1 Department of Microbiology and Cancer Center, University of Virginia Health
System, Charlottesville, VA 22908-0734, USA
2 Department of Health Evaluation Sciences, University of Virginia Health
System, Charlottesville, VA 22908-0734, USA
* Present address: Norris Cotton Cancer Center, Dartmouth Hitchcock Medical
Center, Lebanon, NH 03756
Author for correspondence (e-mail:
ahb8y{at}virginia.edu
)
Accepted 23 April 2002
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Summary |
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Key words: Cas, Fak, Pyk2, Rac1, Yersinia
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Introduction |
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One of the effector molecules encoded on the Y. pseudotuberculosis
virulence plasmid is YopH, a protein tyrosine phosphatase (PTPase) that
inhibits bacterial phagocytosis by host cells
(Rosqvist et al., 1988a;
Guan and Dixon, 1990
).
Substrates of YopH include host cell focal-adhesion-associated proteins such
as Focal Adhesion Kinase (Fak), p130Cas (Crk-associated
substrate; Cas) and paxillin (Black and
Bliska, 1997
; Persson et al.,
1997
; Black et al.,
1998
). Focal adhesion targeting and phosphorylation of these
proteins is critical for the activation of cellular signaling cascades that
regulate the actin cytoskeleton (for reviews, see
Brugge, 1998
;
Vuori, 1998
;
Critchley, 2000
).
YopH-mediated dephosphorylation of Fak, Cas and paxillin is believed to
inhibit the actin cytoskeletal remodeling that is necessary for bacterial
phagocytosis. The observation that YopH disrupts focal adhesions supports this
model (Black and Bliska, 1997
;
Persson et al., 1997
).
Focal adhesions are cellular structures that link transmembrane integrins
to the actin cytoskeleton (Brugge,
1998; Critchley,
2000
). The binding of integrins to extracellular matrix (ECM)
ligands induces integrin clustering and recruitment of the non-receptor
protein tyrosine kinase (PTK) Fak. This results in Fak autophosphorylation and
the creation of a binding site for the Src-homology 2 (SH2) domain of Src
(Schaller et al., 1994
;
Schaller, 1996
). Fak-Src
association increases Src PTK activity and promotes Src-dependent
phosphorylation of additional tyrosine residues on Fak and Fak-associated
proteins, such as Cas and paxillin
(Schaller et al., 1994
;
Calalb et al., 1995
;
Thomas et al., 1998
;
Ruest et al., 2001
).
Integrin-dependent activation of Fak and Src has been implicated in numerous
actin-based cellular processes, including cell cycle progression, adhesion and
migration (Ilic et al., 1997
;
Klemke et al., 1998
;
Cary and Guan, 1999
;
Oktay et al., 1999
).
The Fak family member Pyk2/Cakß/Raftk (Pyk2) shares a high degree of
sequence identity with Fak, particularly in the N-terminus and kinase domain
(Kanner et al., 1994;
Avraham et al., 1995
;
Sasaki et al., 1995
). Like
Fak, Pyk2 contains binding sites for Cas and paxillin as well as an
autophosphorylation site, which can serve as a Src SH2-domain binding site
when it is phosphorylated (Dikic et al.,
1996
). Despite these similarities, Fak and Pyk2 have many distinct
features. Although Fak is expressed fairly ubiquitously, Pyk2 shows a more
restricted distribution, being highly expressed in the brain and hematopoietic
cells (Avraham et al., 1995
;
Sasaki et al., 1995
).
Moreover, although both Fak and Pyk2 have been implicated in signaling to the
actin cytoskeleton, only Fak routinely localizes to focal adhesions
(Sasaki et al., 1995
;
Li et al., 1996
;
Astier et al., 1997a
;
Guan, 1997
). Fak is activated
primarily in response to integrin activation, whereas Pyk2 can be activated in
response to stress, calcium flux or upon ligation of B and T cell receptors
(for a review, see Avraham et al.,
2000
). Certain functions of Fak and Pyk2 may even be antagonistic,
as Fak has been implicated in cell survival whereas Pyk2 signaling can result
in the induction of apoptosis (Frisch et
al., 1996
; Xiong and Parsons,
1997
).
Cas was first identified as a highly tyrosine-phosphorylated protein in
v-Src-and v-Crk-transformed cells (Sakai
et al., 1994). Cas functions as an adapter protein through its
interactions with numerous cellular proteins (for reviews, see
O'Neill et al., 2000
;
Bouton et al., 2001
). It
contains an N-terminal SH3 domain that binds to Fak and Pyk2, a
substrate-binding domain that binds to the adapter molecules Crk and Nck, and
a C-terminus that contains Src-binding sequences. The 15 YXXP motifs located
in the substrate-binding domain serve as potential phosphorylation sites for
cellular PTKs (Songyang et al.,
1994
; Songyang and Cantley,
1998
). Recent work has shown that, although Fak and Pyk2 are both
capable of phosphorylating Cas, Src appears to be responsible for the majority
of Cas phosphorylation (Astier et al.,
1997b
; Ruest et al.,
2001
). Therefore, the role of Cas-Fak or Cas-Pyk2 interactions may
be to bring Cas in close proximity to Src in order to promote its
phosphorylation. Phosphorylation of the Cas substrate-binding domain can
result in the recruitment of Crk and ultimately activation of Rac1, leading to
cytoskeletal remodeling (for reviews, see
Hall, 1998
;
Kiyokawa et al., 1998
;
Bishop and Hall, 2000
;
Ridley, 2000
).
The interplay between Fak, Src, Cas, Crk and Rac1 has been extensively
studied in the actin-dependent process of integrin-dependent migration.
