(Received for publication, September 12, 1995; and in revised form, October 20, 1995)
From the
Treatment of Swiss 3T3 cells with recombinant Pasteurella
multocida toxin (rPMT), a potent intracellularly acting mitogen,
stimulated tyrosine phosphorylation of multiple substrates including
bands of M 110,000-130,000 and M
70,000-80,000. Tyrosine phosphorylation
induced by rPMT occurred after a pronounced lag period (1 h) and was
blocked by either lysosomotrophic agents or incubation at 22 °C.
Focal adhesion kinase (p125
) and paxillin are prominent
substrates for rPMT-stimulated tyrosine phosphorylation. Tyrosine
phosphorylation by rPMT could be dissociated from both protein kinase C
activation and the mobilization of calcium from intracellular stores.
rPMT stimulated striking actin stress fiber formation and focal
adhesion assembly in Swiss 3T3 cells. Cytochalasin D, which disrupts
the actin cytoskeleton, completely inhibited rPMT-induced tyrosine
phosphorylation. In addition, tyrosine phosphorylation of
p125
and paxillin in response to rPMT was completely
abolished when cells were subsequently treated with platelet-derived
growth factor at a concentration (30 ng/ml) that disrupted the actin
cytoskeleton. Our results demonstrate for the first time that rPMT, a
bacterial toxin, induces tyrosine phosphorylation of p125
and paxillin and promotes actin stress fiber formation and focal
adhesion assembly in Swiss 3T3 cells.
The mechanism of action of bacterial toxins has provided
insights into the control of cellular regulatory processes, including
signal transduction and cell
proliferation(1, 2, 3) . Recombinant Pasteurella multocida toxin (rPMT) ()is an
extremely potent mitogen for murine Swiss 3T3 cells, other fibroblast
cell lines, and early-passage cultures and promotes
anchorage-independent growth of Rat-1 cells(4, 5) .
The toxin is a 146-kDa protein that has been purified, cloned, and
sequenced(6, 7, 8, 9, 10, 11, 12, 13) .
The deduced amino acid sequence of PMT shows partial homology with CNF1
and CNF2, produced by some strains of pathogenic Escherichia
coli(14, 15) . It has been proposed that PMT
enters the cells and acts intracellularly to initiate and sustain DNA
synthesis.
Prior to the stimulation of DNA synthesis, rPMT
stimulates the formation of inositol phosphates and mobilizes
Ca from an intracellular pool(16) . Analysis
of the inositol phosphate species generated in response to rPMT
strongly suggests that the toxin stimulates the hydrolysis of
phosphatidylinositol 4,5-bisphosphate by activating cellular
phospholipase C(16) , a major transducer of transmembrane
signaling (17) . rPMT also increases the cellular content of
diacylglycerol, causes translocation of PKC to cellular membranes, and
stimulates the phosphorylation of 80K/MARCKS(4, 18) ,
a prominent substrate of PKC in cultured
fibroblasts(19, 20, 21, 22) . The
stimulation of these early events by rPMT, like its mitogenic
action(5) , requires cellular entry and activation of the
toxin.
Changes in protein tyrosine phosphorylation are known to play
a key role in the action of growth factors and oncogenes but have not
been demonstrated in response to rPMT. Recently, tyrosine
phosphorylation of the cytosolic protein kinase p125(23, 24) and of the cytoskeleton-associated
protein paxillin (25, 26) have been identified as
early events in the action of diverse signaling molecules that mediate
cell growth and differentiation including mitogenic
neuropeptides(27, 28, 29) , the bioactive
lipids LPA and sphingosine (30, 31, 32) ,
extracellular matrix
proteins(33, 34, 35, 36, 37) ,
PDGF at low concentration(38) , and transforming variants of
pp60
(33, 39) . 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 the focal adhesion
plaques(31, 38, 40, 41) , the
distinct sites in the plasma membrane where both p125
and
paxillin are localized(23, 24, 42) . The
effects of rPMT on protein tyrosine phosphorylation, actin stress fiber
formation and focal adhesion assembly were unknown.
