From the Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-5222
Received for publication, October 3, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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YopH is a 468-amino acid protein-tyrosine
phosphatase that is produced by pathogenic Yersinia
species. YopH is translocated into host mammalian cells via a type III
protein secretion system. Translocation of YopH into human epithelial
cells results in dephosphorylation of p130Cas and paxillin,
disruption of focal adhesions, and inhibition of integrin-mediated
bacterial phagocytosis. Previous studies have shown that the N-terminal
129 amino acids of YopH comprise a bifunctional domain. This domain
binds to the SycH chaperone in Yersinia to orchestrate
translocation and to tyrosine-phosphorylated target proteins in host
cells to mediate substrate recognition. We used random mutagenesis in
combination with the yeast two-hybrid system to identify residues in
the YopH N-terminal domain that are involved in substrate-binding
activity. Four single codon changes (Q11R, V31G, A33D, and N34D) were
identified that interfered with binding of the YopH N-terminal domain
to tyrosine-phosphorylated p130Cas but not to SycH. These
mutations did not impair YopH translocation into HeLa cells infected
with Yersinia pseudotuberculosis. Introduction of the V31G
substitution into catalytically inactive (substrate-trapping) forms of
YopH interfered with the ability of these proteins to bind to
p130Cas and to localize to focal adhesions in HeLa cells.
In addition, the V31G substitution reduced the ability of catalytically
active YopH to dephosphorylate target proteins in HeLa cells. These
data indicate that the substrate- and SycH-binding activities of the YopH N-terminal domain can be separated and that the former activity is
important for recognition and dephosphorylation of substrates by YopH
in vivo.
Three Yersinia species (Yersinia pestis,
Yersinia pseudotuberculosis, and Yersinia
enterocolitica) are highly pathogenic for humans. All three harbor
a related 70-kilobase pair plasmid that is essential for
virulence (1). Encoded on this plasmid is a type III protein secretion
system that functions to translocate a set of protein toxins termed
Yops into infected eukaryotic cells. Yops function to prevent
phagocytosis, superoxide production, and cytokine synthesis by
professional phagocytes and other types of host cells (2-5). YopT,
YopE, and YopH target key proteins that regulate the host cell actin
cytoskeleton. YopT modifies and inactivates the small GTP-binding
protein RhoA (6). YopE is a GTPase-activating protein for RhoA, Rac1,
and Cdc42 (7, 8). YopH is a protein-tyrosine phosphatase
(PTP)1 (9) that
dephosphorylates multiple focal adhesion proteins (10-14).
The 468-amino acid YopH protein appears to be composed of two distinct
modular domains. Residues 206-468 comprise the C-terminal PTP
catalytic domain (9). The PTP activity of this domain is essential for
the antiphagocytic function of YopH and Yersinia virulence
(15, 16). Residues 403-410 form a phosphate-binding loop (P-loop)
within the active site (17). Substitution of the nucleophilic Cys at
position 403 with either Ser (C403S) or Ala (C403A) has been shown to
inactivate the enzyme (9). Catalytically inactive forms of YopH can
form stable complexes with substrates in vivo ("substrate
trapping") (18) and localize to focal adhesion complexes in infected
cultured cells (10, 12). The focal adhesion proteins
p130Cas (Cas) (12) (10), focal adhesion kinase (12),
and paxillin (11) have been identified as substrates of YopH in
cultured human epithelial cells. A region within the PTP domain
(residues 223-226) has been shown to be important for targeting of
YopH to focal adhesion complexes (19).
The N-terminal 129 residues of YopH comprise a second modular domain
that is bifunctional. This domain binds to the SycH chaperone in
Yersinia to orchestrate type III-mediated translocation of YopH into host cells (20). A binding site for SycH has been localized
between residues 20 and 69 (20). The N-terminal domain also binds to
Cas and paxillin in vitro in a
phosphotyrosine-dependent manner (11). The efficiency of
substrate dephosphorylation by YopH in vitro is diminished
by removal of the N-terminal domain, suggesting that it is important
for substrate recognition (11). As a first step toward elucidating the
mechanism of substrate recognition mediated by the YopH N-terminal
domain, we have identified several residues that are critical for this activity.
