From the Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0359
Received for publication, November 15, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Internalin B (InlB) is a protein present on the
surface of Listeria monocytogenes that mediates bacterial
entry into mammalian cells. It is thought that InlB acts by binding
directly to the hepatocyte growth factor (HGF) receptor, present on the
surface of host cells. Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton. Here we have compared signaling by HGF and InlB. Whereas
stimulation with equivalent concentrations of HGF and InlB elicits
similar activation of the HGF receptor, we observed striking
differences in downstream activation of MAP kinase. InlB leads to a
greater activation of the Ras-MAP kinase pathway than does HGF. The
leucine-rich repeat region, which was previously shown to be sufficient
for binding and activation of the HGF receptor, lacks the ability to
super-activate the Ras-MAP kinase pathway. Analysis of a series of
deletion mutants suggests that it is the B repeat region between the
leucine-rich repeat and GW domains that endows InlB with an increased
ability to turn on the Ras-MAP kinase pathway. These unexpected
observations suggest that HGF and InlB use alternative mechanisms to
turn on cellular signaling pathways.
The hepatocyte growth factor
(HGF)1 receptor is a
protein-tyrosine kinase that is expressed predominantly on the surface
of epithelial cells. Its natural ligand, HGF or scatter factor, is expressed in fibroblast cells (1). In vivo, HGF and the HGF receptor play key roles in the development of the liver, kidneys, muscle, and neuronal precursors (2). In vitro activation of the HGF receptor stimulates cell division, cell scattering, and the
formation of tubular structures in a three-dimensional matrix (3).
Whereas normal HGF receptor signaling plays a crucial role in embryonic
development, abnormal HGF receptor signaling has been implicated in
both tumor development and metastasis (4).
Activation of the HGF receptor leads to autophosphorylation on several
tyrosine residues. Tyrosine phosphorylation sites regulate kinase
activity and act as binding sites for cellular signaling proteins. The
activated HGF receptor interacts either directly or indirectly with
several signaling molecules including Grb2, Gab1, Shc, phosphoinositide
3 (PI 3)-kinase, Crk, CrkL, and Cbl (5-11). Gab1 is a docking protein
that provides the receptor with binding sites for a variety of other
proteins (12). Grb2 is a cytoplasmic adaptor protein that is involved
in linking Gab1 to the HGF receptor (13). Grb2 also works together with
Sos to link activated receptor protein-tyrosine kinases to Ras
activation (14, 15). Shc is a docking protein that is thought to
collaborate with Grb2 upstream of Ras (16-19). PI 3-kinase is a lipid
kinase that is composed of an 85-kDa regulatory subunit, which contains two SH2 domains and one SH3 domain, and a 110-kDa catalytic subunit (20-22). Activation of PI 3-kinase affects cytoskeletal dynamics and
cell survival. Crk and CrkL are related adaptor proteins that are also
involved in regulating cytoskeletal changes (23). Cbl is a ubiquitin
ligase that regulates receptor internalization and degradation
(24).
The Listeria monocytogenes surface-protein internalin B
(InlB) binds to and activates the HGF receptor. Initiation of host cell
invasion by this intracellular pathogen occurs through the interaction
of InlB with the HGF receptor, leading to host cell protein-tyrosine
phosphorylation and cytoskeletal rearrangements. It has been
established that the activation of both the Ras-MAP kinase pathway and
PI 3-kinase are essential for uptake of L. monocytogenes
into host cells (25, 26). InlB is composed of an amino-terminal
receptor-binding domain followed by a B repeat region (B) and three GW
domains. The receptor-binding domain contains three motifs: an
amino-terminal cap, a leucine-rich repeat (LRR) segment, and an
immunoglobin-like region (IR) (27, 28). The amino-terminal cap
and the LRR domain have been shown to be sufficient to bind and
activate the HGF receptor (29). The GW domains attach InlB
non-covalently to the bacterial cell wall and have been shown recently
to bind to host cell components upon release of InlB from the bacterium
(30, 31).
Both HGF and InlB bind and activate the HGF receptor. Interestingly,
whereas HGF stimulates mitogenesis, cell migration, and tubulogenesis,
InlB induces phagocytosis. This prompted us to compare signaling
downstream of these two HGF receptor ligands. Our results show that HGF
and InlB are very similar in their ability to activate the HGF
receptor, whereas InlB is a stronger activator of the Ras-MAP kinase
pathway. We found that InlB induces tyrosine phosphorylation of a
90-kDa PI 3-kinase-associated protein, whereas HGF did not. Analysis of
InlB deletion mutants showed that, whereas both the IR and B repeat are
necessary for maximal signaling downstream of the HGF receptor, it is
the B repeat region of InlB that is important for the observed
super-activation of the Ras-MAP kinase pathway.