Here we report
that rPMT stimulates tyrosine phosphorylation of multiple proteins
including p125 and paxillin and induces a striking
increase in stress fiber formation and focal adhesion assembly in Swiss
3T3 cells. The mode of action of rPMT differs from that of
neuropeptides, growth factors, and extracellular matrix proteins in
that the toxin appears to enter the cells to trigger protein tyrosine
phosphorylation and cytoskeletal reorganization.
Figure 4: Effect of rPMT on the actin cytoskeleton and focal contacts. Quiescent cultures of Swiss 3T3 cells were washed with DMEM and incubated in DMEM:Waymouth medium 1:1(v/v) containing 20 ng/ml rPMT for the times indicated, and then washed with PBS, fixed in 3.7% paraformaldehyde, and permeabilized with 0.2% Triton X-100. A labeling technique with TRITC-conjugated phalloidin and the monoclonal anti-vinculin antibody was used to compare changes in the formation of stress fibers with those of vinculin at the focal adhesions (shown on the left and right, respectively).
Figure 6:
Effect of rPMT and PDGF on the actin
cytoskeleton. Quiescent cultures of Swiss 3T3 were washed with DMEM and
incubated in DMEM:Waymouth medium 1:1(v/v) with (B and D) or without (A and C) 20 ng/ml rPMT for 4
h and further incubated for 10 min as follows: A, no addition; B, 20 ng/ml rPMT; C, 30 ng/ml PDGF; D, 20 ng/ml rPMT + 30 ng/ml PDGF
,
respectively. Cells were washed with PBS, fixed with 3.7%
paraformaldehyde, and permeabilized with 0.2% Triton X-100. Actin was
stained by incubation with TRITC-conjugated phalloidin (0.25 µg/ml)
in PBS for 10 min at room temperature.
Figure 8: Effect of C3 exoenzyme microinjection on anti-phosphotyrosine staining. Quiescent cultures of Swiss 3T3 cells in 30 mm dishes were washed twice and incubated in DMEM:Waymouth medium 1:1 (v/v). Cells were microinjected (arrows) with 0.5 mg/ml rabbit IgG (CONTROL) or 0.5 mg/ml rabbit IgG + 100 µg/ml C3 (C3 EXOENZYME). Cells were then treated with 20 ng/ml rPMT. After 4 h of incubation, the cells were stained with 4G10 mAb directed against phosphotyrosine residues and visualized by confocal microscopy using Cy5-linked anti-mouse IgG. Microinjected cells were identified by staining with FITC-conjugated anti-rabbit IgG antibody.
Figure 1:
rPMT induces tyrosine phosphorylation
of multiple bands including p125 and paxillin in Swiss
3T3 cells. A, quiescent cultures of Swiss 3T3 cells were
treated in DMEM/Waymouth medium 1:1 (v/v) with (+) or
without(-) 20 ng/ml rPMT for 6 h. Cells were lysed and the
lysates were immunoprecipitated with agarose-linked anti-Tyr(P) mAb,
anti-p125
mAb, 2A7, or anti-paxillin mAb, 165. The
immunoprecipitates were fractionated by SDS-PAGE and further analyzed
by immunoblotting with a mixture of anti-Tyr(P) mAbs. B,
quiescent cultures of Swiss 3T3 cells were treated in DMEM/Waymouth
medium 1:1 (v/v) with 20 ng/ml rPMT for various times (0-24 h) as
indicated. Cells were lysed, and the lysates were immunoprecipitated
using anti-Tyr(P) mAb, mAb 2A7, and mAb 165 and analyzed by
immunoblotting with a mixture of anti-Tyr(P) mAbs, as described above.
The positions of p125
and paxillin are indicated by arrows. The results shown in this and subsequent figures are
representatives autoradiographs of at least three independent
experiments.