Fibroblasts from Fak-/-, Src/Yes/Fyn (SYF)-/- and
Cas-/- mouse embryos are all defective for migration
(Ilic et al., 1995;
Honda et al., 1999
;
Klinghoffer et al., 1999
). The
ability of ectopic Fak to rescue migration in Fak-/- cells requires
its association with both Src and Cas
(Cary et al., 1998
;
Sieg et al., 1999
), providing
evidence that the functions of these molecules are interconnected. Cell
migration can also be enhanced in COS and FG-M pancreatic carcinoma cells by
overexpression of wild-type Cas, but not by expression of a Cas mutant that is
defective for Crk binding (Klemke et al.,
1998
; Cheresh et al.,
1999
; Cho and Klemke,
2000
). Co-expression of Cas with dominant inhibitory Crk or Rac1
molecules blocks this Cas-dependent cell migration pathway, lending further
support to the notion that Cas-Crk-Rac1 signaling plays a key role in this
process.
The host cellular response to Y. pseudotuberculosis infection,
which is largely dependent on invasin-ß1 integrin interactions
(Rosqvist et al., 1988b;
Marra and Isberg, 1997
),
involves many of the same molecules that function in cell migration. Like
integrin binding to fibronectin (FN), uptake of Y. pseudotuberculosis
is coincident with increased tyrosine phosphorylation of several host cell
proteins, including Fak and Cas (Black and
Bliska, 1997
; Persson et al.,
1997
). This phosphorylation appears to be critical for uptake,
since treatment of host cells with PTK inhibitors prevents bacterial uptake
whereas treatment with PTPase inhibitors increases uptake
(Andersson et al., 1996
).
Moreover, bacterial expression of the antiphagocytic PTPase YopH inhibits
phosphorylation of Fak and Cas, and this correlates with an inhibition of
bacterial uptake (Black and Bliska,
1997
; Persson et al.,
1997
).
Previous studies have investigated whether the functions of Fak and Cas are
required for Yersinia uptake. Fak-/- cells have been shown to be
deficient in uptake of invasin-coated beads
(Alrutz and Isberg, 1998) and
HeLa cells expressing a dominant inhibitory variant of Cas were found to be
defective in uptake of Y. pseudotuberculosis
(Weidow et al., 2000
).
Nevertheless, important questions remain regarding the role of Cas and Fak in
the process of Yersinia uptake. For example, although Fak has been implicated
in invasin-mediated phagocytosis (Alrutz
and Isberg, 1998
), it has not yet been determined whether Fak and
its downstream effectors are actually involved in uptake of live Y.
pseudotuberculosis. Additionally, although Cas-Crk-Rac signaling has been
shown to be important for Yersinia uptake in HeLa cells
(Weidow et al., 2000
), the
events upstream of Cas phosphorylation have yet to be fully elucidated.
Since Fak and Cas function coordinately in the process of
integrin-dependent migration, we hypothesized that Yersinia uptake may also
require the concerted activities of both of these molecules. In this study, we
shed light on the molecular pathways that are activated in response to
Yersinia infection. First, we present evidence that both Fak and Cas play
roles in the Yersinia uptake process and that Cas can in fact function in a
novel pathway that is independent of Fak. Interestingly, in spite of this
distinction, Fak and Cas appear to ultimately feed into a common
Rac1-dependent signaling network. Blocking the function of either Fak or Cas
induces similar morphological defects in Yersinia internalization, which are
manifested by incomplete membrane protrusive activity that is consistent with
an inhibition of Rac1 activity. We also provide evidence that Pyk2, which is
expressed at high levels in Fak-/- mouse embryo fibroblasts (MEFs)
relative to other fibroblasts (Sieg et
al., 1998), plays a role in the Cas-dependent Yersinia uptake
pathway that is active in these cells. We show that inhibition of Pyk2 blocks
Cas-dependent Yersinia uptake, and that, while Pyk2 autophosphorylation is
increased in response to infection with a Yersinia strain that does not
express YopH, this response is inhibited in the presence of YopH. These data
suggest that catalytic activity and autophosphorylation of Pyk2 play an
important role in the Cas-dependent Yersinia uptake process in
Fak-/- cells. Finally, we show that Pyk2 also functions in Yersinia
uptake by macrophages, which express Pyk2 at levels comparable to those
observed in Fak-/- cells. Macrophages are important for clearing
Y. pseudotuberculosis infections in vivo. Therefore, Pyk2 may be an
integral part of the host response to Yersinia infections. Taken together,
these data provide new insight into the host cellular signaling networks that
are initiated upon infection with Y. pseudotuberculosis. Importantly,
these findings also contribute to a better understanding of other cellular
processes that involve actin remodeling, such as the host response to other
microbial pathogens, cell adhesion and migration.
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Materials and Methods |
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Antibodies and reagents
Cas monoclonal antibody (mAb) 8G4 has been described previously
(Bouton and Burnham, 1997).
Fak mAb 2A7 was kindly provided by J. T. Parsons (University of Virginia,
Charlottesville, VA). Myc mAb 9E10 was obtained from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). FLAG M5 mAb and FN were purchased from Sigma. A mAb
recognizing Pyk2 was obtained from Transduction Laboratories (San Diego, CA)
and phosphospecific Pyk2-Y402 was purchased from Biosource
International (Camarillo, CA). Polyclonal Yersinia antibodies (SB349) were
kindly provided by James Bliska (SUNY-Stony Brook, NY) and have been
previously described (Black and Bliska,
2000
). Fluoroscein isothiocyanate (FITC)-conjugated goat
anti-rabbit immunoglobulin (Ig), Texas Red (TR)-conjugated goat anti-rabbit Ig
and Cy5-conjugated goat anti-rabbit Ig were purchased from Jackson
Immunoresearch Laboratories, Inc. (West Grove, PA). Cascade blue-conjugated
goat anti-mouse Ig was purchased from Molecular Probes (Eugene, OR).