Cells Lines, Antibodies, and Other Reagents--
Vero cells were
grown in minimal essential medium with Earle's salts containing 10%
fetal bovine serum. Monoclonal antibodies 4G10 against phosphotyrosine,
anti-HGF receptor DO-24, anti-HGF receptor DL-21, anti-phospho-Raf-1
(Ser-338) and a polyclonal serum against p85 were purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal serum
against Raf-1 (E-10) was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Polyclonal antibodies against phospho-p38 MAP kinase and
phospho-MEK 1/2, as well as a monoclonal antibody against
phospho-Erk1/2, were obtained from Cell Signaling Technology (Beverly,
MA). A monoclonal antibody against Ras was obtained from Transduction
Laboratories (Lexington, KY). The MEK1 inhibitor PD98059 was purchased
from New England Biolabs (Beverly, MA). HGF was obtained from Sigma.
Cloning, Expression, and Purification of InlB and InlB
Derivatives--
The LRR domain of InlB was expressed and purified as
described previously (32). Intact InlB was expressed and purified as described previously (31). InlB GST Fusion Proteins--
The GST-Shc SH2, GST-Shc PTB, GST-Grb2
SH2, and GST-p85 N-SH2 domain fusion proteins have been described
previously (33-36). Fusion proteins were isolated as described
(37).
Immunoprecipitation--
Cells were starved overnight in minimal
essential medium with Earle's salts containing 20 mM
Hepes, pH 7.4. Cells were then stimulated for 1 min at 37 °C with
HGF, wild-type, or mutant InlB at a final concentration of 1.5 nM unless otherwise noted. Cells were then rinsed 2 times
with cold phosphate-buffered saline and lysed in 1 ml of 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 500 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and
10 µg/ml leupeptin (PLC-lysis buffer) per 10-cm tissue culture dish.
Lysates were cleared by centrifugation at 10,000 rpm in a
microcentrifuge at 4 °C, incubated with 5 µl of polyclonal
antiserum or 1 µl of monoclonal antibody for 1 h on ice and
subsequently for 1 h with 100 µl of 10% protein A-Sepharose or
anti-mouse IgG-Sepharose for 1 h at 4 °C on an agitator.
Sepharose beads were collected by centrifugation and washed 4 times
with PLC-lysis buffer. Immunoprecipitates were boiled 3 min in 62.5 mM Tris/Cl, pH 6.8, 10% glycerol, 5%
For in vitro binding studies, cell lysates were incubated
for 1 h at 4 °C on a rocker with 10 µl of glutathione-agarose
beads containing 1-2 µg of fusion protein. Beads were washed 4 times with PLC-lysis buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting.
Immunoblotting--
Proteins were transferred to polyvinylidene
difluoride membranes using a Bio-Rad semi-dry blotting apparatus at 50 mA per gel for 60 min at room temperature. For each individual
antibody, immunoblotting was performed per the manufacturer's
protocol. Reactive proteins were visualized by ECL (Amersham Biosciences).
Ras Activation Assay--
GST-RBD was expressed in
Escherichia coli and purified on glutathione-agarose beads.
The beads were washed several times in Tris-buffered saline containing
1% Triton X-100 (TBST) and stored at 4 °C in TBST containing 0.02%
azide. For affinity precipitation, lysates were incubated with GST-RBD
pre-bound to glutathione-agarose (~10 µl of beads containing
~10-20 µg of protein) for 1 h at 4 °C on an agitator.
Bound proteins were resolved by SDS-PAGE on a 12% acrylamide gel and
subjected to Western blotting with an anti-Ras monoclonal antibody.
HGF and InlB Differ in Their Ability to Induce Tyrosine
Phosphorylation of Cellular Proteins--
It has been reported that
InlB binds to and activates the HGF receptor (29). This suggests that
InlB and HGF activate the same signal transduction pathways. To confirm
this, we compared protein tyrosine phosphorylation following
stimulation with either InlB or HGF. Vero cells, which naturally
express the HGF receptor, were grown to confluence, starved overnight
in serum-free medium, and stimulated for 1 min at 37 °C with 1.5 nM HGF or 1.5 nM InlB. Whole cell lysates were
analyzed by anti-Tyr(P) blotting (Fig. 1A). As controls, HGF receptor
immunoprecipitates were analyzed by anti-HGF receptor and anti-Tyr(P)
immunoblotting. The results show that there are several proteins that
become tyrosine-phosphorylated upon stimulation with either HGF or InlB
(Fig. 1A). Most proteins, including the HGF receptor, were
phosphorylated to similar levels in response to either HGF or InlB
(Fig. 1, A and B). Interestingly, one protein,
with an apparent molecular mass of ~40 kDa (Fig. 1A, p40), was more highly tyrosine-phosphorylated
in response to InlB than in response to HGF. These results suggest that
HGF and InlB differ in their ability to turn on cellular signaling cascades.
HGF and InlB Have Different Abilities to Activate the Ras-MAP
Kinase Pathway--
To identify p40, we focused on members of the MAP
kinase family because several of its members, including
p38MAPK, Erk-1 (p44MAPK), and Erk-2
(p42MAPK), have been implicated in signal transduction
downstream of the HGF receptor (38).
To investigate whether p40 is either Erk-1 or Erk-2, cells were
stimulated with HGF or InlB, and whole cell lysates were analyzed by
immunoblotting with an antibody that recognizes the phosphorylated, active forms of Erk-1 and Erk-2 (Fig.
