From the Unité des Interactions
Bactéries-Cellules, 28 rue du Dr. Roux, Institut Pasteur, 75724 Paris, Cedex 15, France and the
INSERM Unité 326, Hôpital Purpan, 31059 Toulouse, France
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ABSTRACT |
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The Gram-positive pathogen Listeria
monocytogenes induces its own internalization into some
non-phagocytic mammalian cells by stimulating host tyrosine
phosphorylation, phosphoinositide (PI) 3-kinase activity, and
rearrangements in the actin cytoskeleton. Entry into many cultured cell
lines is mediated by the bacterial protein InlB. Here we investigate
the role of InlB in regulating mammalian signal transduction and
cytoskeletal structure. Treatment of Vero cells with purified InlB
caused rapid and transient increases in the lipid products of the PI
3-kinase p85-p110, tyrosine phosphorylation of the mammalian adaptor
proteins Gab1, Cbl, and Shc, and association of these proteins with
p85. InlB also stimulated large scale changes in the actin
cytoskeleton (membrane ruffling), which were PI
3-kinase-dependent. These results identify InlB as
the first reported non-mammalian agonist of PI 3-kinase and demonstrate
similarities in the signal transduction events elicited by this
bacterial protein and known agonists such as epidermal growth factor.
Infection of viral, bacterial, or protozoan intracellular
pathogens often involves subversion of mammalian signaling pathways. The first step in the life cycle of an intracellular pathogen is
internalization by the host cell. While some microbial pathogens are
taken up only by professional phagocytes (macrophages or neutrophils), others induce their own uptake ("enter") into cells that are not normally phagocytic, such as epithelial cells underlying mucosal surfaces or endothelial cells inside blood vessels (reviewed in Ref.
1). Entry into non-phagocytic cells can permit traversal of
tissue-specific barriers and promote survival of the pathogen by
providing access to a nutrient-rich environment that is protected from
host antibody or complement.
Listeria monocytogenes is a food-borne bacterial pathogen
that causes severe illnesses leading to meningitis or abortions in
immunocompromised individuals or pregnant women (2). This faculative
intracellular pathogen enters into cells that are non-phagocytic, including epithelial cells and hepatocytes. Entry into such cells is
likely to play an important role in traversing the intestinal, blood-brain, and placental barriers, and in colonization of the liver
(reviewed in Ref. 3).
Entry of L. monocytogenes into non-phagocytic cells involves
specific bacterial surface proteins that exhibit cell tropism. The
bacterial protein InlA (internalin) promotes entry into the intestinal
epithelial cell line Caco-2, whereas the protein InlB mediates
internalization into several other cultured cell lines, including Vero,
HEp-2, HeLa, and some hepatocytes (4-6). InlB has a role in virulence
in the mouse model, as a bacterial mutant ( Entry of L. monocytogenes requires tyrosine phosphorylation
and other signal transduction events that are likely to occur downstream of engagement of E-cadherin or the InlB receptor (3, 9). One
of the ultimate effects of such signaling is to promote rearrangements
in the actin cytoskeleton that drive the entry process. One of the
mammalian signaling proteins that controls bacterial entry is the
phosphoinositide (PI)1
3-kinase p85-p110 (6). Infection with L. monocytogenes
induces accumulation of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 in Vero cells, indicating that this
bacterium activates PI 3-kinase. Activation of p85-p110 is required for
bacterial uptake, since entry is blocked by genetic or pharmacological
inhibition of p85-p110. Bacterial stimulation of PI 3-kinase is
accompanied by interaction of p85 with one or more tyrosine
phosphorylated host proteins, indicating that tyrosine phosphorylation
is involved in signaling mediated by p85-p110. The specific
phosphoproteins that associate with p85 have not been identified.
p85-p110 is a heterodimeric lipid kinase, composed of an 85-kDa
regulatory subunit and a 110-kDa catalytic subunit, which is activated
in response to engagement of cell surface receptors by some growth
factors or cytokines. Activation of p85-p110 is often promoted by
recruitment to the plasma membrane through interaction of SH2 domains
in p85 with tyrosine-phosphorylated proteins (10). Activation of
p85-p110 and some other PI 3-kinases in vivo is characterized by rapid and often transient increases in levels of
PtdIns(3,4)P2 and PtdIns(3,4,5)P3,
phosphoinositides that appear to act as membrane-bound second
messengers to regulate the cellular localization and/or activity of
various signaling proteins (11). p85-p110 controls several critical
processes in mammalian cells, including cell growth, vesicular
trafficking, apoptosis, and organization of cytoskeletal actin.
Although the function of p85-p110 in entry of L. monocytogenes is not understood, an attractive possibility is that
this kinase controls cytoskeletal changes needed for bacterial uptake.
In this report, we identify one of the bacterial factors that activates
PI 3-kinase in mammalian cells. A recombinant InlB protein stimulated
accumulation of the lipid products of p85-p110, tyrosine
phosphorylation of three mammalian adaptor proteins, and the
association of these adaptor proteins with p85. InlB also provoked PI
3-kinase-dependent membrane ruffling. These findings establish InlB as the first reported non-mammalian agonist of p85-p110
and suggest similarities between signal transduction promoted by this
bacterial protein and some mammalian growth factors.
Cell Lines and Media--
The African green monkey kidney cell
line Vero was grown at 37 °C in 5 or 10% C02 in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum,
2 mM glutamine, and non-essential amino acids. All
experiments involving bacterial infections or treatment of cells with
InlB or EGF were performed at 37 °C in 5 or 10% C02.