125I-goat anti-mouse Ig and enhanced chemiluminescence (ECL)
reagents were purchased from NEN Life Sciences (Boston, MA). Horseradish
peroxidase (HRP)-conjugated sheep anti-mouse Ig, HRP-conjugated donkey
anti-rabbit Ig and 125I-conjugated protein A were purchased from
Amersham Pharmacia Biotech (Piscataway, NJ). FuGene 6 transfection reagent was
obtained from Roche Molecular Biochemicals (Indianapolis, IN). PP2 was
purchased from Calbiochem (LaJolla, CA).
Plasmids and transient transfections
The pRK5 plasmids encoding Myc-tagged full-length Cas, Cas-YXXP or
Cas-
SH3 have been previously described
(Burnham et al., 2000
;
Harte et al., 2000
). The
pCAGGS expression vectors encoding Myc-tagged full-length Crk II (pCAGGS-Crk)
or derivatives encoding point mutations (Crk-R38V and Crk-W169L) were
generously provided by Michiyuki Matsuda
(Tanaka et al., 1995
). The
pcDNA3-2AB plasmid encoding FLAG-tagged Fak were generously provided by J.T.
Parsons (University of Virginia, Charlottesville, VA). The expression vector
pCMV encoding Myc-tagged PRNK (residues 781-1009) was kindly provided by
Wen-Chen Xiong (University of Alabama, Birmingham, AL). The pcDNA3-2AB plasmid
encoding FLAG-tagged Rac-N17 and the pRK5 plasmid encoding Myc-tagged Rac-N17
were generously provided by Scott A. Weed (University of Colorado, Denver,
CO). All constructs, except those encoding Rac1 derivatives, were transfected
as per themanufacturer's instructions using FuGene6 transfection reagent (6
µl per 1 µg DNA for J774A.1 and 3 µl per 1 µg DNA for
Fak-/- cells). The media used for transfection of macrophages did
not contain antibiotics. Cotransfections were performed at an approximate
molar equivalent ratio of 1:3 in the order listed. 24 hours post-transfection,
5x105 J774A.1 or 7x105 Fak-/-
cells were plated onto FN-coated coverslips and incubated for an additional 18
hours before infection with Yersinia. For transfection with Rac1 constructs,
2x105 Fak-/- cells were plated directly onto
FN-coated coverslips, transfected 24 hours later and incubated overnight. The
media was changed, and the cells were incubated for an additional 18 hours.
Expression of all ectopic proteins was confirmed by immunoblotting.
Bacterial uptake assay
The Y. pseudotuberculosis strains used for these studies have been
described previously, as have bacterial growth conditions
(Black and Bliska, 1997;
Palmer et al., 1998
).
Multiplicities of infection (MOI) were determined by plating serial dilutions
of final bacterial suspensions. Prior to infection, cells were washed twice
with phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 1.5 mM KH2PO4, pH=7.2),
and antibiotic-free media was added. Cells were infected for 2 hours at an MOI
of approximately 20, unless otherwise indicated, at 37°C in 7.5%
CO2. Cells were washed three times with PBS between all subsequent
steps. Cells were fixed in 3% paraformaldehyde for 20 minutes at room
temperature (RT) before immunostaining [adapted from
(Heesemann and Laufs, 1985
)].
All antibodies were diluted in PBS containing 2% bovine serum albumin (BSA),
and all incubations were at room temperature for 30 minutes. Extracellular
Yersinia were stained by incubation of cells with SB349 (1:500) prior to
incubation with FITC-conjugated goat anti-rabbit Ig (1:1000). Cells were
permeabilized by incubation with 0.4% Triton X-100 in PBS for 2 minutes.
Staining for total (extracellular and intracellular) Yersinia was then
performed by an additional incubation with SB349 (1:500) followed by
incubation with TR-conjugated goat anti-rabbit Ig (1:1000). To detect cells
expressing ectopic proteins, mAbs directed against the ectopic protein were
included in the second incubation with SB349 antibodies, followed by
incubation with TR-conjugated goat anti-rabbit Ig and cascade blue-conjugated
anti-mouse Ig at 1:150. To stain ectopic proteins, FLAG mAb M5 (1:150), Myc
mAb 9E10 (1:300) and Cas 8G4 (1:100) were used. For co-transfections, cells
were stained for the protein encoded by the plasmid that was transfected at
the lower concentration (first construct listed in each figure). Cells were
viewed with a Leica fluorescence microscope and photographed with a cooled
charged-coupled device (CCD) camera controlled by Inovision Isee software.
Bacterial uptake was determined for each experiment using the following
calculation:% uptake=[total cell-associated Yersinia (stained with TR)
extracellular Yersinia (stained with FITC)] / total Yersinia (stained with
TR)x100. For transient transfections, Yersinia were scored only if they
were associated with cells positively stained for ectopic protein
expression.