2A). An anti-Erk-2 blot was
included as a control (Fig. 2B). Both Erk-1 and Erk2 are
activated in response to either HGF or InlB. InlB appears to be a
stronger activator of Erk-1 and Erk-2 than HGF (Fig. 2, A
and C). Stimulation in the presence of PD98059, a potent
inhibitor of MEK, blocks activation of Erk-1 and Erk-2 (Fig.
2A). Anti-Tyr(P) immunoblotting of whole cell lysates from
cells stimulated with InlB or HGF shows that p40 is no longer
phosphorylated when cells are stimulated in the presence of PD98059
(Fig. 2C). These results indicate that p40 represents
Erk-2.
It is possible that differences between HGF and InlB in their ability
to activate Erk-1 and Erk-2 are restricted to a specific ligand
concentration. To investigate this, Vero cells were stimulated with a
range of HGF or InlB concentrations (Fig.
3). Anti-HGF receptor immunoprecipitates
were analyzed by anti-Tyr(P) and anti-HGF receptor immunoblotting,
whereas whole cell lysates were analyzed for the presence of activated
Erk-1 and Erk-2. The results show that receptor phosphorylation levels
off between 0.75 and 1.5 nM HGF or InlB (Fig.
3A) and confirm that InlB is a better activator of Erk-1 and
Erk-2 than HGF at all concentrations tested.
Erk-1 and Erk-2 are activated following a cascade of events known as
the Ras-MAP kinase pathway (39). Finding differences in the extent of
MAP kinase activation following stimulation with HGF or InlB prompted
us to investigate other elements of this pathway. MEK activation was
evaluated by blotting whole cell lysates of control and stimulated
cells with an antibody that recognizes the active form of MEK. The
results show that MEK activation paralleled MAP kinase activation (Fig.
4A). To measure levels of
active Raf, Raf immunoprecipitates were probed with an antibody that
recognizes active Raf (Fig. 4B). As a control the
immunoprecipitates were probed with an anti-Raf antiserum (Fig.
4C). The results show that InlB is a slightly better
activator of Raf than HGF (Fig. 4B). To measure the levels
of active Ras, we used a GST fusion protein containing the Ras-binding
domain (RBD) of Raf. This fusion protein binds only to active,
GTP-bound Ras (40, 41). Lysates from control, HGF-, or InlB-stimulated
Vero cells were incubated with the GST-RBD fusion protein immobilized
on glutathione-agarose beads. Bound proteins were analyzed by anti-Ras
immunoblotting (Fig. 4D). Anti-Ras immunoblots of whole cell
lysates were included as a control (Fig. 4E). The data show
slightly higher levels of Ras activation in response to InlB than in
response to HGF (Fig. 4D). Together, these observations show
that InlB is a better activator of the Ras-MAP kinase pathway than
HGF.
Tyrosine Phosphorylation of an Unidentified 90-kDa Protein in
Response to InlB but Not in Response to HGF--
To find out whether
there are differences in signal transduction events between the HGF
receptor and active Ras, we focused on proteins known to be involved in
Ras activation, including Shc, Grb2, and PI 3-kinase. Analysis of Shc
and Grb2 immunoprecipitates by anti-Tyr(P) blotting showed that all
three isoforms of Shc (p46, p52, and p66) are tyrosine-phosphorylated
in response to either HGF or InlB (results not shown). GST fusion
proteins containing either the Shc PTB domain, the Shc SH2 domain, or
the Grb2 SH2 associated with several unidentified proteins that became
tyrosine-phosphorylated in response to either HGF or InlB. No
qualitative differences in either Shc or Grb2 associated proteins were
observed (results not shown).
Interestingly, a tyrosine-phosphorylated 90-kDa protein that is present
in the PI 3-kinase immunoprecipitates from InlB-stimulated cells is
absent from immunoprecipitates from cells that were stimulated with HGF
(Fig. 5A). To investigate this
further, lysates of HGF- or InlB-stimulated cells were analyzed for the
presence of proteins that bind to the PI 3-kinase amino-terminal SH2
domain in vitro. The results confirmed the presence of a
90-kDa protein present in lysates of InlB-stimulated cells that can
bind to the PI 3-kinase amino-terminal SH2 domain (Fig. 5B).
This protein is absent in lysates of HGF-stimulated cells. Our data
show that stimulation of Vero cells with InlB results in tyrosine
phosphorylation of a 90-kDa protein that is not tyrosine-phosphorylated
in response to HGF.
Sequences Outside the HGF Receptor-binding Domain Contribute to the
Ability of InlB to Turn on MAP Kinase--
InlB is composed of an
amino-terminal LRR domain, an immunoglobin-like region (IR), a B
repeat region, and three carboxyl-terminal glycine and
tryptophan-containing (GW) domains (Fig.