Antibodies, Inhibitors, and Other Materials--
Polyclonal
antiserum against rat p85 Purification of InlB--
We constructed an expression plasmid
by polymerase chain reaction amplification of DNA containing the
inlB coding sequence from the plasmid pPE3 (13) using the
primers 5'-(CTA)GCTAGCGAGACTATCACCGTGTCA-3' and
5'-(CCC)AAGCTTGTTGTAGCTTTTTCGTAGG-3' (underlined
sequences indicate NheI and HindIII sites,
respectively) and Vent DNA polymerase. The purified polymerase chain
reaction product was digested and cloned between the NheI
and HindIII sites in the expression vector pET28 (+)
(Novagen), to yield plasmid pKI22. pKI22, verified by DNA sequence
analysis, allows expression of a recombinant form of InlB that contains
a 6xHis tag at the NH2 terminus of the protein. Cleavage of
the recombinant protein with thrombin is predicted to yield a protein
that is identical to full-length mature InlB (i.e. lacking
the signal peptide) with the amino acids GSHMAS added to the
NH2 terminus. pIK22 was transformed into the
Escherichia coli strain BL21 Measurements of in Vivo Levels of PtdIns(3,4)P2 and
PtdIns (3,4,5)P3--
Approximately 8 × 105 Vero cells were seeded in 10-cm tissue culture dishes
(78.5 cm2 in surface area) and grown in DMEM with 10%
fetal calf serum for approximately 30 h. Cells were then starved
overnight in medium containing DMEM with 0.2% fetal calf serum and
labeled for 5 h by incubation in serum-free DMEM without phosphate
(Sigma) containing 200 µCi/ml 32Pi. Cells
were left untreated or stimulated by addition of InlB or EGF to the
indicated concentrations and incubation for the indicated times. When
appropriate, wortmannin or the solvent Me2SO was added 20 min before treatment with InlB. After stimulation, cells were washed
once with cold phosphate-buffered saline, reactions were stopped by
addition of cold 2.4 N HCl, and cells were recovered from
the dishes by scraping. Lipids were isolated, separated by thin layer
chromatography, deacylated, and quantitated on an high performance
liquid chromatography system with a Whatman Partisphere SAX column as
described (6, 14). We verified that activation of PI 3-kinase was not
due to trace amounts of thrombin that potentially had failed to be
removed by the p-aminobenzamidine resin. Uncut InlB protein
that had not been treated with thrombin also stimulated increases in
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in Vero
cells, indicating that the protein was active in the absence of
thrombin treatment. In addition, 300 ng/ml amounts of the same (cut)
preparation of InlB used for the experiments in Fig. 1 failed to induce
shape changes or aggregation in platelets. (Platelets are extremely sensitive to thrombin and aggregate in response to even trace amounts
of this protease).
Infection of Cell Monolayers with Bacterial
Strains--
Cultures of wt L. monocytogenes strain EGD
(BUG600) or the Immunoprecipitation and Western Blotting--
Approximately
8 × 105 Vero cells were seeded in 75-cm2
tissue culture flasks or 10-cm plates and grown for 40 h. Cells
were then starved for 5 h in serum-free DMEM and treated with InlB or EGF or infected with L. monocytogenes strains as
described above. In some experiments, cells were pretreated with
Me2SO or AG1478 for 20 min prior to addition of InlB or
EGF. After incubation for the indicated times, cells were rinsed with
10 ml of cold phosphate-buffered saline and solubilized by addition of
1.0 ml of ice-cold immunoprecipitation buffer (1% Nonidet P-40, 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM
EDTA, 3 mM sodium orthovanadate, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, leupeptin, pepstatin, and chymostatin). Preclearance and
immunoprecipitations were performed at 4 °C as described (6, 14).
After preclearance, protein concentrations of the lysates were
determined and equal quantities of total protein were used for
immunoprecipitations (typically 0.70 mg total protein per
immunoprecipitation). After the incubation and washing steps,
immunoprecipitates were denatured by boiling in sample buffer for 5 min, and then stored at Analysis of Cytoskeletal Changes Induced by InlB--
4 × 104 Vero cells were seeded onto 22 cm × 22-cm
coverslips, grown for 48 h, and incubated in serum-free DMEM for
5 h. Cells were pretreated for 20 min with Me2SO or
wortmannin as indicated and then incubated with 200 ng/ml InlB for 5 min. Cells were fixed by incubation for 20 min at room temperature in
cytoskeleton buffer (pH 6.8) (16) containing 3% paraformaldehyde, free
amino groups were quenched in cytoskeleton buffer containing 50 mM NH4Cl, the fixed monolayers were
permeabilized by a 5-min incubation in cytoskeleton buffer with 0.4%
Triton X-100, and F-actin was labeled by a 60-min incubation in TBS (pH
7.5) containing FITC-phalloidin (Sigma) and 0.1% gelatin. For analysis
of F-actin in cells infected with L. monocytogenes or
S. typhimurium strain SL1344, 6 × 104 Vero
cells were seeded per coverslip and grown as above. Monolayers were
infected as described and incubated for 5 min (S. typhimurium) or 10 min (EGD). Fixation, permeabilization, and
labeling with FITC-phalloidin were as above. Fixed monolayers were also
labeled for bacteria by incubation with polyclonal antisera raised
against heat-killed L. monocytogenes (R11) or against
lipopolysaccharide of S. typhimurium, followed by secondary
goat anti-rabbit antibodies conjugated to Texas Red (Molecular Probes).
Samples were examined with a laser scanning confocal microscope (Wild
Leitz or Zeiss).
Quantitation of Ruffling--
Vero cells grown on coverslips
were examined for the presence of ruffles (actin-rich membrane folds
shown in Figs. 7 and 8) using a conventional fluorescence microscope
(Nikon Optiphot 2) with an FITC filter. Cells that had no actin-rich
ruffles were scored as negative, whereas cells that had one or more
ruffle were considered to be "ruffle-positive." In each experiment,
approximately 100 cells were examined for each condition. Only 1 ± 2% (mean ± S.D. of five experiments) of control cells had
membrane ruffles, whereas 77 ± 13% of cells had ruffles after
treatment with 3 nM InlB for 5 min. 2 ± 3% (three
experiments) of cells pretreated with wortmannin formed ruffles in
response to InlB.
InlB Stimulates PI 3-Kinase Activity in Mammalian
Cells--
Infection of Vero cells with a bacterial strain deleted for
the inlB gene (
In order to determine whether InlB is sufficient to activate p85-p110,
we tested a recombinant InlB protein (Fig.
1A) for stimulation of
mammalian PI 3-kinase activity. Treatment of Vero cells for 1 min with
soluble InlB at 4.5 nM (300 ng/ml) (a concentration determined to be saturating for the association of p85 in
anti-phosphotyrosine (Tyr(P)) immunoprecipitates (Fig.
2A; see below) caused
increases in the cellular amounts of the two products of p85-p110,
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Fig.
1B). In contrast, InlB treatment had no significant effect
on the levels of the phosphoinositide PtdIns(4,5)P2, which
is not a product of PI 3-kinase (Fig. 1B).