The pattern of
tyrosine-phosphorylated proteins induced by rPMT is strikingly similar
to that stimulated by bombesin and LPA in Swiss 3T3
cells(27, 31) . Recently, the cytosolic tyrosine
kinase p125(23, 24) and the adaptor
protein paxillin (25, 26, 46) have been
identified as prominent tyrosine-phosphorylated proteins in bombesin
and LPA-treated
cells(27, 28, 29, 31) . To determine
whether these cellular proteins were also substrates for rPMT-induced
tyrosine phosphorylation, lysates of Swiss 3T3 cells, incubated with 20
ng/ml rPMT for 6 h, were immunoprecipitated with mAbs that recognize
either p125
or paxillin, and the immunoprecipitates were
analyzed by Western blotting with a mixture of anti-Tyr(P) mAbs. Fig. 1A, shows that rPMT markedly stimulated
p125
and paxillin tyrosine phosphorylation. Thus,
p125
is a component of the broad M
110,000-130,000 band, whereas paxillin is a component of
the diffuse tyrosine-phosphorylated band migrating with an apparent M
70,000-80,000.
Figure 2:
Effect of methylamine, temperature, and
exposure time on rPMT-induced tyrosine phosphorylation. A,
quiescent cultures of Swiss 3T3 cells were incubated for 6 h in
DMEM:Waymouth 1:1(v/v) medium containing 20 ng/ml rPMT or 10 nM bombesin with or without 10 mM CHNH
at 37 °C. Other cultures were incubated with either rPMT or
bombesin under identical conditions except that the temperature of
incubation was 22 °C instead of at 37 °C. Cells were lysed and
the lysates immunoprecipitated with anti-Tyr(P) mAb. The
immunoprecipitates were fractionated by SDS-PAGE and further analyzed
by anti-Tyr(P) immunoblotting. B, the values of the mean of
three independent experiments which are expressed as the percentage of
the maximum stimulation of tyrosine phosphorylation by 20 ng/ml rPMT of
the M
110,000-130,000 band quantified by
scanning densitometry. C, quiescent cultures of Swiss 3T3
cells were treated with 20 ng/ml rPMT for various times (0-5 h)
as indicated. The cells fresh DMEM:Waymouth medium (1:1). The
incubation was terminated at were then washed with DMEM and
subsequently incubated (5-0 h) with the end of the 5 h
period.
To determine whether rPMT
acts through a PKC-dependent pathway, cells were pretreated for 60 min
with the selective PKC inhibitor, GF109203X(50) , at 3.5
µM, a concentration that completely abolished phorbol
12,13-dibutyrate-induced tyrosine phosphorylation of p125 in Swiss 3T3 cells(29) . The cells were subsequently
challenged with 20 ng/ml rPMT for a further 5 h. rPMT-induced tyrosine
phosphorylation of p125
was not significantly reduced (Fig. 3A). In the same experiment, tyrosine
phosphorylation induced by 200 nM phorbol 12,13-dibutyrate for
5 h was completely inhibited by GF109203X (data not shown).
Figure 3:
Effect of GF109203X and thapsigargin on
the stimulation of tyrosine phosphorylation of p125 by
rPMT. A, quiescent Swiss 3T3 cells were preincubated for 1 h
with GF109203X (+) or an equivalent amount of solvent(-) and
then stimulated with 20 ng/ml rPMT for a further 5 h. Cells were lysed
and the lysates immunoprecipitated with mAb 2A7 directed against
p125
and further analyzed by Western blotting with
anti-Tyr(P) mAbs. B, quiescent Swiss 3T3 cells were pretreated
with 30 nM thapsigargin (+) or an equivalent amount of
solvent(-) for 30 min. Cells were then stimulated with 20 ng/ml
rPMT, incubated for a further 6 h and subsequently lysed. The lysates
were immunoprecipitated with mAb 2A7 against p125
and
Western blotted with anti-Tyr(P) mAbs. Anti-Tyr(P) immunoreactivity of
the p125
band was quantified by scanning densitometry. A and B show the values of the mean of three
independent experiments and are expressed as the percentage of the
maximum stimulation by 20 ng/ml rPMT.
To
investigate whether Ca mobilization mediates tyrosine
phosphorylation by rPMT, quiescent cells were pretreated with the tumor
promoter thapsigargin. Thapsigargin depletes Ca
from
intracellular stores by specifically inhibiting the endoplasmic
reticulum Ca
-ATPase (51) . Pretreatment of
cells with 30 nM thapsigargin for 30 min which depletes
Ca
from internal stores in Swiss 3T3 cells (29, 31) had no effect on the subsequent rPMT-induced
tyrosine phosphorylation of p125
(Fig. 3B). Thus, rPMT stimulates tyrosine
phosphorylation of p125
largely through a PKC- and
Ca
-independent pathway.