Construction of Plasmids--
The plasmid pLP15 (21) contains a
DNA fragment coding for YopH fused to a C-terminal M45 epitope tag
inserted between the BamHI and EcoRI sites of
pGEX-2T. Here, pLP15 is designated pGEX2T-YopHM45. A NdeI
site overlaps the initiation codon of the YopH reading frame, and an
XbaI site is present at the point of fusion between the YopH
and M45 sequences in pGEX2T-YopHM45. The plasmid pGEX-YopH1-129M45 was
constructed as follows. A DNA fragment encoding the first 129 residues
of YopH flanked by 5' and 3' restriction sites was synthesized by PCR.
The PCR was performed using pYopH (22) as template and PTP18 (11) and
PTP23 (5'-GATCCCGGGACCCCCTCGCTCCCGACTCTTG-3') as forward and reverse
primers, respectively. The primers were designed to incorporate
BamHI and NdeI restriction sites into the 5' end
of the product and an XbaI site into the 3' end. The PCR
product was digested with BamHI and XbaI. The
pGEX2T-YopHM45 vector was digested with BamHI and
XbaI, and the region coding for full-length YopH was removed
and replaced with the PCR product encoding YopH1-129. This resulted in
a translational fusion between the codons specifying residues 1-129 of
YopH and the 12-amino acid C-terminal M45 epitope tag. The structure of
pGEX-YopH1-129M45 was verified by sequencing.
The plasmids pBDMI and pADMI were derived from the yeast two-hybrid
vectors pGBT9 and pGAD424 (27), respectively, by the insertion
of new polylinker regions. The polylinker regions of pGBT9 or pGAD424
were removed by digestion with EcoRI and SalI. Two complementary oligonucleotides, MIsiteF
(5'-AATTGGGATCCCCGGGAATTCGCGGCCGCG-3') and MIsiteR
(5'-CCCTAGGGGCCCTTAAGCGCCGGCGCAGCT-3'), were annealed and inserted
between the EcoRI and SalI sites in pGBT9 and
pGAD424. The polylinker regions of pBDMI and pADMI were confirmed by
sequencing. A segment of DNA coding for YopH1-129M45 was removed from
pGEX-YopH1-129M45 by digestion with BamHI and
EcoRI. This DNA was inserted between the BamHI
and EcoRI sites of pBDMI, yielding pBD-YopH1-129M45. The
plasmid pAD-Src was constructed from pADMI by the insertion of a DNA
fragment that encodes a mutant form of c-Src (Y416F/Y527F) (23). The
DNA fragment encoding c-Src was removed from pBTM116 (23) by digestion
with BamHI and inserted into the unique BglII site in pADMI. A DNA fragment encoding Cas was removed from
pEBG-p130Cas (24) by digestion with BamHI and
NotI and inserted between the BamHI and
NotI sites of pADMI or pAD-Src, yielding pAD-Cas and pAD-Cas+Src, respectively.
Codon substitutions isolated in the two-hybrid assay (see below) were
introduced into the full-length YopHM45 reading frame by restriction
fragment subcloning. DNA fragments containing the various codon
substitutions were removed from pBD-YopH1-129M45 by digestion with
NdeI and SnaBI and substituted for the
corresponding NdeI-SnaBI fragment of
pGEX2T-YopHM45. A standard restriction fragment subcloning procedure
was used to combine the V31G mutation with either the C403S or the
R409A substitutions. DNA fragments coding for full-length YopH proteins
with the various codon substitutions were removed from the pGEX-2T
backbone by digestion with NdeI and EcoRI and
inserted between the NdeI and EcoRI sites in the plasmid pPROH for expression in Y. pseudotuberculosis. The
plasmid pPROH was generated by the insertion of the yopH
promoter region into the polylinker region of pMMB67HE (25). The
yopH promoter region was amplified by PCR using virulence
plasmid pIB1 as template and the primers PTP26
(5'-CGGATCCGCTGCGCGATGTACTGACCCG-3') and PTP27
(5'-TTACATTAGGAATTCATATGTCCCTCCTTAATTAAATACACGCC-3'). The primers were
designed to incorporate a BamHI site into the 5' end of the
product and NdeI and EcoRI sites into the 3' end.