InlB-mediated increases in PtdIns(3,4)P2 and PtdIns
(3,4,5)P3 in Vero cells were comparable in magnitude to
those caused by EGF (Fig. 1B), a known agonist of p85-p110
in some cultured cells (17). Both the InlB- and EGF-induced
accumulation of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 were transient, being maximal at the
earliest time point examined (1 min) and declining
thereafter.2 Accumulation of
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in Vero cells in response to EGF is blocked by pretreatment of cells with the PI
3-kinase inhibitor wortmannin (19). Similarly, treatment of Vero cells
with wortmannin (50 nM) caused a 100 ± 0% (mean ± S.D. of two experiments) and a 96 ± 4% (three experiments)
inhibition in the InlB-induced increases of PtdIns(3,4)P2
and PtdIns(3,4,5)P3. These results indicate that InlB is an
agonist of PI 3-kinase and that activation of p85-p110 by this
bacterial protein shares some similarities with activation by EGF.
InlB Induces Interaction between p85 and One or More
Tyrosine-phosphorylated Protein(s)--
Stimulation of p85-p110 by
many agonists, including EGF, is accompanied by the interaction of p85
with tyrosine-phosphorylated proteins. Binding of p85 to such proteins,
which are often membrane-associated, may allow recruitment of p85-p110
to the plasma membrane and access to its membrane-bound substrates
(10).
We had demonstrated previously that infection with L. monocytogenes induces the association of p85 with one or more
tyrosine-phosphorylated protein(s) in the mammalian cell. This
association partly depends on inlB and is concomitant with
bacterial-induced accumulation of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 (6). Similarly, treatment of Vero cells
with purified InlB induced the appearance of p85 in
anti-phosphotyrosine (Tyr(P)) immunoprecipitates (Figs. 2 and 3A), but did not cause
detectable tyrosine phosphorylation of p85 itself (Fig. 3B).
These results indicate that InlB stimulates an interaction between p85
and tyrosine-phosphorylated protein(s). Stimulation was optimal at
about 0.75 nM (50 ng/ml) or higher concentrations (Fig.
2A). Association of p85 with phosphoprotein(s) was maximal
at the earliest time point examined (1 min) and declined thereafter
(Fig. 2B). These kinetics correlated well with the kinetics
of accumulation of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 in InlB-treated cells. Taken together,
these results suggest that tyrosine phosphorylation is involved in PI
3-kinase-mediated signaling induced by InlB.
InlB Stimulates Tyrosine Phosphorylation of the Adaptor Proteins
Gab1, Cbl, and Shc and Interaction of These Proteins with p85--
In
order to identify the tyrosine-phosphorylated proteins that interact
with p85, we immunoprecipitated p85 from cells treated with InlB and
detected tyrosine-phosphorylated proteins by Western blotting with
anti-Tyr(P) antibodies. Bands corresponding to proteins of
approximately 110-120 kDa were associated with p85 in lysates from
InlB-treated cells (Fig. 3B, lane 2). We also
detected a less prominent band corresponding to a protein of
approximately 52 kDa. These 110-120- and 52-kDa phosphoproteins were
barely detectable or absent in anti-p85 immunoprecipitates prepared
from untreated cells (Fig. 3B, lane 1).
Incubation of cells with equivalent molar amounts of the invasion
protein InlA (8) or a recombinant (6 × His-tagged) form of the
L. monocytogenes transcription factor PrfA (18) failed to
induce association of tyrosine-phosphorylated proteins with p85,
demonstrating that the effects of InlB were relatively specific (data
not shown).
We tested if the 110-120-kDa phosphoproteins were focal adhesion
kinase (FAK), Gab1, or Cbl, proteins known to interact with p85 upon
stimulation with certain agonists (14, 17, 21-24). Treatment of Vero
cells with InlB stimulated tyrosine phosphorylation of Gab1 and Cbl
(Fig. 3, C and D), but did not cause increased tyrosine phosphorylation of FAK.2 Treatment with InlB also
caused increases in the amount of p85 found in anti-Gab1 and anti-Cbl
immunoprecipitates (Fig. 3, C and D), but not in
anti-FAK immunoprecipitates. We tested if the 52-kDa
tyrosine-phosphorylated protein associated with p85 was an isoform of
the adaptor protein Shc, which is often present as 48-, 52-, and 66-kDa
polypeptides derived from differential translational initiation or
alternative splicing (25). Purified InlB caused an increase in tyrosine
phosphorylation of the three Shc isoforms in Vero cells and an increase
in levels of p85 in anti-Shc immunoprecipitates (Fig. 3E).
These results indicate that InlB stimulates tyrosine phosphorylation of
the adaptor proteins Gab1, Cbl, and Shc and interaction of these
proteins with p85. At present, it is not clear if these three adaptors
form a single complex with p85 or if multiple complexes containing one
or more adaptors and p85 exist.
Reciprocal experiments in which anti-p85 immunoprecipitates were
blotted with anti-Shc antibodies confirmed the interaction between Shc
and p85 in InlB-treated Vero cells (data not shown). In order to
demonstrate the presence of tyrosine-phosphorylated Gab1 in anti-p85
immunoprecipitates, we performed re-immunoprecipitation experiments.
Protein complexes present in anti-p85 precipitates prepared from
InlB-treated cells were dissociated by boiling in the presence of SDS
and the proteins released were re-immunoprecipitated with anti-Gab1
or anti-Cbl antibodies and detected by blotting with anti-Tyr(P)
antibodies (Fig. 4).
Re-immunoprecipitation with antibodies against Gab1 revealed a
~110-kDa phosphoprotein, whereas no protein was detected after
re-precipitation with antibodies against Cbl (compare lanes
5 and 6). These results demonstrate that Gab1 is
present in the anti-p85 immunoprecipitates. The inability to detect Cbl
by this approach could be due to insufficient quantities of this
phosphoprotein or possibly to dissociation of Cbl·p85 complexes by
the anti-p85 antibody.
Gab1, Cbl, and Shc are known to be tyrosine-phosphorylated in response
to EGF and other agonists. In addition, Gab1 and Cbl were recently
found to associate with p85 in response stimulation of some cultured
cells with EGF (17, 22, 24, 26). We found that treatment of Vero cells
with EGF-induced tyrosine phosphorylation of Gab1, Cbl, and Shc and
interaction of these proteins with p85 (Fig. 3, C-E,
lane 3). Thus InlB-mediated signaling via tyrosine phosphorylation has some resemblance to signaling induced by this known
agonist of PI 3-kinase.
