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(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
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) ( Prior to the stimulation of DNA synthesis, rPMT
stimulates the formation of inositol phosphates and mobilizes
Ca 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 Here we report
that rPMT stimulates tyrosine phosphorylation of multiple proteins
including p125
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
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
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
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 CH
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
Figure 3:
Effect of GF109203X and thapsigargin on
the stimulation of tyrosine phosphorylation of p125
To
investigate whether Ca
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
Figure 5:
Cytochalasin D inhibits rPMT-stimulated
tyrosine phosphorylation of multiple bands including
p125
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 [
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
Figure 7:
PDGF blocks tyrosine phosphorylation of
p125
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 rPMT increases the accumulation of the second
messengers diacylglycerol and inositol 1,4,5-trisphosphate, which
activate PKC and mobilize Ca 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 Tyrosine
phosphorylation of p125 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 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 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 In view of these results it was plausible, that p21 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 In conclusion, our results demonstrate,
for the first time, that rPMT stimulates tyrosine phosphorylation of
multiple bands including p125
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 439-445
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
and Paxillin Tyrosine Phosphorylation, Actin Stress
Fiber Formation, and Focal Contact Assembly in Swiss 3T3 Cells (*)
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.
)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. 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.
(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.
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.
Cell Culture
Stock cultures of Swiss 3T3
fibroblasts were maintained in DMEM supplemented with 10% FBS in a
humidified atmosphere containing 10% CO
and 90% air at 37
°C. For experimental purposes, cells were plated either in 30-mm
Nunc Petri dishes at 10
cells/dish, or in 90-mm dishes at 6
10 cells/dish, in DMEM containing 10% fetal bovine
serum and used after 6-8 days when the cells were confluent and
quiescent(43) .Immunoprecipitation
Quiescent cultures of Swiss
3T3 cells (1-2 10
) were washed twice with
DMEM, treated with rPMT or other factors in 10 ml of DMEM/Waymouth 1:1
(v/v) for the times indicated, and lysed at 4 °C in 1 ml of a lysis
buffer solution containing 10 mM Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50
mM NaF, 100 µM Na
VO
, and
1% Triton X-100. Proteins were immunoprecipitated at 4 °C overnight
with agarose-linked mAbs directed against phosphotyrosine, paxillin, or
p125 as indicated. Immunoprecipitates were washed three
times with lysis buffer and extracted for 10 min at 95 °C in 2
SDS-PAGE sample buffer (200 mM Tris/HCl, 6% SDS, 2
mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8) and
analyzed by SDS-PAGE.Western Blotting
Treatment of quiescent cultures
of cells with factors, cell lysis, and immunoprecipitations were
performed as described above. After separation by SDS-PAGE, proteins
were transferred to Immobilon membranes(44) . Membranes were
blocked using 5% nonfat dried milk in PBS, pH 7.2, and incubated for
3-5 h at 22 °C with a mixture of PY20 and 4G10 anti-Tyr(P)
mAbs (1 µg/ml of each). Immunoreactive bands were visualized using I-labeled sheep anti-mouse IgG followed by
autoradiography. Autoradiograms were scanned using an LKB Ultrascan XL
internal integrator. The values expressed represent percentages of the
maximum increase in tyrosine phosphorylation above control values.