Transmission electron microscopy
For transmission electron microscopy (TEM) studies, 7x105
cells were plated onto FN-coated coverslips. The following day, cells were
washed twice with PBS and DMEM containing 10% FCS, and 1 mM sodium pyruvate
was added. Cells were infected at an MOI of approximately 50-500 for 15-60
minutes at 37°C and 5% CO2. Cells were washed three times with
PBS and fixed by addition of 2.5% glutaraldehyde/4% paraformaldehyde for 30
minutes at room temperature. Cells were dehydrated, embedded and sectioned in
the electron microscopy core facility at the University of Virginia. Samples
were viewed by TEM and photographed in collaboration with Jay Brown and
William Newcomb (University of Virginia, Charlottesville, VA).
Immunoblotting
Cells were rinsed twice with PBS and lysed in modified RIPA (50 mM Tris,
150 mM NaCl, 1% Igepal CA-630, 0.5% deoxycholate) containing protease and
phosphatase inhibitors (100 µM leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 0.15 unit/ml aprotinin, 1 mM vanadate) as previously described
(Kanner et al., 1989). Protein
concentrations were determined with the BCA Assay kit (Pierce, Rockford, IL).
40-50 µg of total cell lysate were resolved by 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to
nitrocellulose membranes and immunoblotted as previously described
(Burnham et al., 2000
,
Weidow et al., 2000
).
Statistical methods
In conjunction with the University of Virginia Division of Biostatistics
and Epidemiology, a two-way analysis of variance (ANOVA) was performed on the
log proportion of bacteria observed to be intracellular. After controlling for
variability related to repetitions of experiments, contrasts between test
condition means were used to determine statistical significance. A Bonferroni
correction was then applied to the P values comparing test condition
means to adjust for the number of comparisons
(Miller, 1981;
Rosner, 1995
). Data obtained
from experiments performed together were analyzed independently and maintained
as separate data sets, as depicted in Tables
1,2,3,4,5.
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Results |
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Yersinia uptake is also dependent on Cas-Crk-Rac1 signaling
(Weidow et al., 2000). To
determine whether Cas function is specifically required for Fak-dependent
Yersinia uptake, Fak was expressed in Fak-/- cells together with
either wild-type Cas or a dominant-negative (DN) Cas molecule
(Cas-
YXXP) that was shown to inhibit Y. pseudotuberculosis
uptake by HeLa cells. Cells co-expressing Fak and wild-type Cas exhibited
Yersinia uptake levels that were similar to those observed in Fak-expressing
cells (Table 2; Group A).
Statistical analysis of these data indicated that the level of uptake promoted
by these cells was greater than mock-transfected cells (P value
versus Mock; P<0.0001) but not different from Fak-transfected
cells (P value versus Fak; P=1.0000). Co-expression of Fak
and Cas-
YXXP also promoted Yersinia uptake, although the level of
uptake was somewhat reduced compared with uptake levels observed in the
presence of Fak or Fak plus wild-type Cas. This reduction may be due to the
fact that ectopic Fak was expressed at slightly lower levels in cells
co-transfected with Cas-
YXXP plasmids (data not shown). Regardless, the
finding that Cas-
YXXP did not fully inhibit Fak-mediated uptake may
suggest that functions that involve the Cas substrate-binding domain, such as
Cas-Crk signaling, are not absolutely required for Yersinia uptake under these
conditions.
To determine whether Fak-dependent Yersinia uptake was in fact independent
of Crk, we asked whether expression of DN Crk molecules affected Fak-mediated
uptake. Overexpression of Crk molecules containing point mutations that block
the function of either the Crk SH2 (Crk-R38V) or the Crk SH3 (Crk-W169L)
domain have previously been shown to inhibit uptake of Y.
pseudotuberculosis by HeLa cells
(Weidow et al., 2000). To
determine whether expression of these molecules also affects Fak-mediated
Yersinia uptake, invasion assays were performed in Fak-/- cells
co-transfected with constructs encoding Fak and wild-type Crk, Crk-R38V or
Crk-W169L. Fak-mediated uptake was slightly reduced in the presence of
wild-type Crk compared with cells expressing Fak alone, but this decrease was
not found to be statistically significant
(Table 2, Group B). Similar
levels of uptake were observed in cells coexpressing Fak and the mutant Crk
molecules, indicating that there was no functional difference between
expression of these molecules and expression of wild-type Crk. It is possible
that this failure to effectively inhibit Fak-mediated uptake may have resulted
from insufficient expression of the DN constructs. This does not appear to be
the case, however, since similar expression levels of both Crk-R38V and
Crk-W169L (data not shown) were able to effectively inhibit a second Yersinia
uptake pathway in these cells (see below,
Table 4). Taken together, these
data suggest that Crk is not required for Fak-dependent Yersinia uptake by
Fak-/- MEFs.
Rac1 has also been implicated in Yersinia uptake, and our previous study
indicated that it functioned downstream of Cas in this process
(Weidow et al., 2000;
Alrutz et al., 2001
;
Werner et al., 2001
;
Wiedemann et al., 2001
). To
determine if Rac1 is necessary for efficient Fak-mediated uptake,
Fak-/- cells were co-transfected with constructs encoding Fak and
DN Rac1 (Rac1-N17). Expression of DN Rac1 was found to effectively block
Fak-mediated Yersinia uptake (Table
2, Group C), indicating that uptake of Yersinia into
Fak-expressing Fak-/- MEFs requires Rac1. Collectively, these data
suggest that Rac1 activity is critical for Fak-dependent Yersinia uptake, but
Cas-Crk interactions do not appear to play a major role in this process.
Membrane protrusive activity coincident with Yersinia uptake requires
Fak and Cas
Y. pseudotuberculosis is internalized by host cells through a
process that involves extension of membrane protrusions around the bacteria
(Fallman et al., 1997;
Isberg et al., 2000
). To
determine whether Fak expression is required for these cytoskeletal changes,
Fak+/+ and Fak-/- cells were infected with YP17/pVector
and processed for electron microscopy. The majority of cell-associated
Yersinia were observed to be fully engulfed by Fak+/+ cells under
the conditions of this assay (Fig.