The PCR product was digested with BamHI and
EcoRI, inserted between the BamHI and
EcoRI sites of pMMB67HE, and verified by sequencing.
Identification of Codon Changes in the N-terminal Domain of YopH
That Interfere with Binding to Cas in the Two-hybrid System--
The
DNA sequence coding for the first 129 residues of YopH was subjected to
random mutagenesis using a PCR under suboptimal conditions (26). The
PCR was performed using pBD-YopH1-129M45 as template and the
oligonucleotides 5'PBD2H (5'-CCGTCACAGATAGATTGGCTTCAGTGG-3') and
3'PBD2H (5'-CCTGAGAAAGCAACCTGACCTACAGGA-3') as forward and reverse
primers, respectively. The resultant PCR product contained the
YopH1-129M45 coding region flanked on either side by ~100 base pairs
of sequence derived from pBDMI. The PCR product was introduced into
pBDMI via homologous recombination in yeast cells. For this purpose,
pBDMI was digested with BamHI, and the linear plasmid was
mixed with the PCR product and supercoiled pAD-Cas+Src. The mixture was
used to transform the yeast two-hybrid reporter strain Y153 to
Trp and Leu prototrophy on synthetic dextrose media lacking Trp
and Leu (SD-Trp-Leu) (27).
Mutations that interfered with binding of the YopH N-terminal domain to
tyrosine-phosphorylated Cas were identified by screening ~1500
Trp+ Leu+ colonies of Y153 on filters for reduced expression of
Antibodies--
Hybridoma supernatant containing monoclonal
anti-M45 antibody was provided by Dr. P. Hearing (State University of
New York at Stony Brook). Anti-M45 recognizes the 12-amino acid epitope SRDRLPPFETET. Anti-M45 was purified from the supernatant using protein
A-Sepharose as described (28). Anti-M45 was used at a dilution of
1:1000 for immunoblotting and at a final concentration of 0.5 µg/ml
for immunofluorescence labeling. Immunoprecipitations were performed
with 1.0 µg of M45 per sample. Monoclonal anti-phosphotyrosine (pTyr)
antibody 4G10 was purchased from Upstate Biotechnology. The anti-pTyr
antibody was used at a dilution of 1:1000 for immunoblotting and at
final concentration of 5 µg/ml for immunofluorescence labeling. Monoclonal anti-Cas (P27820) was purchased from Transduction
Laboratories and used at a dilution of 1:1000 for immunoblotting.
Immunoprecipitations were performed with 1.0 µg of anti-Cas per
sample. The rabbit anti-YopH antibody SB360 was prepared in a
commercial facility (Calico Biologicals, Inc.) using a purified
glutathione S-transferase-YopH fusion protein as antigen.
SB360 was used at a dilution of 1:1000 for immunoblotting. Anti-mouse
and anti-rabbit IgG conjugated to horseradish peroxidase were purchased
from Sigma and used at dilutions of 1:1000 or 1:15,000, respectively.
Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated
F(ab')2 goat anti-mouse secondary antibody was purchased
from Jackson ImmunoResearch Laboratories, Inc. and used at a dilution
of 1:250.
Bacterial and HeLa Cell Cultures and Infection
Conditions--
The Y. pseudotuberculosis serogroup III
strains YP17 (yopHyopE) and YP19 (yopHyopEyopB)
and their growth conditions have been described
previously (21). Both strains carry a
naturally occurring deletion in the plasmid-borne yopT gene
and are devoid of YopT activity.2 Expression
plasmids derived from pPROH that produce wild-type or mutant YopH
proteins were introduced into these strains from E. coli by
conjugation (10). For infection assays, bacteria were grown overnight
at 26 °C with shaking in Luria broth containing 100 µg/ml
ampicillin. Bacteria were subcultured into Luria broth containing 100 µg/ml ampicillin and 2.5 mM CaCl2 to an
A600 value of 0.1. Cultures were shaken at
37 °C for 2 h. Bacteria were pelleted by centrifugation and
resuspended in warm (37 °C) Hanks' balanced salt solution to an
A600 value of 1.0 (~1 × 109
colony-forming units/ml).