6A). It is known that the LRR
domain by itself can bind to the HGF receptor. LRR can be isolated as a
non-physiological disulfide-linked dimer (LRRd) or as a
monomer (LRRm).2
Dimeric LRR activates the HGF receptor, whereas the monomer is unable
to do so.2 To find out whether the LRR domain is sufficient
for InlB-mediated signaling, we compared the signaling capabilities of
full-length InlB with those of LRRd. Vero cells were
stimulated with InlB, LRRd, and HGF and analyzed for HGF
receptor phosphorylation and MAP kinase activation (Fig. 6). Our data
show that stimulation with 1.5 nM HGF, InlB, or
LRRd results in very similar levels of HGF receptor
activation, as measured by receptor tyrosine phosphorylation (Fig. 6,
B and C). Interestingly, we found that there is a
dramatic difference between InlB and either LRRd or HGF in
their abilities to activate Erk-2 (Fig. 6, D and
E). LRRd resembled HGF in its ability to
activate Erk-2, whereas InlB was found to be a more potent activator.
This suggests that portions of InlB besides the LRR domain are required
for ability of InlB to super-activate the Ras-MAP kinase pathway.
The IR and B Repeats Play an Important Role in Signaling by
InlB--
To find out which regions of InlB are involved in MAP kinase
super-activation, we generated a series of carboxyl-terminal deletion
mutants. Progressive deletion from the carboxyl terminus affected
receptor activation.2 This made it difficult to separate
receptor activation from MAP kinase activation. To test the importance
of the IR, B, and GW domains to the ability of the InlB to activate
cell signaling, mutant proteins with internal deletions were generated
(Fig. 7A). Vero cells were
stimulated with the various mutants and analyzed for HGF receptor and
MAP kinase activation (Fig. 7, B and D). Control
blots showing receptor levels and MAP kinase levels were included (Fig.
7, C and E). Our data show that the InlB mutant lacking the first GW repeat (InlB InlB is a protein that is expressed on the surface of
L. monocytogenes and that initiates host cell
entry by binding to the HGF receptor (29, 42). Binding of InlB to the
HGF receptor stimulates phagocytosis (3, 43). In contrast, stimulation with HGF results in mitogenesis, cell scattering, and tubulogenesis (44, 45). This prompted us to compare signaling in response InlB and
HGF.
Several members of the MAP kinase family, including Erk-1, Erk-2, and
stress-activated p38 MAPK, are activated in response to HGF
(46). We found that InlB is a better activator of Erk-1 and Erk-2 than
HGF. This is of interest for two reasons. First, because InlB and HGF
are both thought to signal exclusively through the HGF receptor, we
expected MAP kinase activation to correlate with receptor activation.
Our data show that this is not the case (Figs. 3, 6, and 7). Second, MAP kinase activation has been shown to be essential for InlB-mediated host cell invasion (25). Our data raise the possibility that InlB uses
a distinct signaling mechanism, one that differs from that used by HGF,
to turn on MAP kinase.
To explain the differences in activation of the Ras-MAP kinase pathway,
we examined proteins that are thought to act proximal in the HGF
receptor signal transduction process. Phosphorylation of Shc- and
Grb2-associated proteins in response to HGF has been reported
previously (7, 47). We did not observe qualitative differences between
InlB and HGF in their abilities to stimulate the phosphorylation of
Shc- and Grb2-associated proteins (results not shown). It is worth
noting that the identity of some of these associated proteins remains
to be determined.
It has been shown previously (26, 48) that activation of the HGF
receptor leads to tyrosine phosphorylation of p85, the regulatory
subunit of PI 3-kinase. There are several tyrosine-phosphorylated proteins that co-immunoprecipitated with the 85-kDa subunit of PI
3-kinase in response to either InlB or HGF. These include a 120-kDa
protein that most likely represents Gab1, a 110-kDa protein, p85, and a
55-kDa protein. In addition, there is a 90-kDa protein that is
tyrosine-phosphorylated in response to InlB but not in response to
HGF. In vitro binding experiments showed that this protein
binds to the amino-terminal SH2 domain of p85. It is tempting to
speculate that the difference in the ability to activate the Ras-MAP
kinase pathway between InlB and HGF is a result of the difference in
ability to stimulate phosphorylation of this 90-kDa protein. A variety
of downstream targets for PI 3-kinase has been identified (49).
However, we are unaware of the identification of a 90-kDa protein known
to bind to the amino-terminal SH2 domain of p85. Identification and
further characterization of this protein will be necessary to find out
whether this protein performs an essential function during
InlB-dependent signal transduction. We are currently using
an affinity purification approach to identify this protein.
InlB is composed of an amino-terminal domain that contains an
amino-terminal cap, several LRR, and an immunoglobin-like region (IR). These three motifs comprise a single structural domain that is
followed by a B repeat region (B) and a carboxyl-terminal domain that
contains three tandem GW repeats (31, 50) (Fig. 6A). The LRR
domain was found previously (29) to mediate binding and activation of
the HGF receptor. The GW domain has been shown to mediate the
association of InlB with the bacterial surface (50, 51). When we
compared InlB and LRRd, we found that both proteins are
similar in their ability to turn on the HGF receptor but different in
their ability to turn on MAP kinase. InlB is a stronger activator of
MAP kinase.