2A). In contrast, Fak-/- cells exhibited short membrane
protrusions near the bacteria, but full bacterial internalization was rarely
observed (Fig. 2B). These data
suggest that the defect in Y. pseudotuberculosis uptake exhibited by
Fak-/- cells may arise from an inability to fully extend membrane
protrusions at the site of bacterial adherence. Interestingly, macrophages
expressing DN Rac1 exhibited a similar morphological defect in Fc-mediated
phagocytosis (Massol et al.,
1998
), providing support for the theory that Fak-/-
cells may be deficient for Yersinia uptake as a consequence of an inability to
fully activate Rac1.
|
Rac1 has also been implicated in Cas-mediated uptake of Y.
pseudotuberculosis in HeLa cells
(Weidow et al., 2000). To
determine whether the Cas-dependent mechanisms of Yersinia uptake might be
morphologically similar to those involving Fak, HeLa cells stably expressing
either vector (HeLa/RK5) or Cas-
YXXP (HeLa/Cas-
YXXP) were
examined by electron microscopy following infection with YP17/pVector. Whereas
numerous Yersinia were observed to be fully engulfed by HeLa/RK5 cells
(Fig. 2C), cells that expressed
DN Cas-
YXXP were rarely observed to contain fully internalized bacteria
(Fig. 2D). Instead, short
membrane protrusions in the vicinity of the bacteria were evident. This defect
was strikingly similar to the phenotype exhibited by Fak-deficient cells,
suggesting that Fak and Cas may feed into common Rac1-dependent signaling
networks that regulate membrane protrusions during the course of Yersinia
uptake.
Cas promotes Y. pseudotuberculosis uptake independently of
Fak
Both Fak and Cas have the ability to independently signal to Rac1. Since
Cas-Crk signaling did not appear to function downstream of Fak in
Fak-/- fibroblasts to promote Yersinia uptake, we hypothesized that
Cas might have the ability to function independently of Fak during this
process. To address this hypothesis, Fak-/- cells were transfected
with a construct encoding wild-type Cas in the absence of ectopic Fak.
Interestingly, overexpression of Cas in Fak-/- cells reproducibly
promoted Yersinia uptake to a level that was statistically greater than the
level exhibited by mock-transfected cells
(Table 3). Moreover,
Cas-mediated uptake in Fak-/- cells was found to be dose-dependent
(Fig. 3), suggesting that Cas
may be a limiting factor for Yersinia uptake under conditions in which Fak is
not expressed.
|
To determine whether Cas-Crk-Rac1 signaling plays a role in
Fak-independent, Cas-mediated Yersinia uptake, we first asked whether
phosphorylation of Cas is required for Yersinia uptake. It has been previously
shown that Src kinases are primarily responsible for phosphorylation of Cas in
its substrate-binding domain (Ruest et
al., 2001) and that phosphorylation of this domain promotes
Cas-Crk association (Sakai et al.,
1994
; Burnham et al.,
1996
). To determine whether Src kinase activity is necessary for
Cas-mediated Yersinia uptake, Fak-/- cells were first transfected
with a construct encoding wild-type Cas and then pretreated for 10 minutes
with a Src-family kinase inhibitor (PP2)
(Hanke et al., 1996
;
Zhu et al., 1999
) prior to
infection with YP17/pVector. PP2 pretreatment of Cas-transfected cells
resulted in a complete inhibition of uptake relative to the uptake levels
exhibited by untreated, Cas-transfected, cells
(Table 4, Group A). These data
suggest that the activity of a PP2-sensitive kinase, most probably a
Src-family kinase, is necessary for Cas to promote Yersinia uptake.
Next we investigated whether the substrate-binding domain was necessary for
Cas-mediated uptake in the absence of Fak. Fak-/- cells were
transfected with a plasmid encoding the Cas derivative that is deleted for
this domain, Cas-YXXP. Invasion assays performed on these cells showed
that expression of Cas-
YXXP promoted uptake above the level observed in
mock-transfected cells (Table
4, Group B; P=0.0082), although not to the level of cells
expressing wild-type Cas. These data indicate that signaling via the
substrate-binding domain of Cas is not absolutely required for
Fak-independent, Cas-dependent Yersinia uptake.
Two observations led us to investigate whether Crk functioned in this
Cas-dependent process. First, expression of Cas-YXXP in
Fak-/- MEFs did not promote Yersinia uptake to the same extent as
wild-type Cas (see above). Second, expression of DN Crk molecules inhibits
Yersinia uptake by HeLa cells (Weidow et
al., 2000
). Fak-/- cells were cotransfected with
constructs encoding Cas and either wild-type or DN variants of Crk.
Co-expression of Cas and Crk promoted uptake to a level that was similar to
the level of uptake observed when Cas alone was expressed
(Table 4, Group C). In
contrast, co-expression of Cas with either Crk-R38V or Crk-W169L resulted in a
significant decrease in Cas-dependent Yersinia uptake. In fact, the level of
uptake observed in these cells was no different from that observed in
mock-transfected cells (P=1.0000). The inhibitory effect of these DN
Crk molecules appeared to be specific for Cas because expression of these same
molecules at equivalent levels (data not shown) failed to significantly
inhibit Fak-dependent Yersinia uptake (see
Table 2, Group B). These data
suggest that at least one component of the Cas-dependent Yersinia uptake
pathway in Fak-/- cells may involve Crk.