HeLa cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf
serum (Life Technologies, Inc.) and 1 mM sodium pyruvate in
a 5% CO2 humidified incubator at 37 °C. For the
immunofluorescence experiments, 1 × 105 HeLa cells
were seeded in 1 ml of media onto sterile glass coverslips placed in a
24-well tissue culture plate and cultured overnight. For the
translocation, immunoprecipitation, and in vivo
dephosphorylation assays, 2 × 106 HeLa cells in 10 ml
of media were seeded into 100-mm tissue culture dishes and cultured
overnight. HeLa cells were overlaid with fresh media 30 min prior to
bacterial infection. Cells were left uninfected or infected with
bacteria grown as described above at a multiplicity of infection of
50:1 at 37 °C in a 5% CO2 incubator.
Immunofluorescence Assays--
All steps following a 2-h
infection (see above) were performed at room temperature. Coverslips
were washed twice with phosphate-buffered saline (PBS) containing 1 mM Na3VO4, fixed with 4%
paraformaldehyde for 10 min, and then permeabilized with 0.2% Triton
X-100 for 10 min. Coverslips were washed twice with PBS containing 1%
bovine serum albumin (BSA) and then incubated for 1 h with primary
antibody (anti-pTyr or anti-M45) diluted in PBS containing 3% BSA.
Coverslips were washed with PBS and then incubated for 1 h with
TRITC-conjugated secondary antibody diluted in PBS containing 3% BSA.
Coverslips were washed well with PBS before mounting on slides in 10%
Airvol (Air Products, Inc.), 100 mM Tris, pH 8.5, 25%
glycerol, and 2.5% DABCO anti-fade (Sigma). The stained cell samples
were analyzed by epifluorescence microscopy using a Zeiss AxioPlan 2 microscope equipped with a 100× Plan-NeoFluar (numerical
aperture 1.4) oil immersion objective. Epifluorescent images were
captured with a Spot CCD camera and Adobe Photoshop, version 5.5 software running on a Macintosh G4 computer.
Translocation Assay--
After a 2-h infection, the dishes were
placed on ice and washed twice with 10 ml of ice-cold PBS. Cells were
lysed in 0.5 ml of Nonidet P-40 lysis buffer (150 mM NaCl,
50 mM Tris, pH 8.0, 1% Nonidet P-40, 1 mM
Na3VO4, and 10 mM NaF) for 15 min
on ice with occasional rocking. Cells were scraped into microcentrifuge tubes and centrifuged for 10 min at 12,000 × g at
4 °C. The supernatants were transferred to new tubes, and protein
concentrations were determined using the Bio-Rad protein assay. Samples
of 20 µl containing 5 µg of total cell protein each were separated
on 10% SDS polyacrylamide gels and analyzed by immunoblotting using
anti-M45.
Immunoprecipitation Assay--
After 2 h of infection the
dishes were placed on ice and washed twice with 10 ml of ice-cold PBS
containing 1 mM Na3VO4. Cells were
lysed in 0.5 ml of ice-cold Triton X-100 lysis buffer (10 mM Tris, pH 7.6, 150 mM NaCl, 5 mM
EDTA, 10% glycerol, 1% Triton X-100, 1 mM
Na3VO4, 10 mM NaF, 200 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride, 20 µM leupeptin, and 1 µM
pepstatin) for 15 min on ice with occasional rocking. Cells were
scraped into microcentrifuge tubes and centrifuged for 10 min at
12,000 × g at 4 °C. The supernatants were
transferred to new tubes, and protein concentrations were determined
using a Bio-Rad protein assay. Cell lysates were adjusted to contain
450 µg of protein in a volume of 1 ml. 50 µl of a 50% suspension
of protein A-Sepharose beads (Amersham Pharmacia Biotech) was
added to each lysate sample, and the tubes were incubated for 30 min at
4 °C with rotation as a pre-clearing step. After the protein
A-Sepharose beads were removed by centrifugation, the supernatants were
transferred to new tubes and mixed with anti-M45. The tubes were
incubated for 3 h at 4 °C with rotation. Immune complexes were
recovered by addition of 50 µl of 50% protein A-Sepharose, followed
by incubation for 3 h at 4 °C with rotation. The beads were
pelleted by centrifugation, washed three times with 1 ml of 4 °C
lysis buffer, resuspended in 60 µl of 1× Laemmli sample buffer, and
boiled for 5 min. Immunoprecipitated proteins were separated on 7.5%
SDS polyacrylamide gels under reducing conditions and analyzed by
immunoblotting with anti-Cas, anti-pTyr, or anti-YopH antibodies as
described below.