InlB Does Not Regulate PI 3-Kinase or Bacterial Entry through
Activation of the EGF Receptor--
Although signal transduction
elicited by InlB and EGF share some similarities (Figs. 1B
and 3), InlB does not promote activation of PI 3-kinase or bacterial
entry through activation of the EGF-R. Treatment of Vero cells with
InlB did not result in detectable tyrosine phosphorylation of the EGF-R
(Fig. 5A). In addition, signal
transduction promoted by InlB was pharmacologically disassociated from
that promoted by EGF. Treatment of Vero cells with the EGF-R inhibitor
AG1478 (20) inhibited EGF-induced autophosphorylation of the EGF-R,
tyrosine phosphorylation of Gab1, and activation of PI 3-kinase. In
contrast, AG1478 did not block the tyrosine phosphorylation of Gab1 or
activation of PI 3-kinase caused by InlB (Fig. 5; data not shown).
L. monocytogenes Stimulates Interaction of p85 with Multiple
Adaptor Proteins--
The experiments in Fig. 3 show that, in the
absence of bacteria, purified InlB induces tyrosine phosphorylation of
Gab1, Cbl, and Shc. It was important to determine whether InlB controls
phosphorylation of these adaptor proteins during bacterial entry into
mammalian cells.
A kinetic study indicated that p85 appeared in anti-Tyr(P)
immunoprecipitates approximately 5-10 min after infection with L. monocytogenes (Fig.
6A) and remained stable in
amount for at least 50 min thereafter.2 Under these
conditions, bacterial entry is not synchronous and occurs at a
relatively constant rate from 10 to 90 min after infection. Based on
these kinetics, we chose 10 min as the time point of infection at which
to examine tyrosine phosphorylation of Gab1, Shc, and Cbl. As expected,
infection of Vero cells with wt L. monocytogenes resulted in
increased tyrosine phosphorylation of Gab1, Cbl, and Shc and
association of these proteins with p85 (Fig. 6, B-D). The
effect caused by bacteria was weaker than that provoked by treatment of
cells with saturating amounts of InlB protein. In contrast, infection
with the InlB Stimulates PI 3-Kinase-dependent Membrane
Ruffling--
Many agonists of PI 3-kinase provoke large changes in
the actin cytoskeleton known as membrane ruffling (27-29). Treatment of Vero cells with soluble InlB (3 nM) caused ruffling that
was most apparent 5-10 min after treatment and greatly diminished after 30-60 min. Incubation with InlB for 5 min resulted in
approximately 75% of the cell population having one or more actin-rich
ruffle (Fig. 7B), whereas
cells that were not incubated with InlB (Fig. 7A) had
essentially no detectable ruffling (ruffles were observed in less than
1% of the cells in the population). InlB-induced ruffling was PI
3-kinase-dependent, since it was inhibited by treatment of
cells with wortmannin (Fig. 7C). As expected, incubation of
Vero cells with EGF induced membrane ruffling that was sensitive to
wortmannin or AG1478, whereas InlB-mediated ruffling was not sensitive
to AG1478 (data not shown). Incubation of cells with wortmannin did not
cause a general paralysis of cytoskeletal responses, since
"ruffle-like" changes in F-actin provoked by the bacterial pathogen
S. typhimurium were not blocked by this treatment (Fig. 7E). Taken together, these results indicate that soluble
InlB stimulates changes in the actin cytoskeleton through a mechanism dependent on p85-p110.
L. monocytogenes Does Not Induce Membrane Ruffling during
InlB-mediated Entry--
Entry of L. monocytogenes into
Vero cells and other mammalian cells requires re-organization of the
actin cytoskeleton, since uptake is impaired by treatment of cells with
cytochalasin D (3, 6). The fact that soluble InlB causes PI
3-kinase-dependent changes in the actin cytoskeleton led us
to examine whether we could detect similar changes during uptake of
L. monocytogenes. Interestingly, infection of Vero cells
with wt L. monocytogenes did not result in membrane ruffling
(Fig. 7F). In fact, we were unable to detect any obvious
accumulation in F-actin near adherent bacteria. We confirmed that
bacterial entry was occurring in these conditions at a relatively
constant rate from 10 to 90 min after infection by measuring bacterial
internalization in parallel with a commonly used antibody labeling
technique (12). We also verified that bacterial infection does not
somehow inhibit ruffling induced by InlB. Cells were infected with
L. monocytogenes for 10 min and then treated with purified
InlB for 5 min, resulting in the formation of membrane ruffles (data
not shown). Taken together, these results suggest that changes in the
actin cytoskeleton that accompany InlB-mediated entry are too small in
scale and/or transient to permit detection by laser scanning confocal
microscopy. InlB present on the surface of L. monocytogenes
appears to cause different cytoskeletal changes than those promoted by
InlB that is freely diffusible in solution.
Our results establish that the L. monocytogenes protein
InlB is an agonist of mammalian p85-p110. To our knowledge, InlB is the
first reported non-mammalian polypeptide activator of this PI
3-kinase, which is known to be regulated by several growth factors or
hormones, including insulin, plateletderived growth factor,
N-formyl-methionyl-leucyl-phenylalanine, thrombin, and EGF. InlB-mediated activation of p85-p110 has features in common with
that promoted by these known agonists. Like EGF and insulin, InlB
affects p85-p110 in part through tyrosine phosphorylation of adaptor
proteins and interaction of these proteins with p85. In fact, InlB
regulates phosphorylation of some of the same adaptor proteins utilized
by EGF and insulin, including Gab1, Cbl, and Shc (17, 22, 24-26). InlB
also resembles these growth factors in its ability to stimulate
membrane ruffling through p85-p110.
It is clear that InlB is not the only L. monocytogenes
factor that regulates mammalian PI 3-kinase activity. Infection of Vero
cells with the The sequence determinants in InlB that promote activation of p85-p110
and bacterial entry remain to be defined. InlB contains an
NH2-terminal region with eight 22-amino acid repeats that
are rich in leucine residues (4, 13, 30), making it a member of the
functionally diverse leucine-rich repeat family of proteins (31). We
are currently testing whether the leucine-rich repeat region of InlB is
sufficient for activation of mammalian PI 3-kinase and bacterial uptake.
