125, and paxillin. p125
is a cytosolic tyrosine kinase that lacks SH2 and SH3 domains but
associates with other proteins including v-Src and
paxillin(38) . The recent molecular cloning and sequencing of
paxillin revealed that this protein contains multiple domains that can
participate in protein-protein interactions(35, 39) .
A coordinate increase in tyrosine phosphorylation of p125
and paxillin is elicited by a variety of extracellular agents
that modulate cell growth and differentiation (see Introduction for
references). Our results suggest that p125
and paxillin
could also play a role in the signaling pathways induced by SPC.
SPC
is known to release Ca from intracellular stores, and
changes in [Ca
]
can lead to
tyrosine phosphorylation of a M
125,000
band(31) . However, SPC-stimulated tyrosine phosphorylation of
p125
is completely independent of intracellular
Ca
release or Ca
influx. As shown
in the accompanying paper(6) , SPC rapidly activates PKC. Since
direct activation of PKC has been shown to stimulate tyrosine
phosphorylation of p125
, SPC could activate tyrosine
phosphorylation of p125
through a PKC-dependent
mechanism. However, neither down-regulation of PKC with PDB nor the PKC
inhibitor GF 109203X blocked SPC-stimulated tyrosine phosphorylation of
p125
. Thus, SPC induces tyrosine phosphorylation of
p125
by a pathway largely independent of both
Ca
mobilization and PKC activation.
The increases
in tyrosine phosphorylation of p125 and paxillin in
response to SPC were accompanied by a novel and dramatic reorganization
of the actin cytoskeleton. Specifically, SPC evoked a striking
formation of peripheral actin microspikes followed by the development
of actin stress fibers. Both events were associated with the assembly
of focal adhesion plaques, which were localized at early times at the
ends of actin microspikes and later at the ends of actin stress fibers.
Thus, focal adhesion assembly in response to SPC occurs prior to actin
stress fiber formation and is also linked to the formation of
microspikes at the plasma membrane.
Tyrosine phosphorylation of
p125 and paxillin and the cytoskeletal changes in
response to SPC can be clearly distinguished from effects induced by
sphingosine. Tyrosine phosphorylation of p125
and
paxillin as well as actin rearrangement elicited by sphingosine
developed gradually, reaching a maximum by 60 min(14) . In
sharp contrast, SPC induced a maximum increase in p125
tyrosine phosphorylation and visible changes in the actin
cytoskeleton and focal contact assembly after 1 min of its addition.
Furthermore, tyrosine phosphorylation of paxillin by SPC is
characterized by a mobility shift resulting in the appearance of slower
migrating forms, a process mediated by PKC(9) . Indeed,
paxillin contains multiple potential sites for PKC
phosphorylation(35, 39) . Sphingosine-induced tyrosine
phosphorylation of paxillin is not accompanied by any measurable
mobility shift in accord with the very small stimulation of PKC by
sphingosine in intact Swiss 3T3 cells(14) . Thus, SPC and
sphingosine induce different signaling pathways in Swiss 3T3 cells.
Interestingly, exogenously added sphingosine 1-phosphate induces
tyrosine phosphorylation of p125
and paxillin with
kinetics similar to that of SPC. (
)
Tyrosine
phosphorylation of p125 and paxillin in response to
bombesin and other agents is critically dependent on the integrity of
the actin cytoskeleton(8) . Given the similar kinetics of
SPC-stimulated tyrosine phosphorylation and cytoskeletal changes, it
was of interest to establish whether these events were also linked in
SPC-treated Swiss 3T3 cells. Pretreatment of quiescent Swiss 3T3 cells
with cytochalasin D led to a complete disruption of the actin
cytoskeleton and abolished tyrosine phosphorylation of p125
stimulated by SPC. Thus, the integrity of the actin cytoskeleton
is essential for SPC-induced tyrosine phosphorylation. This conclusion
was substantiated by experiments using PDGF at a high concentration (30
ng/ml), which disrupted actin stress fibers in response to SPC. At this
concentration, PDGF also profoundly decreased SPC-induced tyrosine
phosphorylation of p125
, revealing a novel cross-talk
between SPC and PDGF.
Recent findings indicate that both
cytoskeletal changes and tyrosine phosphorylation of p125 and paxillin induced by bombesin require functional p21
protein(21, 27) . These findings suggested the
existence of a pathway activated by seven transmembrane domain
receptors in which p21
is upstream of both cytoskeletal
responses and tyrosine phosphorylation of specific proteins. In view of
the results described above, it was plausible that the small G protein
p21
could also be involved in SPC-induced cytoskeletal
changes. Microinjection of C. botulinum C3 exoenzyme, which
ADP-ribosylates and inactivates p21
function, prevented
actin stress fiber formation, focal contact assembly, and tyrosine
phosphorylation in response to SPC. Thus, p21
is upstream
of both cytoskeletal changes and tyrosine phosphorylation in response
to SPC.
Recently, a possible link between p125 tyrosine phosphorylation and the MAPK pathway has been suggested.
Several groups demonstrated that integrin-mediated cell adhesion
activates MAPK by a pathway critically dependent on the integrity of
the actin cytoskeleton (40, 41) . Schlaepfer et
al.(42) showed that integrin engagement stimulates an
interaction between p125
and the adapter protein Grb2,
suggesting a possible link between p125
and the
p21
/Raf/MAPK pathway. In view of the data provided in the
accompanying paper (6) on SPC-induced MAPK activation and here
on p125
tyrosine phosphorylation, it was important to
define whether these events are causally related. Several lines of
evidence demonstrate a dissociation of p125
tyrosine
phosphorylation from MAPK activation in response to SPC in Swiss 3T3
cells. SPC-induced MAPK activation is dependent on the activity of PKC
and a pertussis toxin-sensitive G
protein. In contrast,
tyrosine phosphorylation of p125
was largely independent
of PKC and not affected by treatment with pertussis toxin. Crucially,
tyrosine phosphorylation of p125
in response to SPC was
completely prevented by disruption of the actin cytoskeleton with
cytochalasin D, whereas MAPK activation by SPC was unaffected by an
identical treatment with cytochalasin D. Thus, p125
tyrosine phosphorylation and MAPK activation are independently
regulated in SPC-treated cells.
In conclusion, our results
demonstrate, for the first time, that SPC stimulates tyrosine
phosphorylation of p125 and paxillin. Furthermore, SPC
induces a unique pattern of reorganization of the actin cytoskeleton
and focal adhesion assembly in Swiss 3T3 cells. The integrity of the
polymerized actin network and functional p21
are
essential for SPC-induced tyrosine phosphorylation.