In Vivo Dephosphorylation Assay--
HeLa cells were left
uninfected or infected for 15, 30, 60, or 120 min. Dishes were placed
on ice and washed twice with 10 ml of ice-cold PBS containing 1 mM Na3VO4. Cells were lysed in 0.5 ml of ice-cold Triton X-100 lysis buffer for 15 min on ice with
occasional rocking. Cells were scraped into microcentrifuge tubes and
centrifuged for 10 min at 12,000 × g at 4 °C. The
supernatants were transferred to new tubes, and protein concentrations
were determined using the Bio-Rad protein assay. Supernatant samples containing 50 µg of protein in a volume of 20 µl were mixed with an
equal volume of 2× Laemmli sample buffer and boiled for 3 min. The
resulting samples were separated on 7.5% SDS polyacrylamide gels under
reducing conditions and analyzed by immunoblotting with anti-pTyr or
anti-YopH as described below.
Immunoblotting--
Proteins separated in SDS polyacrylamide
gels were electrophoretically transferred to nitrocellulose filters
(Schleicher & Schüll). Unless indicated all subsequent steps were
performed at room temperature. The nitrocellulose filters were blocked
in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 1% BSA for 1 h. Filters were incubated with primary antibody (see above) diluted in TBST for 1 h. Filters were washed four times in TBST and then incubated for 1 h with the appropriate secondary antibody diluted (1:1000 for anti-mouse and 1:15,000 for anti-rabbit) in TBST.
The filters were washed four times in TBST and developed using the
Renaissance (PerkinElmer Life Sciences) chemiluminescence system. In some cases, the blots were stripped of bound antibodies by
incubation in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol at 50-55 °C for 30 min. After the
filter was extensively washed in TBST, the immunoblotting procedure was
then repeated starting with the blocking step.
Interaction of the YopH N-terminal Domain with
Tyrosine-phosphorylated Cas in the Yeast Two-hybrid System--
A
modified yeast two-hybrid system (23) was used to detect association of
the YopH N-terminal domain with tyrosine-phosphorylated Cas. A yeast
strain carrying a lacZ reporter gene under control of the
Gal4p transcription factor was transformed with two expression vectors.
One vector produced the DNA binding domain of Gal4p fused to residues 1 to 129 of YopH adjoined to a C-terminal M45 epitope tag
(BD-YopH1-129M45). The second vector produced a modified form of the
Src tyrosine kinase and the activation domain of Gal4p fused to Cas
(AD-Cas). Based on previous studies (23), ectopic expression of Src was
expected to result in tyrosine phosphorylation of the AD-Cas fusion
protein in yeast cells. Identification of Amino Acid Substitutions in the YopH N-terminal
Domain That Interfere with Binding to Tyrosine-phosphorylated
Cas--
Random mutagenesis was used to identify amino acid
substitutions in the YopH N-terminal domain that interfere with binding to tyrosine-phosphorylated Cas in the two-hybrid system. The DNA sequence encoding the first 129 residues of YopH was amplified using a
PCR and Taq DNA polymerase under suboptimal
conditions (26). The resulting DNA product was inserted into a Gal4p
binding domain fusion vector in a yeast reporter strain using in
vivo recombination. Approximately 1500 yeast colonies were
screened on filters for reduced
We tested each of the mutant BD-YopH1-129 proteins by two-hybrid assay
for interaction with an AD-SycH fusion protein to determine whether any
of the amino acid substitutions interfered with binding to the
chaperone. With the exception of the Q11R mutation, all of the
substitutions resulted in higher levels of Amino Acid Substitutions in the YopH N-terminal Domain That Reduce
Binding to Cas Do Not Interfere with YopH Translocation into HeLa
Cells--
We next examined the effect of the substitutions in the
N-terminal domain on translocation of YopH into HeLa cells. The Q11R, V31G, A33D, and N34D codon changes were inserted into the full-length yopH gene carried on a bacterial expression vector
(pPYopHM45), and the resulting plasmids were introduced into a
yopEyopHyopT mutant Y. pseudotuberculosis strain
(YP17). HeLa cells were infected with the bacterial strains for 2 h and lysed in 1% Nonidet P-40. Soluble fractions of the lysates were
prepared by centrifugation. Samples of the soluble fractions were
analyzed by immunoblotting with the M45 antibody to measure the amount
of translocated YopH protein in the soluble fractions. As seen in Fig.