Focal adhesions are
subcellular structures which are formed at regions of close contact
between cells and their underlying substratum. Several proteins are
specifically localized in focal adhesions including vinculin, paxillin,
talin, and -actinin(52) . Here we demonstrate that
addition of rPMT to quiescent Swiss 3T3 cells induced localization of
vinculin into focal adhesions, first visible after 1 h and reaching a
maximum after 8 h (Fig. 4, right). Addition of 25
µM cycloheximide did not prevent the localization of
vinculin into focal contacts induced by rPMT (results not shown). The
relative amount of vinculin in focal adhesions, as judged by the
intensity of immunofluorescent staining, increased in parallel with the
association of stress fibers at these sites of the plasma membrane.
Thus, rPMT induced actin stress fiber formation and focal adhesion
assembly in Swiss 3T3 cells with kinetics that closely parallels rPMT
induced tyrosine phosphorylation of p125
and paxillin.
Figure 5:
Cytochalasin D inhibits rPMT-stimulated
tyrosine phosphorylation of multiple bands including
p125. A, quiescent cultures of Swiss 3T3 cells
were treated for 1 h in the presence of various concentrations of
cytochalasin D as indicated and then 20 ng/ml rPMT was added to the
cell cultures and incubated for a further 6 h. The cells were then
lysed and the lysates immunoprecipitated using anti-Tyr(P) mAb or 2A7
mAb directed against p125
. The immunoprecipitates were
fractionated by SDS-PAGE and further analyzed by immunoblotting using a
mixture of anti-Tyr(P) mAbs. The position of p125
is
indicated by an arrow. B, quiescent cells, labeled with 50
µCi/ml of [
P]P
for 12 h, were
treated with (+) or without(-) 1.2 µM
cytochalasin D for 1 h. Then, some of the cultures received 20 ng/ml
rPMT and all cultures were incubated for a further 6 h. Cells were
subsequently lysed and the lysates were immunoprecipitated with
anti-80K/MARCKS antibody and further analyzed by
SDS-PAGE.
The inhibitory effect of
cytochalasin D may, in theory, have resulted from interference with the
entry and activation of PMT. To examine this possibility we tested the
ability of rPMT to activate PKC and phosphorylate 80K/MARCKS in cells
treated in the absence or presence of cytochalasin D. Quiescent Swiss
3T3 cells labeled with [P]P
for 12
h, were pretreated with 1.2 µM cytochalasin D for 1 h and
then challenged with 20 ng/ml rPMT for further 6 h. Fig. 5B shows that treatment with cytochalasin D at a concentration that
completely inhibited rPMT-induced tyrosine phosphorylation did not
prevent rPMT mediated stimulation of 80K/MARCKS phosphorylation.
As shown previously in Fig. 5, rPMT-induced tyrosine phosphorylation requires the
integrity of the actin cytoskeleton. Given that 30 ng/ml PDGF also
destabilized the actin stress fibers we decided to investigate the
effects of PDGF on tyrosine phosphorylation induced by rPMT. Quiescent
Swiss 3T3 cells were treated with 20 ng/ml rPMT for 4 h and then 5
ng/ml or 30 ng/ml PDGF were added to the cells for further 10 min. As
shown in Fig. 7, rPMT induced tyrosine phosphorylation of either
p125 or paxillin was markedly inhibited by addition of 30
ng/ml PDGF but not by 5 ng/ml PDGF.
Figure 7:
PDGF blocks tyrosine phosphorylation of
p125 and paxillin induced by rPMT. Quiescent cultures of
Swiss 3T3 were incubated with or without rPMT for 4 h. Subsequently, 5
or 30 ng/ml PDGF
was added (+) to rPMT pretreated
cells, as well as nontreated cells, for an additional 10 min. Cells
were lysed and the lysates immunoprecipitated with 2A7 mAb against
p125
(A) or 165 mAb against paxillin (B). The immunoprecipitates were analyzed by SDS-PAGE followed
by immunoblotting using anti-Tyr(P) mAbs. Values shown are expressed as
a percentage of the maximum tyrosine phosphorylation of p125
or paxillin stimulated by 20 ng/ml
rPMT.