As Rac1 functions downstream of Cas-Crk in HeLa cells during the process of
Yersinia uptake (Weidow et al.,
2000), we also investigated whether DN Rac1 could inhibit
Cas-dependent Yersinia uptake in Fak-/- cells. Yersinia uptake was
significantly impaired in cells co-transfected with plasmids encoding Cas and
Rac1-N17 (Table 4, Group D).
Collectively, these data indicate that the Cas-Crk-Rac1 pathway, which has
previously been shown to function in HeLa cells to promote Yersinia uptake,
can also function independently of Fak in MEFs to promote uptake of Y.
pseudotuberculosis. However, because inhibition of Crk or Rac1, but not
Cas-
YXXP, completely blocked Cas-mediated uptake, these data suggest
that Crk and Rac1 may also play other roles in this process that are
independent of the substrate-binding domain of Cas.
Cas and Pyk2 coordinately function to promote Fak-independent Y.
pseudotuberculosis uptake
Cas can signal to Rac1 through mechanisms that are independent of Crk
binding to the substrate-binding domain. Many of these involve the Cas SH3
domain. To determine whether this domain is required for Cas-dependent Y.
pseudotuberculosis uptake, Fak-/- cells were transfected with
a Cas construct in which the SH3 domain was deleted (Cas-SH3).
Overexpression of Cas-
SH3 failed to promote uptake above the level
exhibited by mock-transfected cells (Table
4, Group E), suggesting that functions of Cas that involve its SH3
domain play a critical role in Cas-mediated Yersinia uptake in the absence of
Fak.
One protein that binds to the Cas SH3 domain is Pyk2, and Cas/Pyk2
complexes have been detected in Fak-/- cells
(Ueki et al., 1998) (data not
shown). Consequently, we investigated whether the ability of Cas to promote
Y. pseudotuberculosis uptake in the absence of Fak requires Pyk2
function. Fak-/- cells were co-transfected with constructs encoding
Cas and a C-terminal derivative of Pyk2, termed PRNK, that can potentially
serve as an inhibitor of Pyk2 (Xiong et
al., 1998
). PRNK expression did in fact reduce Cas-mediated uptake
to a level that was similar to that exhibited by mock-transfected cells
(Table 4, Group F). These data
suggest that Pyk2 function may be required for Cas to promote Y.
pseudotuberculosis in the absence of Fak.
Pyk2 functions in Yersinia uptake by macrophages
Although Fak-/- cells are a useful tool to investigate
Fak-independent signaling pathways, most other fibroblasts do not express
significant levels of Pyk2 (Sieg et al.,
1998). By contrast, macrophages naturally express Pyk2 at levels
comparable to Fak-/- MEFs (data not shown), and these cells play an
important role in clearing Y. pseudotuberculosis infections in vivo.
Therefore, we investigated whether Pyk2 also plays a role in Yersinia uptake
in macrophages. J774A.1 macrophages were first transfected with a construct
encoding PRNK and then infected with YP17/pVector to measure Yersinia uptake.
Although mock-transfected macrophages internalized approximately 53% of
attached Yersinia, cells expressing PRNK only internalized an average of 37%.
This decrease in Yersinia uptake was shown to be statistically significant
(Table 5; P=0.0012).
These data suggest that, in addition to playing a role in Cas-dependent
Yersinia uptake in Fak-/- MEFs, Pyk2 function may also be required
to promote Y. pseudotuberculosis uptake by macrophages.
To explore the mechanism of Pyk2 function in Yersinia uptake, we investigated whether Pyk2 underwent changes in autophosphorylation in response to infection. Fak-/- cells (Fig. 4, lanes 1-4) and macrophages (lanes 5-8) were infected with various Yersinia strains, and lysates were immunoblotted with activation-specific antibodies directed against the autophosphorylation site of Pyk2, tyrosine 402 (Y402). Infection with YP17/pVector resulted in increased levels of Y402 phosphorylation in both cell types, although the increase observed in Fak-/- cells was reproducibly lower than that observed in macrophages (upper panels). Infection with YP17/pYopH resulted in decreased levels of Y402 phosphorylation, whereas infection with YP17/pYopH-C403S (a Y. pseudotuberculosis strain expressing a catalytically-inactive, substrate-trapping mutant of YopH) augmented Y402 phosphorylation. Immunoblotting for Pyk2 indicated that equivalent levels of Pyk2 were present in each case (lower panels). Thus, infection of both Fak-/- MEFs and macrophages with Y. pseudotuberculosis was found to induce Pyk2 autophosphorylation. The finding that this site can also serve as a substrate for the antiphagocytic effector YopH further supports a role for Pyk2 activation in the process of Yersinia uptake.
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Discussion |
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Earlier studies measuring uptake of invasin-coated beads into
Fak-/- MEFs and Y. pseudotuberculosis into HeLa cells
indicated that both Fak and Cas may play a role in Yersinia uptake
(Alrutz and Isberg, 1998;
Weidow et al., 2000
). Our data
extend these initial reports by providing evidence that Fak-/- MEFs
are defective in uptake of live Y. pseudotuberculosis and that this
defect can be rescued by ectopic expression of Fak. This Fak-dependent uptake
pathway requires Rac1, but Cas-Crk signaling appears to be dispensable.