3, YopH proteins containing the Q11R,
V31G, A33D, and N34D substitutions were detected in the soluble
fractions at levels comparable with wild-type YopH. YopH was not
detected in the soluble fraction when HeLa cells were infected with a
yopB mutant of YP17 (YP19/pPYopHM45) that is defective for
Yop translocation (lane 1). These results indicated that
YopH proteins containing the Q11R, V31G, A33D, and N34D substitutions are not defective for translocation into HeLa cells. Similar results were obtained when translocation of mutant YopH proteins was analyzed by immunofluorescence microscopy (data not shown; see Fig.
4, A and B). Thus,
amino acid substitutions in the YopH N-terminal domain that interfere
with binding to Cas do not interfere with YopH translocation.
The N-terminal of YopH Domain Is Required for Efficient Substrate
Trapping in Vivo--
The V31G substitution was engineered into a
catalytically inactive form of YopHM45 (YopHC403SM45) to determine
whether the N-terminal domain is important for substrate trapping
in vivo. The V31G mutation was selected for this purpose,
because it strongly interfered with substrate-binding activity in the
two-hybrid system (Fig. 1A). A plasmid encoding the mutant
protein was introduced into Y. pseudotuberculosis YP17, and
the resulting strain was used to infect HeLa cells. Initially, the HeLa
cells were processed for immunofluorescence microscopy using the M45
antibody to examine the effect of the V31G substitution on localization
of inactive YopH to focal adhesions. Analysis by epifluorescence
microscopy showed that the V31G substitution strongly reduced, but did
not eliminate, localization of inactive YopH to focal adhesions (Fig. 4, compare panels C and E). Infected HeLa cells
were also labeled with an anti-pTyr antibody to demonstrate that focal
adhesions were not disrupted under the conditions of the infection
(Fig. 4G).
Detergent lysates of the infected HeLa cells were prepared and
subjected to immunoprecipitation with M45 antibody to determine the
amount of Cas that was directly bound to the mutant YopH proteins. The
immune complexes were analyzed by immunoblotting with either anti-Cas
antibody (Fig. 5A) or
anti-pTyr antibody (Fig. 5B). The results obtained with the
anti-pTyr antibody were more informative because of higher sensitivity.
The V31G substitution had a dramatic inhibitory effect on binding of
Cas to inactive YopH (Fig. 5B, compare lanes 2 and 3). However, the V31G substitution did not abolish
coprecipitation of Cas with inactive YopH (Fig. 5B,
lane 3), suggesting that the PTP domain contributes, as
well, to substrate-trapping activity. The filter analyzed in Fig.
5B was stripped of the anti-pTyr antibody and analyzed by
immunoblotting with anti-YopH as a control for recovery of inactive
YopH protein in the immunoprecipitation (Fig. 5C).
To determine whether Cas was binding to the P-loop in the PTP domain,
the V31G substitution was introduced into a catalytically inactive form
of YopH in which Arg-409 in the P-loop motif was changed to Ala
(R409A). Arg-409 appears to stabilize the transition state of the
enzyme and is thus critical for YopH catalytic activity (29). In
addition, Arg-409 plays an important role in substrate binding (29),
and therefore the R409A substitution was predicted to interfere with
binding of tyrosine-phosphorylated Cas to the P-loop. The R409A
substitution by itself did not interfere with localization of YopH to
focal adhesions (Fig. 4D) or with binding of YopH to Cas
(Fig. 5B, lane 4), indicating that the N-terminal domain is the major determinant of substrate binding in
vivo. When the V31G and the R409A substitutions were combined,
localization of YopH to focal adhesions was abolished (Fig.
4F) and Cas-binding activity was further decreased but not
eliminated (Fig. 5B, lane 5). These results
indicated that the P-loop containing the C403S substitution does bind
tyrosine-phosphorylated Cas in vivo but that this
substrate-binding activity is substantially weaker than that of the
N-terminal domain.