The results presented here show for the first time that rPMT,
an intracellularly acting bacterial toxin, induces tyrosine
phosphorylation of multiple substrates in Swiss 3T3 cells. In this
study, we identified two substrates, p125 and paxillin,
which were tyrosine-phosphorylated in response to rPMT. p125
is a cytosolic tyrosine protein kinase localized in focal
adhesions that lacks SH2 and SH3 domains but associates with other
proteins including v-Src and
paxillin(23, 24, 46) . Paxillin, a M
70,000 protein, is a major phosphotyrosyl
protein in chicken embryo and like p125
, is localized to
focal adhesions(41, 42) . Recent molecular cloning
revealed that paxillin is a multidomain protein that may function as an
adaptor capable of associating with p125
, Crk, and
Src(25, 26, 46) . A coordinate increase in
tyrosine phosphorylation of p125
and paxillin is induced
by a variety of molecules that regulate cell growth and
differentiation(27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
Our results suggest that p125
and paxillin tyrosine
phosphorylation could also play a role in the signaling pathways
stimulated by rPMT.
rPMT increases the accumulation of the second
messengers diacylglycerol and inositol 1,4,5-trisphosphate, which
activate PKC and mobilize Ca from intracellular
stores, respectively (49) , and direct PKC activation is known
to stimulate tyrosine phosphorylation of p125
and
paxillin(27, 29) . However, neither a selective
inhibitor of PKC nor depletion of intracellular Ca
stores blocked the rPMT-stimulated tyrosine phosphorylation of
p125
. Thus, rPMT induces tyrosine phosphorylation of
p125
by a pathway largely independent of PKC activation
and Ca
mobilization.
The effects of many bacterial
toxins require them to bind and enter cells via endocytotic
pathways(1, 57, 58) . Activation of some
bacterial toxins is believed to occur during their transit through the
endosomal/lysosomal compartments. In these acidic compartments, the
toxin may be activated by proteolysis or by conformational changes.
Interestingly, rPMT becomes susceptible to proteolysis at lysosomal pH
values(59) . Membrane-permeant weak bases that increase the pH
of the acidic intracellular compartments block the process of
activation. The characteristic lag period in the action of many toxins
is a manifestation of the complex events of entry and activation.
Several lines of evidence indicate that the stimulation of protein
tyrosine phosphorylation by rPMT also requires cell entry and
activation of the toxin. For example, rPMT induced tyrosine
phosphorylation of multiple substrates including p125 and
paxillin after a pronounced (1 h), cycloheximide-insensitive, lag
period and its effect was selectively blocked by the lysosomotrophic
agent methylamine or by reducing the temperature of incubation to 22
°C, which prevents vesicular trafficking(48) . Other
responses induced by rPMT, including polyphosphoinositide hydrolysis,
Ca
mobilization, PKC activation, and commitment to
DNA synthesis exhibit similar
characteristics(5, 16, 18) . Recently,
colloidal gold-labeled rPMT has been shown to be rapidly internalized
into endocytic vesicles in toxin-sensitive cell lines(60) .
Together, these findings suggest that rPMT enters the cells via
endosomal/lysosomal compartments and then initiates events leading to
the activation of signal transduction pathways, including p125
and paxillin tyrosine phosphorylation.
Tyrosine
phosphorylation of p125 and paxillin stimulated by
bombesin, LPA, sphingosine, and PDGF is closely related to changes in
the organization of the actin microfilament network induced by these
ligands in Swiss 3T3 cells(29, 31, 38) . The
cytoskeletal changes induced by LPA and bombesin require functional
p21
protein(40) . In addition, bombesin and LPA
stimulate tyrosine phosphorylation by a pathway critically dependent on
the integrity of the actin
cytoskeleton(28, 29, 31) . These findings
raised the possibility that tyrosine phosphorylation of p125
and paxillin, actin stress fiber formation, focal adhesion
assembly and p21
function may lie in a novel signal
transduction pathway. Having established that rPMT induces p125
and paxillin tyrosine phosphorylation, it was, therefore, of
interest to determine whether this toxin can also induce changes in the
organization of the actin cytoskeleton. In addition, the polymerization
of the actin cytoskeleton has been postulated to play an important role
in the endocytosis of many bacteria by animal cells(61) .