The primary recognized function of InlB is in mediating entry of
L. monocytogenes into non-phagocytic mammalian cells (4, 5,
12, 34). Our previous results indicated that InlB-mediated entry and
activation of p85-p110 involve interaction of p85 with one or more
unknown tyrosine-phosphorylated host protein(s) (6). In this report, we
identify Gab1, Cbl, and Shc as at least some of the phosphoproteins
that are likely to be important for bacterial uptake mediated by PI
3-kinase. Gab1, Cbl, and Shc lack kinase or other known activities and
are believed to act as cytosolic "adaptors" that couple p85-p110
and other signaling proteins to surface receptors engaged by a variety
of cytokines or growth factors (35). Of these three adaptors, Gab1 may
play the most important role in signaling during InlB-mediated entry.
The Formation of Gab·p85 and Cbl·p85 complexes in response to EGF or
other agonists is driven by interaction of the SH2 domains in p85 with
SH2-binding sites of the type YxxM present in Gab1 and Cbl (17, 22, 24,
36). It seems likely that Gab1·p85 and Cbl·p85 complexes induced by
InlB also involve such interactions. Shc lacks YxxM sites and is
therefore unlikely to directly interact with the p85 SH2 domains.
However, the formation of Shc·p85 complexes in response to InlB could
involve interaction of Shc with a YxxM-containing protein that is
itself associated with p85 (perhaps Gab1 or Cbl) and/or a previously
described direct interaction between Shc and the p85 SH3 domain
(37).
How do Gab1, Cbl, or Shc form complexes with p85 in response to
interaction of the host cell with InlB? We hypothesize that InlB
engages a receptor on the surface of the mammalian cell, an idea
supported by the fact that purified InlB binds to intact Vero cells or
other cell lines (12, 34). Our current model is that binding of InlB to
this receptor leads to tyrosine phosphorylation of Gab1, Cbl, and Shc
and association of these adaptor proteins with p85. Complexes
containing Gab1, Cbl, or Shc and p85-p110 may be recruited to a
cytoplasmic region in the InlB receptor, resulting in membrane
translocation of p85-p110 and access to its membrane-bound
substrate, PtdIns(4,5)P2.
The identity of the putative InlB receptor is not known. Despite some
similarities with EGF-mediated signaling, signaling promoted by InlB
does not require tyrosine phosphorylation of the EGF-R. InlB-mediated
signaling may also be independent of other known receptor tyrosine
kinases, such as the insulin, insulin-like growth factor-1, or
platelet-derived growth factor receptor, since we do not detect
tyrosine-phosphorylated proteins of the expected sizes (95 or 180 kDa)
in anti-Tyr(P) or anti-p85 immunoprecipitates from InlB-treated cells
(see Fig. 3, A and B). It is possible that the
InlB receptor lacks intrinsic kinase activity and is coupled to a
cytosolic tyrosine kinase. Clearly, further work is needed to identify
the receptor(s) and tyrosine kinase(s) involved in InlB-mediated entry.
How does activation of PI 3-kinase lead to bacterial uptake?
Experiments with cytochalasin D indicate that an intact actin cytoskeleton is needed for internalization of L. monocytogenes and most other bacterial pathogens (reviewed in
Refs. 1, 3, and 38). Interestingly, purified InlB elicits PI
3-kinase-dependent changes in the actin cytoskeleton
(membrane ruffling), raising the possibility that p85-p110 may be
involved in cytoskeletal changes that mediate uptake. However, we were
unable to detect ruffling or other types of accumulation of F-actin
near adherent bacteria. Therefore, the potential role of PI 3-kinase in
cytoskeletal changes during entry of L. monocytogenes could
not be tested. It is not clear why InlB stimulates membrane ruffling
when presented to cells as a diffusible ligand, but not when associated
with L. monocytogenes. It is possible that the gross
cytoskeletal changes caused by soluble InlB are due to ligand diffusion
and engagement of receptors over a large surface, whereas InlB present
on bacteria clusters a more limited number of receptors and induces
more local changes that are less readily detectable. Alternatively, the
lack of ruffling seen with L. monocytogenes could be due to
an insufficient quantity of InlB interacting with the mammalian cell surface.
Understanding how p85-p110 mediates internalization of L. monocytogenes will require the identification of proteins that act downstream of this lipid kinase in bacterial entry. Several proteins have been demonstrated recently to bind and respond to the lipid products of PI 3-kinase, including the phospholipase PLC
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
inlB) deleted
for the inlB gene is defective in colonization of the liver
(4, 5, 7). InlA promotes bacterial entry by interacting with its
mammalian receptor, the cell-cell adhesion molecule E-cadherin (8). The
mammalian receptor for InlB is not known.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(06-195), polyclonal antibodies against a
peptide in human Gab1(06-579), and monoclonal antibodies against
phosphotyrosine (Tyr(P)) (clone 4G10; 16-101) used for
immunoprecipitation were purchased from Upstate Biotechnology. Affinity-purified polyclonal antibodies raised against peptides in
human Cbl (sc-170) and human epidermal growth factor receptor (EGF-R)
(sc-03) were from Santa Cruz Biotechnology. Rabbit preimmune IgG was
from Sigma. Monoclonal anti-Tyr(P) antibodies (clone RC20) coupled to
peroxidase (E120H), the protein A-horseradish peroxidase conjugate, and
affinity-purified polyclonal anti-Shc antibodies (S14630) were from
Transduction Laboratories. Western blotting indicated that the anti-Shc
antibodies recognized the three known Shc isoforms (p46, p52, and p66)
in Vero cells. Protein A-Sepharose CL-4B beads were from Amersham
Pharmacia Biotech. The BCA kit used to determine protein concentrations
of InlB preparations and solubilized mammalian lysates was from Pierce.
Recombinant human epidermal growth factor (EGF) was from Calbiochem or
Upstate Biotechnology. Wortmannin was purchased from Biomol or Sigma, and the tyrosine kinase inhibitor AG1478 was from Calbiochem. At the
concentrations used, these inhibitors had no effect on the viability of
Vero cells, as determined by staining with trypan blue.
DE to yield strain BUG1713.
Growth of this strain, induction of the InlB protein, cell lysis with a
French press, and purification of the recombinant 6xHis InlB protein on
nickel columns were as described (12). Since the protein was not
sufficiently pure after passage on the nickel column, it was further
purified with a HiTrap SP column (Amersham Pharmacia Biotech). The
protein was loaded onto the column in buffer containing 50 mM HEPES (pH 7.9), 0.2 M NaCl and eluted by a
continuous 90-ml salt gradient from 0.2 to 1.0 M NaCl. Pure
fractions were concentrated using Centriprep and Centricon 30 (Amicon)
devices, the NH2-terminal 6 × His tag sequence was
removed by cleavage with thrombin (Novagen), and cleaved InlB was
separated from uncut protein by passage over a nickel column as
described (12). Approximately 4 mg of cleaved InlB protein was obtained
from 1.2 liters of an induced bacterial culture. Thrombin was removed
from preparations of the cut protein by three successive incubations
with an agarose resin coupled to the protease inhibitor
p-aminobenzamidine (Sigma, A7155), and InlB preparations
were stored as 1-2 mg/ml solutions in 10-µl aliquots at
80 °C.