To find out why InlB is a better activator of MAP kinase than either
LRRd or HGF, we analyzed a series of carboxyl-terminal InlB
deletion mutants for their ability to stimulate cell signaling to the
Ras-MAP kinase pathway. Because progressive deletion of the carboxyl
terminus affected the abilities of these mutant proteins to activate
the HGF receptor, it was impossible to look at downstream
signaling.2 Therefore, mutant proteins with internal
deletions were generated and tested for their abilities to activate
cell signaling. Deletion of the first GW repeat (GW1) resulted in a
protein that is unaffected in its ability to activate the HGF receptor
or the Ras-MAP kinase pathway. This demonstrates that internal
deletions can be made in InlB without completely destroying its
biological activity. Neither GW1 nor the B repeat region of InlB seems
necessary for maximal activation of the HGF receptor. Comparing the
abilities of these proteins to mediate signaling downstream of the HGF
receptor, however, shows that the mutant that is missing both GW1 and
the B repeats region is severely compromised in its ability to activate the Ras-MAP kinase pathway. The IR appears to be essential for HGF
receptor activation because the protein that is missing this region, as
well as GW1 and the B repeats, has lost the ability to turn on the HGF
receptor. An alternative explanation is that the IR is essential for
normal folding of InlB. However, we do know that folding of LRR is
independent of IR, as LRRd itself is active (Fig. 6). Our
results show that it is possible to separate activation of the HGF
receptor from the super-activation of MAP kinase. Furthermore, it
appears that it is the B repeat that is essential for the increased
ability of the InlB to turn on MAP kinase.
One possible explanation for the observed differences in the ability to
activate MAP kinase is that the kinetics for the ligand-receptor interaction differs between HGF and InlB. This has been observed for
the T cell receptor, where downstream signaling is affected by the
kinetics of the interaction between receptor and ligand (52). However,
it should be noted that the activation of the T cell receptor requires
the interaction of two different cell types and is the result of
several interactions among various proteins present on the surface of
the interacting cells (53, 54).
An alternative explanation is that the B repeat region of InlB could
act by recruiting an unrelated receptor into the signaling complex.
InlB thus may signal in a manner analogous to the nerve growth factor
(NGF). Two unrelated receptors for NGF have been identified (55). One
is a receptor protein-tyrosine kinase and is called the NGF receptor or
Trk (56). The second one is called p75 and is a member of the tumor
necrosis factor receptor family (57). The NGF receptor can act as a
receptor for NGF by itself. Co-expression of p75 increases NGF binding,
and it influences the ability of NGF to stimulate NGF receptor
autophosphorylation (58-60). This second receptor could activate
distinct signaling pathways that are different from those activated by
the HGF receptor but act in synergy with the signaling downstream of
the HGF receptor to lead to the super-activation of MAP kinase that is
seen in response to InlB.
What is the identity of the second receptor? It has been reported
that both gC1qR and glycosaminoglycans are membrane-bound host-cell
components that interact with InlB and are important for InlB-mediated
invasion of L. monocytogenes (30, 61). However, it has been
established that InlB interacts with both of these molecules via its GW
domains and not the B repeat region (30, 31). The identification of a
second receptor that can interact with the B repeat of InlB would be of
importance to understanding how InlB mediates host-cell signaling.
In summary, we have found that there are differences in the abilities
of InlB and HGF to turn on the Ras-MAP kinase pathway. Analysis of
deletion mutants shows that the LRR, the IR, and the B repeat region
are all necessary for signaling mediated by InlB, yet it is the B
repeat region that is responsible for the signaling differences between
InlB and HGF in their abilities to activate MAP kinase. We believe that
the LRR is involved in binding to the HGF receptor, whereas one or more
of the other domains could interact with a second receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GW1 (residues 36-392 and 464-630), InlB
B-GW1 (residues 36-321 and 464-630), and InlB
IR-B-GW1
(residues 36-248 and 464-630) were cloned into pet28b (Novagen) by
PCR from the intact InlB expression plasmid using standard protocols.
InlB
GW1, InlB
B-GW1 and InlB
IR-B-GW1 were purified using a
protocol identical to that used for intact InlB. Protein concentrations
were determined using the absorption at 280 nm and the calculated
extinction coefficients.
-mercaptoethanol, 5 mM dithiothreitol, 2.3% SDS, and 0.025% bromphenol blue
(SDS-sample buffer) and resolved by SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
InlB and HGF differ in their abilities to
cause phosphorylation of cellular proteins. Whole cell lysates
(WCL) of control (lane 1) and HGF- (lane
2) or InlB (lane 3)-stimulated Vero cells were analyzed
by anti-Tyr(P) immunoblotting (A). Anti-HGF receptor
immunoprecipitates (IP) of control (lane 1) and
HGF- (lane 2) or InlB (lane 3)-stimulated Vero
cells were analyzed by anti-Tyr(P) (B) and anti-HGF receptor
(C) immunoblotting.
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Fig. 2.
InlB is a more potent activator of Erk-1 and
Erk-2 than HGF. Vero cells were stimulated in the absence
(lanes 1-3) or presence (lanes 4-6) of PD98059.
Whole cell lysates of control (lanes 1 and 4),
HGF (lanes 2 and 5), and InlB-stimulated
(lanes 3 and 6) cells were probed with an
antiserum against activated Erk-1 and Erk-2 (A), an antibody
against Erk-2 (B), and an antibody against Tyr(P)
(C) (P.Tyr blot).