Here we report that rPMT elicits dramatic cytoskeletal responses in
quiescent Swiss 3T3 cells. Specifically, rPMT induces striking
formation of actin stress fibers and focal adhesion plaques in these
cells. This is the first time that rPMT has been shown to induce
accumulation of actin stress fibers and to promote focal adhesion
assembly in any cell type. PMT shows amino acid sequence homology with
the NH-terminal region of CNF1 and CNF2 produced by
pathogenic E. coli strains(14, 15) .
Interestingly, the E. coli toxins have been shown to induce
reorganization of actin cytoskeleton which has been postulated to block
cytokinesis leading to multinucleated cells. In contrast, rPMT induces
actin reorganization that does not interfere with cell division since
rPMT promotes striking proliferation in fibroblast cell
lines(4, 5) . It would be interesting, therefore to
compare the cytoskeletal responses induced by these toxins in the same
cell type.
The kinetics of the cytoskeletal responses induced by
rPMT closely parallel the time course of rPMT stimulated tyrosine
phosphorylation. Pretreatment of quiescent Swiss 3T3 with cytochalasin
D completely disrupted the actin cytoskeleton and blocked the tyrosine
phosphorylation of p125 stimulated by rPMT. Thus, the
integrity of the actin cytoskeleton is essential for rPMT induced
tyrosine phosphorylation.
Recent results from our laboratory
revealed that PDGF at high concentrations caused disruption of the
actin cytoskeleton(31, 38) . As tyrosine
phosphorylation and actin stress fiber formation induced by rPMT appear
to be closely linked in Swiss 3T3 cells, we examined a possible
cross-talk between PDGF and rPMT on actin stress fiber organization and
p125 tyrosine phosphorylation. PDGF at a high
concentration (30 ng/ml) profoundly inhibited rPMT induced tyrosine
phosphorylation of p125
establishing a novel cross-talk
between PDGF and rPMT on tyrosine phosphorylation. This can be
explained by the ability of PDGF to interfere with rPMT induced actin
stress fiber organization and focal adhesion assembly in rPMT-treated
cells.
In view of these results it was plausible, that p21 could also be involved in rPMT induced cytoskeletal changes and
tyrosine phosphorylation. Microinjection of C3 C. botulinum exoenzyme, which ADP-ribosylates and inactivates p21
function, prevented tyrosine phosphorylation of focal adhesion
proteins in response to rPMT. Thus, p21
is upstream of
tyrosine phosphorylation in response to rPMT.
Most normal cells
require contact with an adhesive substratum to proliferate and
oncogenic transformation removes this requirement for
adherence(62) . Integrin-mediated signals, including tyrosine
phosphorylation of focal adhesion proteins (34, 35, 36, 37) have been
implicated in promoting anchorage-dependent
growth(41, 63, 64) . It is conceivable that
growth factors and oncogenes that induce anchorage-independent growth
mimic integrin-mediated signals. Interestingly, we have previously
reported that rPMT is a potent inducer of anchorage-independent colony
formation in certain target cells(4) . A salient feature of the
results presented here is that the striking increase in p125 and paxillin tyrosine phosphorylation, actin stress fiber
formation and focal adhesion assembly induced by rPMT remain
undiminished even after 24 h of incubation. It is tempting to speculate
that rPMT circumvents the requirement for integrin-mediated signals
generated in adherent cells as a result of its striking and persistent
effects on the organization of the cytoskeleton and the tyrosine
phosphorylation of focal adhesion proteins, a proposition that deserves
further experimental work.
In conclusion, our results demonstrate,
for the first time, that rPMT stimulates tyrosine phosphorylation of
multiple bands including p125 and paxillin. Furthermore,
rPMT induces striking increase in the formation of actin stress fiber
and focal adhesion assembly in Swiss 3T3 cells. The integrity of the
polymerized actin network and functional p21
are
essential for rPMT-induced tyrosine phosphorylation. To our knowledge,
this is the first time, that an intracellularly acting bacterial toxin
has been shown to induce protein tyrosine phosphorylation in animal
cells.