Aliquots were thawed, diluted to 1000-fold concentrated stock solutions
in DMEM, and used immediately for experiments involving cell
stimulation. Unused protein was generally discarded, although we have
observed that InlB retains some activity after several rounds of
freeze-thawing.
inlB isogenic derivitive (BUG1047) were
grown in brain heart infusion broth (Difco) at 37 °C to an
0D600 of 0.80-1.0. Aliquots were then taken, washed three
times in DMEM, diluted to approximately 107 bacteria/ml,
and bacteria were added to cell monolayers at a multiplicity of
infection of about 50:1 (bacteria:mammalian cell). The Salmonella
typhimurium strain SL1344 was grown and prepared for infection as
described (8), except that 0.3 M NaCl was included in the
LB medium. SL1344 was added to Vero cells at an multiplicity of
infection of about 20:1. Contact between L. monocytogenes or
S. typhimurium and cell monolayers was initiated by
centrifugation (1 min at 200 × g), internalization was
allowed to proceed for the indicated times, and cells were then taken
for processing for immunoprecipitation or immunofluorescence.
20 °C until analysis by SDS-polyacrylamide
gel electrophoresis. "Re-immunoprecipitation" experiments were
performed as described (15) with some modification. After
immunoprecipitation with anti-p85 antibodies, immune complexes were
dissociated by boiling for 2 min in buffer containing 50 mM
Tris-HCl (pH 7.5), 0.5% SDS, and 5 mM dithiothreitol.
Protein A-Sepharose beads were then removed by centrifugation, and the supernatant was diluted 30-fold into buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 3 mM sodium orthovanadate, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and
10 µg/ml aprotinin, leupeptin, pepstatin, and chymostatin. The
diluted solutions were incubated with the appropriate antibodies for
approximately 12 h, followed by a 1-h incubation with protein A-Sepharose beads, washing, and storage at
20 °C. Proteins present in immunoprecipitates were separated on 9.0% (Shc
immunoprecipitations) or 7.5% (all other immunoprecipitations)
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes (Hybond C, Amersham Pharmacia Biotech) with a
semidry apparatus. Membranes were blocked overnight by incubation in
Tris-buffered saline (TBS) with 0.1% Tween 20 containing 3% bovine
serum albumin (for immunoblotting with anti-Tyr(P) antibodies) or 5%
non-fat milk (for all other antibodies). For the anti-Tyr(P) blots,
membranes were simply incubated for 1 h at room temperature in
TBS-Tween containing anti-Tyr(P) antibodies directly coupled to
horseradish peroxidase (RC20-HRPO). For all other blots, membranes were
incubated for 1 h in blocking buffer with primary antibody,
followed by washing and a second 1-h incubation in blocking buffer
containing a protein A-horseradish peroxidase conjugate. After
extensive washing of the membranes, proteins were detected by using the
ECL or ECL Plus chemiluminescent systems (Amersham) and exposure to
film (Hyperfilm MP, Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
inlB) results in smaller
increases in PtdIns(3,4,5)P3 than is observed upon
infection with wt (
inlB+) L. monocytogenes, indicating that InlB is needed for full activation of p85-p110 (6).
However, the fact that low-level accumulation of
PtdIns(3,4,5)P3 is detectable in cells infected with the
inlB mutant suggests that bacterial factors in addition
to InlB contribute to stimulation of p85-p110. On the basis of these
results, it was not known whether InlB itself is an agonist of p85-p110
or whether this bacterial protein only plays an accessory role in PI
3-kinase activation.
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Fig. 1.
Purified InlB stimulates PI 3-kinase activity
in mammalian cells. A, purification of recombinant InlB
protein. Full-length InlB containing an NH2-terminal 6 × His tag was purified on nickel and HiTrap SP columns, and the 6 × His tag was removed by proteolysis as described. To assess purity, 6 µg of the final cleaved protein (right lane) was
visualized on a 12% SDS-polyacrylamide gel stained with Coomassie
Blue. The left lane contains molecular weight markers of the
indicated sizes. B, PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 accumulate in cells treated with InlB. Vero
cells were left untreated (U), treated with ~4.5
nM (300 ng/ml) purified InlB for 1 min (+InlB),
or incubated with 17 nM (100 ng/ml) EGF for 1 min
(+EGF). Reactions were then stopped and the
concentrations of the phosphoinositides PtdIns(3,4)P2,
PtdIns(3,4,5)P3, and PtdIns(4,5)P2 were
determined. Values are the average (±S.E.) of three (EGF; all
phosphoinositides), five (InlB; PtdIns(3,4,)P2), or six
(InlB; PtdIns(3,4,5)P3 and PtdIns(4,5)P2)
experiments and are expressed as the relative amount of the particular
phosphoinositide, using the counts/min in untreated control cells as
the reference (value of 1). In these experiments, the average
counts/min values for PtdIns(3,4)P2,
PtdIns(3,4,5)P3, and PtdIns(4,5)P2 in control
cells were 5.6 × 103, 4.3 × 103,
and 1.5 × 106, respectively. The average counts/min
for these phosphoinositides in cells treated with InlB were 7.1 × 104, 5.3 × 104, and 1.5 × 106, respectively.
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Fig. 2.
InlB stimulates the interaction of p85 with
one or more tyrosine-phosphorylated protein(s). A, Vero
cells were left untreated (U) or treated with increasing
concentrations of InlB for 1 min. B, cells were left
untreated or treated with 3 nM (200 ng/ml) purified InlB
for various times. Cells were then solubilized, and
tyrosine-phosphorylated proteins were immunoprecipitated with
anti-Tyr(P) antibodies, and p85 (the regulatory subunit of p85-p110)
was detected by immunoblotting. Results similar to those in
A and B were obtained in two additional
experiments.
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Fig. 3.
InlB stimulates tyrosine phosphorylation of
Gab1, Cbl, and Shc and interaction of these proteins with p85.