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Fig. 3.
The ability of InlB to activate Erk-1/2 to a
greater extent than HGF is not
concentration-dependent. Anti-HGF receptor
immunoprecipitates from Vero cells that were stimulated with increasing
concentrations of either HGF or InlB for 1 min at 37 °C were
analyzed by anti-Tyr(P) (A) and anti-HGF receptor
(B) immunoblotting. Whole cell lysates were analyzed by
immunoblotting with an antibody that recognizes active Erk-1 and Erk-2
(C) and an antibody against Erk-2 (D).
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Fig. 4.
InlB is a better activator of the Ras-MAP
kinase pathway than HGF. Whole cell lysates of control (lane
1), HGF- (lane 2), and InlB-stimulated (lane
3) Vero cells were analyzed by immunoblotting with an antibody
against active MEK (A). Anti-Raf immunoprecipitates from
these same cells were probed with an antibody against active Raf
(B) and an antiserum against Raf (C). Lysates of
control, HGF-, and InlB-stimulated Vero cells were incubated with the
Ras-binding domain of Raf immobilized on agarose beads. Bound proteins
were analyzed by anti-Ras immunoblotting (D). Whole cell
lysates from these same cells were probed by anti-Ras immunoblotting
(E).
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Fig. 5.
An unidentified tyrosine-phosphorylated
protein associates with PI 3-kinase in response to InlB but not in
response to HGF. Immunoprecipitates (IP) of the p85
subunit of PI 3-kinase from control (lane 1), HGF-
(lane 2), and InlB-stimulated (lane 3) Vero cells
were analyzed by anti-Tyr(P) immunoblotting (A). Lysates
from control, HGF-, and InlB-stimulated Vero cells were incubated with
a GST fusion protein containing the amino-terminal SH2 domain of p85
immobilized on agarose beads. Bound proteins were analyzed by
anti-Tyr(P) immunoblotting (B).
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Fig. 6.
The LRR domain dimer resembles HGF in its
ability to turn on MAP kinase. A schematic diagram of InlB and the
LRR domain dimer (LRRd) is shown (A). Anti-HGF
receptor immunoprecipitates from control (lane 1), HGF-
(lane 2), InlB- (lane 3), and LRRd-
stimulated (lane 4) Vero cells were analyzed by anti-Tyr(P)
(B) and anti-HGF receptor (C) immunoblotting.
Whole cell lysates were analyzed by immunoblotting with an antibody
that recognizes active Erk-1 and Erk-2 (D) and an antibody
against Erk-2 (E).
GW1) is unaffected in its ability to turn on either the HGF receptor or MAP kinase (Fig. 7). A second mutant that lacks both the B repeat and the first GW repeat
(InlB
B-GW1) is unaffected in its ability to turn on the receptor but
has lost the ability to super-activate MAP kinase (Fig. 7). Finally, a mutant that lacks the IR, the B repeat, and the first GW repeat (InlB
IR-B-GW1) loses the ability to turn on the HGF receptor (Fig.
7A). These data clearly show that the IR and B repeat are important for signaling by InlB.
View larger version (36K):
[in a new window]
Fig. 7.
Regions outside of the LRR domain contribute
to the ability of InlB to activate the HGF
receptor and the Ras-MAP kinase pathway. A schematic diagram of
all InlB deletion mutants is shown (A). Anti-HGF receptor
immunoprecipitates (IP) from control (lane 1),
HGF- (lane 2), InlB- (lane 3), InlB GW1-
(lane 4), InlB
B-GW1- (lane 5), and
InlB
IR-B-GW1 (lane 6)-stimulated Vero cells were analyzed
by anti-Tyr(P) (P.Tyr blot) (B) and anti-HGF
receptor (C) immunoblotting. Whole cell lysates
(WCL) were analyzed by immunoblotting with an antibody that
recognizes active Erk-1 and Erk-2 (D) and an antibody
against Erk-2 (E).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by a grant from the American Heart Association (to P. G.) and National Institutes of Health Grant R01 AI47163 (to P. G.).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.
Supported by National Institutes of Health Training Grants NCI T32
CA09523 and 5T 329M07240.
§ Supported by National Institutes of Health Training Grant GM07240.
¶ W. M. Keck Distinguished Young Scholar in Medicine.
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, University of California San Diego, 9500 Gilman Dr.,
La Jolla, CA 92093-0359. Tel.: 858-822-2024; Fax: 858-534-7042; E-mail:
geer@ucsd.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211666200
2 M. Banerjee, J. Copp, M. Marino, T. Chapman, P. van der Geer, and P. Ghosh, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: HGF, hepatocyte growth factor; InlB, internalin B; MAP, mitogen-activated protein; PI, phosphatidylinositol; LRR, leucine-rich repeat; SH2, Src homology 2; B, B repeat region; IR, immunoglobin-like region; GST, glutathione S-transferase; RBD, Ras-binding domain; NGF, nerve growth factor; MEK, MAP kinase/extracellular signal-regulated kinase kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sonnenberg, E., Weidner, K. M., and Birchmeier, C. (1993) Exs (Basel) 65, 381-394 |
2. | Birchmeier, C., and Gherardi, E. (1998) Trends Cell Biol. 8, 404-410[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Trusolino, L.,
Pugliese, L.,
and Comoglio, P. M.