Quiescent and confluent cultures of
Swiss 3T3 cells in 30-mm dishes were washed twice in phosphate-free
DMEM and incubated at 37 °C with this medium containing 50
µCi/ml carrier-free [P Labeling of Cells and Analysis of
80K/MARCKS Phosphorylation
P]P
. After
12 h, various factors were added for the indicated times. The cells
were subsequently lysed, and the lysates were immunoprecipitated with
specific anti-80K/MARCKS antibody(45) .Immunostaining of Cells
Quiescent Swiss 3T3 cells
were washed twice with DMEM and incubated for the indicated time in
DMEM/Waymouth medium 1:1 (v/v) at 37 °C with the indicated
concentration of rPMT or other factors. For actin staining, cells were
washed once with PBS, fixed in 4% paraformaldehyde in PBS for 10 min at
room temperature, and permeabilized with PBS containing 0.2% Triton
X-100 for 8 min at room temperature. The cells were then incubated with
TRITC-conjugated phalloidin (0.25 µg/ml) in PBS for 10 min at room
temperature and visualized utilizing a Zeiss Axiophot
immunofluorescence microscope. In experiments in which quiescent Swiss
3T3 cells were labeled with both TRITC-conjugated phalloidin and
anti-vinculin antibody, the cells were fixed and permeabilized as
described above and then stained with a mixture of TRITC-conjugated
phalloidin (0.25 µg/ml) and anti-vinculin antibody (dilution 1:100)
for 30 min at room temperature. Cells were subsequently washed three
times in PBS and then incubated with FITC-labeled anti-mouse IgG as a
second antibody at a dilution of 1:100 for another 30 min at room
temperature.Microinjection
For C3 exoenzyme microinjection
experiments Swiss 3T3 cells were plated in 30-mm dishes at 10 cells/dish in DMEM containing 10% fetal bovine serum and used
after 6-8 days when the cells were confluent and quiescent. To
facilitate localization of microinjected cells, a circle was scored on
the bottom of the dishes, and in each experiment approximately 60
adjacent cells in the circle were microinjected. The efficiency of
injection was determined by co-injecting rabbit immunoglobulin at 0.5
mg/ml followed by staining of the cells with FITC-conjugated
anti-rabbit IgG antibody. Cells were stained with 4G10 mAb directed
against phosphotyrosine residues and visualized by confocal microscopy
using Cy5-linked anti-mouse IgG.Confocal Microscopy
Confocal imaging was performed
using a Bio-Rad MRC 600 laser scanning head fitted to a Nikon Optiphot
microscope. A 40 numerical aperture/1.4 planapochromat oil
immersion lens (Nikon) was used for all imaging. FITC- and
TRITC-conjugated phalloidin fluorochromes were excited at 488 and 568
nm, respectively, using a krypton/argon mixed gas laser (Bio-Rad). Two
filter blocks were used, K1 and K2. K1 is a double dichromic filter
enabling excitation at the wavelengths of 488 and 568 nm, whereas the
K2 filter is a 560-nm dichromic combined with 522-nm green emission and
585-nm red emission filters. Images were collected using the Kalman
filter. Care was taken to ensure that the TRITC-conjugated phalloidin
channel was sufficiently bright relative to the fluorescein signal to
minimize the contribution of bleed-through from the green channel into
the red channel (approximately 10%). Correction of images for
bleed-through and other processing was carried out using COMOS and SOM
programs (Bio-Rad) run on a Compaq Deskpro 66M 486 computer (66 MHz).
Data are presented as projections of sequential optical sections. For
Z-series, optical sections were recorded at 0.5 µm. Final images
were photographed directly from the VDU screen (see Fig. 4, Fig. 6, and Fig. 8).
; 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.
Materials
Phorbol 12,13-dibutyrate, bombesin,
cytochalasin D, TRITC-conjugated phalloidin, monoclonal anti-vinculin
antibody, and FITC-linked anti-mouse IgG were obtained from Sigma. The
specific PKC-inhibitor GF109203X and thapsigargin were obtained from
Calbiochem-Novabiochem Ltd. Nottingham, UK. Agarose-linked anti-Tyr(P)
mAb was purchased from Oncogene Science Inc., Manhasset, NY. PY20
anti-Tyr(P) mAb and the mAb directed against paxillin (mAb 165) were
from ICN, High Wycombe, UK. 4G10 anti-Tyr(P) mAb was from Upstate
Biotechnology, Inc., Lake Placid, NY. mAb 2A7 directed against
p125 was from TCS Biologicals Ltd., Buckingham, UK.
p125
immunoblotting was performed with mAb from
Transduction Laboratories, Lexington, KY. The anti-Tyr(P) mAb PY72 was
obtained from the hybridoma development unit, Imperial Cancer Research
Fund.
I-labeled sheep anti-mouse IgG (15 mCi/mg),
carrier-free [
P]P
, and recombinant
BB homodimer PDGF were from Amersham Corp., UK. rPMT was prepared as
described previously(13) . All other reagents were of the
highest grade commercially available. The C3 Clostridium botulinum exoenzyme was a gift from Dr. N. Morii and Professor S. Narumiya,
Department of Pharmacology, Kyoto University Faculty of Medicine,
Sakyo-ku 606, Japan.