Vero cells were incubated with 3 nM InlB for 1 min
(lane 2), incubated with 17 nM (100 ng/ml) EGF
for 1 min (lane 3), or left untreated (lane 1),
followed by solubilization and immunoprecipitation (IP) with
the indicated antibodies. Association of p85 with
tyrosine-phosphorylated proteins was analyzed by probing anti-Tyr(P)
immunoprecipitates with anti-p85 antibodies (A, lower
panel) and by immunoblotting anti-p85 immunoprecipitates with
anti-Tyr(P) antibodies (B, upper panel). The
arrowhead in B indicates the ~110-kDa
phosphoprotein(s) that co-immunoprecipitated with p85, and the
small arrow shows the ~52-kDa protein that (based on its
size) may be an isoform of Shc. Note that the anti-Tyr(P) blot in
B does not contain a signal at 85 kDa, indicating that p85
is not detectably tyrosine-phosphorylated. In the upper
panels in C-E, tyrosine phosphorylation of Gab, Cbl,
or Shc was detected by blotting with anti-Tyr(P) antibodies. The
positions of phosphorylated Gab1 (~110 kDa) or Cbl (~110 kDa) are
indicated by arrowheads. Positions of the phosphorylated Shc
isoforms (~46, 52, and 66 kDa) in E are also indicated.
Small arrows highlight ~46- and 52-kDa phosphoproteins in
anti-Gab1 immunoprecipitates, which may be Shc proteins. The ~170-kDa
phosphoprotein observed in lane 3 of A-E is
likely to be the EGF-R. In lane 2 of A, a weak
signal corresponding to an unknown tyrosine-phosphorylated protein of
~130-140 kDa is detected from lysates of InlB-treated cells. This
phosphoprotein was evident in some but not all experiments involving
treatment with InlB (e.g. it is not obvious in Fig.
6A, lane 4), possibly because of interference
from the strong Tyr(P) signals of proteins at 110-120 kDa. In the
second panels in A-E, the membranes were stripped and p85
was detected by blotting with anti-p85 antibodies. For anti-Cbl and
anti-Shc immunoprecipitations, the membranes were stripped a second
time and probed with anti-Cbl or anti-Shc antibodies (lower
panels) to verify that equivalent amounts of Cbl or Shc were in
the immunoprecipitates loaded in the different lanes. Similar third
blots with anti-Gab1 antibodies were not routinely performed on
anti-Gab1 immunoprecipitates, since the antibody often failed to
interact with ~110 kDa Gab1 after two rounds of membrane stripping.
However, similar experiments in which anti-Gab1 immunoprecipitates were
directly probed with anti-Gab1 antibodies showed that InlB treatment
did not result in an increase of immunoprecipitable Gab1. Controls with
preimmune rabbit IgG demonstrated that the tyrosine phosphorylation of
proteins and co-immunoprecipitation of p85 in A-E were
specific for the Gab1, Cbl, and Shc antibodies.2 The
results in A-E are representative of at least three
experiments.
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Fig. 4.
Tyrosine-phosphorylated Gab1 is present in
anti-p85 immunoprecipitates from InlB-treated cells. Vero cells
were left untreated (lanes 1, 3, and 4) or
treated with 3 nM InlB for 1 min (lanes 2, 5, 6, and 7). Solublized lysates were then prepared by addition of
immunoprecipitation buffer and 3.5 mg (untreated cells) or 5.0 mg
(InlB-treated cells) of lysate was used for immunoprecipitation with
anti-p85 antibodies. Proteins co-immunoprecipitated with p85 were
dissociated by boiling in the presence of 0.5% SDS, 5 mM
dithiothreitol, and amounts of the immunoprecipitates corresponding to
0.3 mg of starting material were saved for loading in lanes
1 and 2. The remainder was diluted into
re-immunoprecipitation buffer and amounts corresponding to 1.5 mg of
starting material were used for each re-precipitation with antibodies
( ) against Gab1 (lanes 3 and 5), Cbl
(lanes 4 and 6), or with control preimmune rabbit
IgG (lane 7). Tyrosine-phosphorylated proteins in the
final immunoprecipitates were detected with anti-Tyr(P) antibodies.
Anti-p85 immunoprecipitates that had been re-immunoprecipitated with
anti-Gab1 antibodies (lane 5) contained a
tyrosine-phosphorylated protein of ~110 kDa with a mobility
equivalent to that of the major phosphoprotein in anti-p85
immunoprecipitates that had not been subject to re-immunoprecipitation
(lane 2) (all of the lanes in Fig. 4 are from the same
SDS-polyacrylamide gel). Similar results were obtained in two other
experiments.
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Fig. 5.
InlB-mediated signaling does not involve the
activated EGF-R. A, InlB does not stimulate tyrosine
phosphorylation of the EGF-R. Vero cells were pretreated for 20 min
with 0.1% Me2SO (lanes 1-3) or with 250 nM of the EGF-R-specific inhibitor AG1478 (lane
4). Cells were then left untreated (lane 1), treated
with 3 nM InlB for one min (lane 2), or treated
with 17 nM EGF for one min (lane 3). Addition of
purified InlB did not induce tyrosine phosphorylation of the EGF-R
(compare lanes 1 and 2), whereas addition of EGF
did (lane 3). The decreased reactivity to the anti-EGF-R
antibody seen in lane 3 is probably due to tyrosine
phosphorylation of the carboxyl-terminal tail in the EGF-R (the
antibody recognizes a peptide in this COOH-terminal region).
B, InlB-induced tyrosine phosphorylation of Gab1 does not
require activation of the EGF-R. Vero cells were treated for 20 min
with 0.1% Me2SO (lanes 1-3) or with 250 nM AG1478 (lanes 4 and 5) and then
left alone (lane 1) or incubated with 3 nM InlB
(lanes 2 and 4) or 17 nM EGF for 1 min (lanes 3 and 5). The EGF-R specific inhibitor
AG1478 blocked EGF-induced tyrosine phosphorylation of Gab1 (lane
5), but had no effect on InlB-induced tyrosine phosphorylation of
Gab1 (lane 4). The results in A and B
are representative of three experiments.
inlB mutant resulted in almost no induction in
tyrosine phosphorylation of Gab1 or association of this protein with
p85 (Fig. 6B). Somewhat unexpectedly, infection with the
inlB strain caused tyrosine phosphorylation of Cbl and
Shc about as efficiently as did the wt strain (Fig. 6, C and
D). Taken together, these results indicate that endogenous InlB promotes the tyrosine phosphorylation of at least Gab1 during infection with L. monocytogenes. In addition to InlB,
L. monocytogenes must produce at least one other factor that
contributes to phosphorylation of Cbl and Shc. This unidentified factor
may be responsible for the low level but significant activation of PI
3-kinase that occurs upon infection of Vero cells with the
inlB mutant (6) (see "Discussion").