(1998)
FASEB J.
12,
1267-1280 |
4. | Rong, S., Segal, S., Anver, M., Resau, J. H., and Vande Woude, G. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4731-4735[Abstract] |
5. | Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., dalla Zonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994) Cell 77, 261-271[Medline] [Order article via Infotrieve] |
6. | Weidner, K. M., Di Cesare, S., Sachs, M., Brinkmann, V., Behrens, J., and Birchmeier, W. (1996) Nature 384, 173-176[CrossRef][Medline] [Order article via Infotrieve] |
7. | Pelicci, G., Giordano, S., Zhen, Z., Salcini, A. E., Lanfrancone, L., Bardelli, A., Panayotou, G., Waterfield, M. D., Ponzetto, C., Pelicci, P. G., and Comoglio, P. M. (1995) Oncogene 10, 1631-1638[Medline] [Order article via Infotrieve] |
8. |
Graziani, A.,
Gramaglia, D.,
Cantley, L. C.,
and Comoglio, P. M.
(1991)
J. Biol. Chem.
266,
22087-22090 |
9. | Garcia-Guzman, M., Dolfi, F., Zeh, K., and Vuori, K. (1999) Oncogene 18, 7775-7786[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Sakkab, D.,
Lewitzky, M.,
Posern, G.,
Schaeper, U.,
Sachs, M.,
Birchmeier, W.,
and Feller, S. M.
(2000)
J. Biol. Chem.
275,
10772-10778 |
11. |
Fixman, E. D.,
Holgado-Madruga, M.,
Nguyen, L.,
Kamikura, D. M.,
Fournier, T. M.,
Wong, A. J.,
and Park, M.
(1997)
J. Biol. Chem.
272,
20167-20172 |
12. | Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K., and Wong, A. J. (1996) Nature 379, 560-564[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Nguyen, L.,
Holgado-Madruga, M.,
Maroun, C.,
Fixman, E. D.,
Kamikura, D.,
Fournier, T.,
Charest, A.,
Tremblay, M. L.,
Wong, A. J.,
and Park, M.
(1997)
J. Biol. Chem.
272,
20811-20819 |
14. | Schlessinger, J. (1993) Trends Biochem. Sci. 18, 273-275[CrossRef][Medline] [Order article via Infotrieve] |
15. | Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature 363, 83-85[CrossRef][Medline] [Order article via Infotrieve] |
16. | Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature 360, 689-692[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Smit, L.,
de Vries-Smits, A. M.,
Bos, J. L.,
and Borst, J.
(1994)
J. Biol. Chem.
269,
20209-20212 |
18. | Lioubin, M. N., Myles, G. M., Carlberg, K., Bowtell, D., and Rohrschneider, L. R. (1994) Mol. Cell. Biol. 14, 5682-5691[Abstract] |
19. | Salcini, A. E., McGlade, J., Pelicci, G., Nicoletti, I., Pawson, T., and Pelicci, P. G. (1994) Oncogene 9, 2827-2836[Medline] [Order article via Infotrieve] |
20. |
Carpenter, C. L.,
Duckworth, B. C.,
Auger, K. R.,
Cohen, B.,
Schaffhausen, B. S.,
and Cantley, L. C.
(1990)
J. Biol. Chem.
265,
19704-19711 |
21. | Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. (1991) Cell 65, 83-90[Medline] [Order article via Infotrieve] |
22. | Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz, L. F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 65, 91-104[Medline] [Order article via Infotrieve] |
23. | Feller, S. M. (2001) Oncogene 20, 6348-6371[CrossRef][Medline] [Order article via Infotrieve] |
24. | Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE http://www.stke.org/cgi/content/full/OC_sigtrans; 2001/PE40 |
25. |
Tang, P.,
Sutherland, C. L.,
Gold, M. R.,
and Finlay, B. B.
(1998)
Infect. Immun.
66,
1106-1112 |
26. |
Ireton, K.,
Payrastre, B.,
and Cossart, P.
(1999)
J. Biol. Chem.
274,
17025-17032 |
27. | Marino, M., Braun, L., Cossart, P., and Ghosh, P. (1999) Mol. Cell 4, 1063-1072[Medline] [Order article via Infotrieve] |
28. | Schubert, W. D., Gobel, G., Diepholz, M., Darji, A., Kloer, D., Hain, T., Chakraborty, T., Wehland, J., Domann, E., and Heinz, D. W. (2001) J. Mol. Biol. 312, 783-794[CrossRef][Medline] [Order article via Infotrieve] |
29. | Shen, Y., Naujokas, M., Park, M., and Ireton, K. (2000) Cell 103, 501-510[Medline] [Order article via Infotrieve] |
30. | Jonquieres, R., Pizarro-Cerda, J., and Cossart, P. (2001) Mol. Microbiol. 42, 955-965[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Marino, M.,
Banerjee, M.,
Jonquieres, R.,
Cossart, P.,
and Ghosh, P.