rPMT Stimulates Tyrosine Phosphorylation in Swiss 3T3
Cells
To examine the effect of rPMT on tyrosine phosphorylation,
quiescent cultures of Swiss 3T3 cells were treated with 20 ng/ml rPMT
for 6 h, conditions known to result in the stimulation of DNA synthesis (5) . The cells were lysed, the lysates were incubated with
agarose linked anti-Tyr(P) mAb, and the resulting immunoprecipitates
were analyzed by Western blotting with a mixture of PY20 and 4G10
anti-Tyr(P) mAbs. As shown in Fig. 1A, rPMT markedly
stimulated the tyrosine phosphorylation of a group of bands migrating
with an apparent M of 110,000-130,000 and
70,000-80,000. The ability of rPMT to induce tyrosine
phosphorylation of these proteins was completely abolished by PMT
antiserum in a selective manner (Table 1).
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.
(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.rPMT Enters the Cells to Elicit Tyrosine
Phosphorylation
Several lines of evidence presented here
indicate that rPMT has to enter cells to induce tyrosine
phosphorylation of multiple proteins including p125 and
paxillin. (i) Neuropeptides and LPA elicit maximum tyrosine
phosphorylation of p125
and paxillin within minutes of
addition to the cell cultures. In contrast, there is a lag period of 1
h between the addition of 20 ng/ml PMT and a detectable increase in
tyrosine phosphorylation of these proteins (Fig. 1B).
This lag period did not reflect a requirement for de novo protein synthesis as cycloheximide at 25 µM, a
concentration that inhibits protein synthesis in Swiss 3T3
cells(47) , did not prevent the increase in protein tyrosine
phosphorylation in response to 20 ng/ml rPMT. In addition, we verified
that similar amounts of p125
were recovered after
different times of treatment with rPMT (results not shown).
Interestingly, the enhanced tyrosine phosphorylation of several
proteins including p125
and paxillin induced by rPMT
persisted for at least 24 h (Fig. 1B). (ii) The
lysosomotrophic agent methylamine, a membrane-permeant weak base known
to inhibit lysosomal processing, completely blocked tyrosine
phosphorylation of the M
110,000-130,000 and M 70,000-80,000 bands in response to rPMT.
The inhibitory effect of methylamine was selective because it did not
prevent the increase in tyrosine phosphorylation induced by bombesin in
parallel cultures (Fig. 2, A and B). (iii) The
entry of many bacterial toxins into the cell cytoplasm is
temperature-dependent(48) . As shown in Fig. 2, A and B, treatment with 20 ng/ml rPMT for 6 h at 22 °C
failed to stimulate tyrosine phosphorylation in Swiss 3T3 cells. In
contrast, bombesin induced tyrosine phosphorylation of M 110,000-130,000 and M 70,000-80,000 bands in parallel cultures of these cells
incubated at 22 °C. (iv) Many bacterial toxins that enter the cells
cannot be removed by extensive washing(1) . Fig. 2C demonstrates that the increase in tyrosine phosphorylation in
rPMT-treated cells persisted after removal of the toxin from the
extracellular medium. A transient exposure of cells to the toxin for
3-h stimulated maximum tyrosine phosphorylation. Thus, the toxin
appears to enter the cells via an endosomal/lysosomal pathway where it
is processed and then released into the cytosol in an active form.
NH 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.
Role of PKC and Ca
rPMT induces a striking
mobilization of Ca on
rPMT-induced Tyrosine Phosphorylation
and stimulation of PKC in Swiss
3T3 cells (16, 18, 49) . It is known that PKC
activation leads to increased tyrosine phosphorylation of p125
and paxillin (28, 29) . Consequently, we
examined whether these signaling pathways could be involved in
rPMT-induced tyrosine phosphorylation.
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).
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.
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.
rPMT Stimulates Actin Stress Fiber and Focal Contact
Formation
Given the localization of p125 and
paxillin to focal contacts, which form at the end of actin stress
fibers, we examined the effect of rPMT on actin cytoskeleton
organization and focal adhesion assembly. Actin filaments were
visualized with TRITC-labeled phalloidin, and vinculin was detected by
immunofluorescence with a anti-vinculin mAb. As shown in Fig. 4,
quiescent Swiss 3T3 cells have few actin stress fibers. Addition of 20
ng/ml rPMT to quiescent Swiss 3T3 cells induced a striking increase in
the formation of actin stress fibers. Formation of new stress fibers
was detected after a lag period of approximately 1 h and reached a
maximum after 8 h when the cells contained numerous densely packed
stress fibers (Fig. 4, left).