Surprisingly, ectopic expression of Cas was also found to rescue the defect in Yersinia uptake exhibited by Fak-/- cells. This is one of the first examples of Cas functioning independently of Fak in a biological process. Although the mechanism through which ectopic Cas promotes uptake is not known, endogenous levels of Cas are clearly not sufficient to promote Yersinia uptake in the absence of Fak, since Fak-/- cells are defective in this process even though they express the same amount of Cas as do Fak+/+ MEFs (data not shown). This suggests that ectopic Cas drives Yersinia uptake either by supplementing the function of limiting amounts of endogenous Cas or by activating novel pathway(s) that are independent of the function of endogenous Cas. Although it is difficult to distinguish between these two possibilities, the finding that ectopic Cas promoted uptake in a dose-dependent fashion lends support for the theory that Cas may be a limiting factor in this Fak-independent Yersinia uptake pathway.
Investigation of the molecular requirements of Cas-dependent Yersinia
uptake in Fak-/- cells showed that multiple Cas-binding proteins
play a role in this process. The first of these molecules, Src, is implicated
through studies using the PTK inhibitor PP2. Pretreatment of cells with PP2
abolished Cas-dependent Yersinia uptake, indicating that Src family kinases
are required. The second Cas-binding protein that is implicated in this
pathway is the small adapter molecule Crk, which binds to sites within the
substrate-binding domain of Cas that become phosphorylated by Src
(Sakai et al., 1994;
Ruest et al., 2001
). We have
previously shown that Yersinia infection induces Cas-Crk complexes in cells
that express endogenous Fak and that signaling downstream of Crk is critical
for Yersinia uptake by these cells (Weidow
et al., 2000
). The finding that expression of DN Crk molecules
blocked Cas-dependent Yersinia uptake in Fak-/- MEFs indicates that
Cas-Crk signaling may also play a role in this process when Fak is not
expressed.
Cas-Crk signaling has been implicated in a number of integrin-dependent
processes, including cell migration and Yersinia uptake
(Klemke et al., 1998;
Cheresh et al., 1999
;
Cho and Klemke, 2000
;
Weidow et al., 2000
). In these
cases, the binding of Crk to Cas is thought to result in activation of the
small GTPase Rac1 through recruitment of Crk-binding proteins such as DOCK180
and C3G (for review, see Feller,
2001
). Our data are consistent with the involvement of Rac1 in
Cas-dependent Yersinia uptake since expression of a DN Rac1 mutant effectively
blocked this process. Thus, the Cas-Crk-Rac1 cascade, which has previously
been shown to promote Yersinia uptake in cells that express Fak, appears to
also function in the absence of Fak.
The role of Crk in Fak-dependent Yersinia uptake pathways is less clear, as
expression of DN Crk constructs at levels equivalent to those that blocked
Cas-dependent uptake failed to inhibit Fak-mediated uptake by
Fak-/- MEFs. In spite of the apparent divergence in Fak- and
Cas-dependent Yersinia uptake processes with respect to the requirement for
Crk, however, both pathways appear to ultimately lead to Rac1 activation.
Visualization of the Yersinia uptake process by electron microscopy in cells
either lacking Fak or expressing a DN inhibitor of Cas indicated that both
cell types exhibit a common morphological defect in uptake. We propose that it
is the activation of Rac1, through distinct Fak- and Cas-dependent pathways,
that leads to the establishment of membrane protrusions involved in bacterial
internalization. Reports showing that Fak can activate Rac1 independently of
Cas-Crk are consistent with this hypothesis
(Cary and Guan, 1999;
Schlaepfer et al., 1999
;
Hauck et al., 2000
). In those
cases where Fak or Cas signaling is compromised, the resultant decrease in
Rac1 activation is likely to lead to decreased membrane protrusive activity
and inhibition of Yersinia uptake. In this regard, Yersinia uptake appears to
be similar to the process of Fc-mediated phagocytosis in macrophages, where
expression of DN Rac1 correlates with a failure to fully extend and fuse
membrane protrusions as well as an inhibition of particle uptake
(Massol et al., 1998
).
In addition to Cas-Crk-Rac signaling networks, we present evidence that
Pyk2 plays a role in Yersinia uptake in both Fak-/- cells that
express aberrantly high levels of Pyk2 and in macrophages that express both
Fak and Pyk2. The full-length C-terminus of Pyk2, PRNK, has been shown to
inhibit various functions of Pyk2
(Ivankovic-Dikic et al., 2000,
Watson et al., 2001
). In this
study, we show that a shorter variant of PRNK (residues 680-1009) has
inhibitory activity with respect to Cas-dependent Yersinia uptake in
Fak-/- cells. This inhibition by PRNK may occur via several
distinct mechanisms. For example, PRNK may compete with Pyk2 for localization
to critical subcellular compartments or it may sequester Pyk2 binding
partners. However, direct interactions between PRNK and Cas may not be
involved, since the PRNK construct used in this study contains only a low
affinity Cas-binding motif and it therefore may not associate with Cas at a
level sufficient to inhibit Cas signaling
(Xiong et al., 1998
). In any
case, PRNK-mediated displacement of Pyk2 away from functional signaling
complexes could ultimately lead to an inhibition of Yersinia uptake.
The requirement for Pyk2 in the process of Yersinia uptake appears to be a
common feature of Fak-/- cells and macrophages. In both cell types,
Pyk2 autophosphorylation becomes elevated upon Yersinia infection with strains
that do not express YopH, and YopH efficiently dephosphorylated this site.