(2002)
EMBO J.
21,
5623-5634 |
32. | Braun, L., Nato, F., Payrastre, B., Mazie, J. C., and Cossart, P. (1999) Mol. Microbiol. 34, 10-23[CrossRef][Medline] [Order article via Infotrieve] |
33. | Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104[Medline] [Order article via Infotrieve] |
34. | van der Geer, P., Wiley, S., Lai, V. K.-M., Olivier, J. P., Gish, G., Stephens, R., Kaplan, D., Shoelson, S., and Pawson, T. (1995) Curr. Biol. 5, 404-412[Medline] [Order article via Infotrieve] |
35. | Suen, K. L., Bustelo, X. R., Pawson, T., and Barbacid, M. (1993) Mol. Cell. Biol. 13, 5500-5512[Abstract] |
36. | Reedijk, M., Liu, X., van der Geer, P., Letwin, K., Waterfield, M. D., Hunter, T., and Pawson, T. (1992) EMBO J. 11, 1365-1372[Abstract] |
37. |
Barnes, H.,
Larsen, B.,
Tyers, M.,
and van Der Geer, P.
(2001)
J. Biol. Chem.
276,
19119-19125 |
38. |
Awasthi, V.,
and King, R. J.
(2000)
Am. J. Physiol.
279,
L942-L949 |
39. | Robinson, M. J., and Cobb, M. H. (1997) Curr. Opin. Cell Biol. 9, 180-186[CrossRef][Medline] [Order article via Infotrieve] |
40. | de Rooij, J., and Bos, J. L. (1997) Oncogene 14, 623-625[CrossRef][Medline] [Order article via Infotrieve] |
41. | Taylor, S. J., and Shalloway, D. (1996) Curr. Biol. 6, 1621-1627[Medline] [Order article via Infotrieve] |
42. | Dramsi, S., Biswas, I., Maguin, E., Braun, L., Mastroeni, P., and Cossart, P. (1995) Mol. Microbiol. 16, 251-261[Medline] [Order article via Infotrieve] |
43. |
Cossart, P.,
and Lecuit, M.
(1998)
EMBO J.
17,
3797-3806 |
44. | Furge, K. A., Zhang, Y. W., and Vande Woude, G. F. (2000) Oncogene 19, 5582-5589[CrossRef][Medline] [Order article via Infotrieve] |
45. | Vande Woude, G. F., Jeffers, M., Cortner, J., Alvord, G., Tsarfaty, I., and Resau, J. (1997) CIBA Found. Symp. 212, 119-130[Medline] [Order article via Infotrieve] |
46. | Chang, L., and Karin, M. (2001) Nature 410, 37-40[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Fixman, E. D.,
Fournier, T. M.,
Kamikura, D. M.,
Naujokas, M. A.,
and Park, M.
(1996)
J. Biol. Chem.
271,
13116-13122 |
48. |
Rahimi, N.,
Tremblay, E.,
and Elliott, B.
(1996)
J. Biol. Chem.
271,
24850-24855 |
49. | Duronio, V., Scheid, M. P., and Ettinger, S. (1998) Cell. Signal. 10, 233-239[CrossRef][Medline] [Order article via Infotrieve] |
50. | Braun, L., Dramsi, S., Dehoux, P., Bierne, H., Lindahl, G., and Cossart, P. (1997) Mol. Microbiol. 25, 285-294[Medline] [Order article via Infotrieve] |
51. | Jonquieres, R., Bierne, H., Fiedler, F., Gounon, P., and Cossart, P. (1999) Mol. Microbiol. 34, 902-914[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Williams, C. B.,
Engle, D. L.,
Kersh, G. J.,
Michael White, J.,
and Allen, P. M.
(1999)
J. Exp. Med.
189,
1531-1544 |
53. | Brown, M. J., and Shaw, S. (1999) Curr. Biol. 9, R26-R28[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Germain, R. N.
(2001)
J. Biol. Chem.
276,
35223-35226 |
55. | Friedman, W. J., and Greene, L. A. (1999) Exp. Cell Res. 253, 131-142[CrossRef][Medline] [Order article via Infotrieve] |
56. | Kaplan, D. R., Hempstead, B. L., Martin, Z. D., Chao, M. V., and Parada, L. F. (1991) Science 252, 554-558[Medline] [Order article via Infotrieve] |
57. | Chao, M. V. (1994) J. Neurobiol. 25, 1373-1385[Medline] [Order article via Infotrieve] |
58. | Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F., and Chao, M. V. (1991) Nature 350, 678-683[CrossRef][Medline] [Order article via Infotrieve] |
59. | Barker, P. A., and Shooter, E. M. (1994) Neuron 13, 203-215[Medline] [Order article via Infotrieve] |
60. | Verdi, J. M., Birren, S. J., Ibanez, C. F., Persson, H., Kaplan, D. R., Benedetti, M., Chao, M. V., and Anderson, D. J. (1994) Neuron 12, 733-745[Medline] [Order article via Infotrieve] |
61. |
Braun, L.,
Ghebrehiwet, B.,
and Cossart, P.
(2000)
EMBO J.
19,
1458-1466 |
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