-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.
Cytochalasin D Inhibits rPMT-stimulated Tyrosine
Phosphorylation of Multiple Bands Including
p125
The striking effects of PMT on stress fiber
formation and focal adhesion assembly shown in Fig. 4prompted
us to examine whether the integrity of the actin filament network is
necessary for rPMT stimulated tyrosine phosphorylation. Quiescent Swiss
3T3 cells were pretreated for 1 h with increasing concentrations of
cytochalasin D and then stimulated with 20 ng/ml rPMT for 6 h.
Cytochalasin D blocked rPMT-induced tyrosine phosphorylation of all
phosphorylated bands including tyrosine phosphorylation of p125 in a similar, concentration-dependent manner (Fig. 5A). A complete inhibition of p125
tyrosine phosphorylation was achieved at the concentration of 1.2
µM cytochalasin D.
. 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.
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.Effect of High Concentrations of PDGF on rPMT-induced
Actin Stress Fiber Formation and p125
Recent data from our laboratory have shown that
PDGF at high concentrations (30 ng/ml) abolishes bombesin- and
LPA-induced actin stress fiber and focal
contacts(31, 38) . This prompted us to investigate the
effect of PDGF on rPMT induced stress fiber formation. Quiescent Swiss
3T3 cells were treated with 20 ng/ml rPMT for 4 h, and then 30 ng/ml
PDGF was added for 10 min. Cells were then fixed and stained with
TRITC-conjugated phalloidin. As shown in Fig. 6B, rPMT
caused a marked increase in stress fiber formation, whereas 30 ng/ml
PDGF caused disruption of the actin stress fibers and retraction of
cell bodies (Fig. 6C). Interestingly, addition of 30
ng/ml PDGF to rPMT-treated cells reduced the number of actin stress
fibers (Fig. 6D) and focal contacts (results not shown)
induced by rPMT. The remaining stress fibers in rPMT- and PDGF-treated
cells lost their unidirectional arrangement as tightly packed bundles.
In contrast, PDGF at 5 ng/ml did not disrupt the actin reorganization
induced by rPMT (results not shown). Tyrosine
Phosphorylation
or paxillin was markedly inhibited by addition of 30
ng/ml PDGF but not by 5 ng/ml PDGF.
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.
Microinjection of C. botulinum C3 Exoenzyme Inhibits
rPMT-induced Tyrosine Phosphorylation at Focal Contacts
The rho gene product p21 has been implicated in the
neuropeptide-stimulated formation of focal adhesions, actin stress
fibers, and in tyrosine phosphorylation of p125
and
paxillin(40, 53, 54, 55) . To
investigate the role of p21
in the rPMT-stimulated
tyrosine phosphorylation of focal adhesion-associated proteins we
utilized the C. botulinum C3 exoenzyme which ADP-ribosylates
Asn
of p21
and thereby prevents its
function(56) . Recombinant C3 exoenzyme was microinjected at a
concentration of 100 µg/ml into confluent and quiescent Swiss 3T3
cells, and the cultures were further treated with 20 ng/ml rPMT for 4
h. Cells were then fixed, permeabilized and stained for tyrosine
phosphorylated proteins which are predominantly localized at the focal
contacts in rPMT treated cells (Fig. 8). The tyrosine
phosphorylation of focal adhesion proteins in response to rPMT was
profoundly inhibited in cells microinjected with C3 exoenzyme
(indicated by the arrows in Fig. 8, right). In
parallel experiments, Swiss 3T3 cells were microinjected only with
immunoglobulin and then stimulated with 20 ng/ml rPMT. These cells
display the typical pattern of tyrosine phosphorylated proteins
demonstrating that microinjection itself did not interfere with
rPMT-induced tyrosine phosphorylation of focal adhesion proteins (Fig. 8, left).
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.
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.
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.
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) .
-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. stimulated by rPMT. Thus, the
integrity of the actin cytoskeleton is essential for rPMT induced
tyrosine phosphorylation.
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.
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.
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.
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.
), p125 focal adhesion
kinase; PKC, protein kinase C; TRITC, tetreamethylrhodamine B
isothiocyanate; PAGE, polyacrylamide gel electrophoresis; FITC,
fluorescein isothiocyanate; LPA, lysophosphatidic acid.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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