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Fig. 6.
L. monocytogenes stimulates
tyrosine phosphorylation of Gab1, Cbl, and Shc and interaction of these
proteins with p85. Vero cells were left uninfected (lane
1) or infected with wt L. monocytogenes strain EGD
(lane 2) or the inlB mutant (lane
3) for 10 min. For comparison, cells were treated with 3 nM recombinant InlB protein for 1 min (lane 4).
Gab1, Cbl, or Shc were immunoprecipitated from solubilized lysates, and
tyrosine phosphorylation or association of these proteins with p85 was
evaluated by blotting with anti-Tyr(P) or anti-p85 antibodies as
described in the legend to Fig. 4 and under "Experimental
Procedures." The results are representative of three
experiments.
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Fig. 7.
Purified InlB induces membrane ruffling in
Vero cells. Vero cells were pretreated with 0.2%
Me2SO (A, B, D) or 100 nM wortmannin (C, E). Cells were then
left alone (A) or incubated with 3 nM InlB for 5 min (B and C). After treatment with InlB or EGF,
cells were fixed, stained with FITC-phalloidin to detect F-actin, and
examined with a confocal microscope using the × 63 objective.
InlB induced the formation of actin-rich membrane folds or ruffles that
were sensitive to wortmannin treatment. In D and
E, cells were infected with S. typhimurium strain
SL1344 for 5 min and then fixed. F-actin was labeled with
FITC-phalloidin (green), bacteria were stained with a
Salmonella-specific antiserum (red), and the
labeled cells were examined with a confocal microscope using the × 100 objective. As observed previously (38, 41), infection with
S. typhimurium provoked the formation of actin-rich
structures that had some resemblance to membrane ruffles
(D). Formation of these structures in Vero cells was not
sensitive to wortmannin treatment (E), as reported
previously for COS cells (41). In F, cells were infected
with wt L. monocytogenes strain EGD for 10 min followed by
labeling of F-actin (green) and bacteria (red).
In contrast to purified InlB or S. typhimurium, L. monocytogenes failed to induce the formation of ruffles. A kinetic
study showed that ruffling was absent at all time points of infection
with L. monocytogenes that were examined (i.e. 1, 5, 10, 20, 30, 45, and 60 min postinfection) (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
inlB mutant results in approximately 30% of the increase in cellular PtdIns(3,4,5)P3 that normally
occurs upon infection with wt L. monocytogenes (6). Thus,
the
inlB mutant is only partly defective in stimulating
host PI 3-kinase activity. In this work, we show that the
inlB mutant is partly defective in inducing tyrosine
phosphorylation of Gab1, but is still able to cause phosphorylation of
the adaptors Cbl and Shc. It seems likely that this tyrosine
phosphorylation of Cbl and Shc is responsible for the residual
activation of p85-p110 caused by the
inlB strain.
Although the bacterial factor responsible for residual stimulation of
PI 3-kinase is not known, it appears to be relatively specific to
L. monocytogenes. Infection with the non-pathogenic
Listeria species Listeria innocua did not result in tyrosine phosphorylation of Gab, Cbl, or Shc or interaction of these
proteins with p85. The invasion protein InlA was not responsible for
the tyrosine phosphorylation of Cbl and Shc caused by the
inlB mutant, since an L. monocytogenes mutant
strain deleted for both the inlA and inlB genes
(4) was fully capable of inducing phosphorylation of these two
adaptors.2 Further work is needed to identify the bacterial
and mammalian components involved in the residual stimulation of PI
3-kinase.
inlB bacterial mutant, which is defective for entry
into Vero cells, is also defective in promoting tyrosine
phosphorylation of Gab1, but is still able to promote phosphorylation
of Cbl and Shc. Although these results are consistent with the idea
that a complex composed of phosphorylated Gab1 and p85 is needed for
entry of L. monocytogenes, definitive evidence for the role
of Gab1 or the other adaptor proteins will require genetic approaches
that specifically interfere with the function of each individual protein.
1, some atypical isoforms of protein kinase C, the serine-threonine kinases PDK
and Akt, the integrin-binding protein Grp1, and the Arf exchange factor
ARNO (11). Another signaling protein that interacts with PtdIns(3,4)P2 and PtdIns(3,4,5)P3 is the GTPase
Rac (19). Rac acts downstream of p85-p110 to mediate membrane ruffling
in Swiss 3T3 fibroblasts (39) and is also required for ruffling and
Fc-
receptor-mediated phagocytosis in macrophages (40). It will be
interesting to determine whether Rac or other GTPases are involved in
InlB-mediated bacterial entry into non-professional phagocytes or in
ruffling induced by InlB.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge L. Braun for advice in constructing the InlB expression plasmid, and L. Braun, H. Gantelet, and P. Steffen for help with protein purification. We also thank R. Hellio for his expertise in confocal microscopy, Prof. H. Chap for his interest and support of this project, and Dr. I. Lasa for helpful advice. Drs. V. David and R. Hurme are thanked for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from Biomed (BMH 4 CT 96-0659), Direction Générale de l'Armement, Ministére de la Defense (97/069), Ministére de l'Education Nationale et de la Recherche Scientifique et Technologique (Pr. Microbiologie 98), and by Institut Pasteur.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of fellowships from the Jane Coffin Childs Memorial Fund for Medical Research and from La Fondation Recherche et Partage.
¶ To whom correspondence should be addressed (to K. I. or P. C.). Present address (for K. I.): Dept. of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8. Tel.: 416-946-5356; Fax: 416-978-6885; E-mail: k.ireton{at}utoronto.ca. For P. C.: Unité des Interactions Bactéries-Cellules, 75724 Paris Cedex 15, France. Tel.: 33-1-45-68-88-41; Fax: 33-1-45-68-87-06; E-mail: pcossart{at}pasteur.fr.
2 K. Ireton, B. Payrastre, and P. Cossart, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphoinositide; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Tyr(P), phosphotyrosine; EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; SH, Src homology; FITC, fluorescein isothiocyanate; TBS, Tris-buffered saline; WTM, wortmannin; DMEM, Dulbecco's modified Eagle's medium; wt, wild type.
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