Interestingly, the level of induction of Pyk2 autophosphorylation appeared to
be less pronounced in Fak-/- MEFs than in macrophages. Pyk2
localizes to focal adhesions and becomes activated in response to adhesive
signals in Fak-/- cells (Du et
al., 2001). This adhesion-dependent activation may have functional
consequences for the process of Yersinia uptake by these cells, perhaps by
providing a sub-threshold level of Pyk2 activity that can synergize with
overexpressed Cas to promote uptake. The downstream effects of Pyk2
phosphorylation in Fak-/- cells probably include recruitment of Src
family kinases to the autophosphorylation site, activation of Src and
subsequent phosphorylation of other Src- and/or Pyk2-binding proteins such as
Cas. We propose that, at least in Fak-/- cells, phosphorylation of
Cas through this mechanism creates Crk-binding sites. Subsequent recruitment
of Crk and perhaps DOCK180 to the complex can then lead to activation of Rac1
and uptake of Y. pseudotuberculosis. It remains to be determined
whether this Pyk2-Cas pathway also functions in macrophages or whether it is
only active when Fak is not expressed.
Although a Pyk2-Cas-Crk pathway may be responsible for a portion of the
Yersinia uptake observed in Fak-/- cells, there are indications
that other Cas-dependent pathways also contribute to this process. The finding
that Cas-YXXP was able to promote Fak-independent Yersinia uptake,
albeit to a lesser extent than wild-type Cas, indicates that functions
independent of the substrate-binding domain of Cas are involved. Evidence
suggests that these functions are predominantly associated with the Cas SH3
domain because Cas molecules that do not contain this domain were found to be
completely deficient in promoting Yersinia uptake. Numerous proteins in
addition to Pyk2 bind to the SH3 domain of Cas, including Fak, PTP-PEST,
PTP-1B and C3G (for a review, see O'Neill
et al., 2000
; Bouton et al.,
2001
). Thus, although phosphorylation of Cas and subsequent
binding to Crk may provide a positive signal for uptake, dephosphorylation of
Cas by PTP-PEST or PTP-1B may serve to downregulate this pathway. C3G may also
play a central role in this process. This protein, which is a guanine
nucleotide exchange factor (GEF) for Rap1, was initially found to associate
with Cas in a yeast two-hybrid screen
(Kirsch et al., 1998
). In
yeast, the orthologue of Rap1 has been shown to indirectly activate Cdc42,
which can then activate Rac1 (Bos,
1998
; Bos et al.,
2001
). It is tempting to speculate, therefore, that C3G binding to
the SH3 domain of Cas could lead to activation of Rap1 in mammalian cells,
ultimately resulting in activation of Cdc42 and/or Rac1. This pathway could
account for how Cas-
YXXP may be competent to promote Yersinia uptake
even though it cannot signal to Crk, because Cas-
YXXP/C3G complexes
could still signal to Cdc42 and/or Rac1. It could also explain why
Cas-
SH3 is completely deficient in promoting uptake because deletion of
the SH3 domain would block both the C3G-Cas and Pyk2-Cas-Crk pathways that
lead to Cdc42 and/or Rac1 activation.
Although we have not directly addressed the specific requirements for
invasinß1-integrin association in the Yersinia uptake pathways
defined above, it is well established that these interactions serve as the
predominant initiator of Yersinia uptake into host cells
(Rosqvist et al., 1988b;
Isberg, 1989
;
Marra and Isberg, 1997
). Many
of the molecular features of Yersinia uptake that have been revealed in this
study are common to other integrin-dependent processes such as cell adhesion
and migration, as well as Fc-mediated phagocytosis
(May and Machesky, 2001
). In
addition to these common elements, there are also likely to be aspects of
Yersinia uptake that are distinct from other integrin-mediated events. One
molecule that may function in this manner in Fak-/- cells is Pyk2,
which seems to link ß1 integrin signals initiated by Yersinia attachment
to Src, Cas, Crk and Rac1. The mechanism of activation of Pyk2 by ß1
integrins is unclear, particularly because Pyk2 does not contain the
N-terminal sequences present in Fak that have been shown to bind to ß1
integrin tails (Schaller et al.,
1995
; Klingbeil et al.,
2001
). However, in Fak-/- cells, Pyk2 can localize to
focal adhesions (Du et al.,
2001
). This may occur through its association with paxillin
(Hiregowdara et al., 1997
),
which is capable of binding to both ß1 and
4
integrins (Schaller and Parsons,
1995
; Liu et al.,
1999
; Liu and Ginsberg,
2000
). Focal adhesion localization of Pyk2 under these conditions
may be important for the Yersinia-dependent activation of Pyk2, as well as
propagation of the Pyk2-Cas pathway that promotes Yersinia uptake in the
absence of Fak. The mechanism of Pyk2 activation during the course of Yersinia
uptake into macrophages, which also express Fak, is even less clear. However,
this process may also involve ß1 integrin ligation, as it has been
previously shown that Pyk2 can become activated upon cell adhesion in cells
that express Fak (Lakkakorpi and Vaananen,
1996
; Hiregowdara et al.,
1997
; Ma et al.,
1997
; Brinson et al.,
1998
; Hatch et al.,
1998
; Li et al.,
1998
; Litvak et al.,
2000
; Du et al.,
2001
; Watson et al.,
2001
).
Several important questions regarding the role of Pyk2 in the process of Yersinia uptake into macrophages must now be addressed. First, the molecular mechanisms by which Pyk2 activation leads to Yersinia uptake remain to be elucidated. Within the framework of this question, it remains to be determined whether Cas and Pyk2 function in concert to promote Yersinia uptake by macrophages. If so, what are the roles of Cas-binding partners such as Crk and C3G? Finally, it is important to determine whether Fak and Pyk2 play independent, redundant or competing roles in this process, since both molecules are expressed in these cells. These questions form the basis for future studies designed to address the molecular mechanisms involved in Yersinia uptake by macrophages.
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Acknowledgments |
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