The Substrate-binding Activity of the YopH N-terminal Domain Is
Required for Efficient Substrate Dephosphorylation in Vivo--
We
next examined the effect of the V31G substitution on dephosphorylation
of substrates by YopH in infected HeLa cells. Lysates prepared from
HeLa cells infected for 15, 30, 60, or 120 min with YP17 strains
producing either YopH or YopHV31G were analyzed by immunoblotting with
anti-pTyr antibody. Two heavily phosphorylated protein bands, of 130 and 68 kDa, were detected in lysates of uninfected HeLa cells (Fig.
6A, lanes 1 and
6). The 68-kDa band has previously been shown to correspond
to paxillin (11). The efficiency of paxillin dephosphorylation over
time by wild-type YopHM45 was greater than that observed for
YopHV31GM45 (Fig. 6A, compare lanes 2 and
7). The filter analyzed in Fig. 6A was stripped of the anti-pTyr antibody and analyzed by immunoblotting with anti-YopH. The results indicated that differences in levels of YopH
protein production could not account for differences in rates of
substrate dephosphorylation (Fig. 6B). Because Cas
comigrates with focal adhesion kinase, Cas was immunoprecipitated from
HeLa cell lysates and analyzed by anti-pTyr immunoblotting to directly monitor its rate of dephosphorylation. As shown in Fig.
7, the efficiency of Cas
dephosphorylation was greater for wild-type YopH than for YopHV31G
(compare lanes 2 and 7). Analysis of the same
filter with anti-Cas antibodies confirmed that equivalent amounts of
Cas were recovered from each of the lysate samples. These data indicate
that the substrate-binding activity of the YopH N-terminal domain is
required for efficient dephosphorylation of substrates in
vivo.
The goal of this study was to identify residues in the YopH
N-terminal domain that are important for substrate-binding activity but
not SycH-binding activity. We used random mutagenesis to isolate single
amino acid changes in the N-terminal domain that interfere with binding
to tyrosine-phosphorylated Cas in a yeast two-hybrid system. Four
single codon changes (Q11R, V31G, A33D, and N34D) and one double codon
change (G41S and D106G) were identified in the screen. Separation of
the G41S and D106G mutations indicated that the G41S change was largely
responsible for the mutant phenotype.
It has been determined by truncation experiments that a region between
residues 20 and 69 of YopH is critical for binding to SycH (30, 31).
All of the codon changes that resulted in reduced binding of the YopH
N-terminal domain to Cas, except for Q11R, fell within this putative
binding site for SycH. This may indicate that tyrosine-phosphorylated
proteins and SycH bind to an overlapping region of the YopH N-terminal
domain. Surprisingly, all of the single codon changes, except for Q11R,
appeared to result in tighter binding of SycH to the YopH N-terminal
domain in the two-hybrid system. Binding experiments carried out
in vitro with purified components will be required to
confirm that SycH binds tighter to the mutant N-terminal domain,
because other variables inherent to the two-hybrid system could also
result in increased expression of It has recently been reported that the Yersinia
yop virulon regulators LcrQ/YscM1 and YscM2, which
share significant sequence similarity with the YopH N-terminal domain,
also bind to SycH (33). By sequence alignment of YopH1-129 with LcrQ
and YscM1, it is possible to predict which residues are involved in
SycH-binding activity. None of the residues identified in our screen as
being critical for substrate-binding activity in the YopH N-terminal domain are conserved among the three proteins. This observation further
suggests that distinct residues in the YopH N-terminal domain mediate
interaction with tyrosine-phosphorylated substrates or SycH.
The three-dimensional structure of the
YopH N-terminal domain has recently been determined to a resolution of
2.2 Å.3 It is a single, highly compact domain
composed of 4 The role of the N-terminal domain in substrate recognition in
vivo was addressed by the introduction of the V31G substitution into the full-length YopH reading frame. Initially, the V31G
substitution was introduced into two different catalytically inactive
forms of YopH, one in which the P-loop in the PTP domain retained the capacity to bind substrate (C403S) and the other in which binding of
substrate to the P-loop is blocked (R409A). This strategy allowed us to
independently assess the contributions of the N-terminal domain and the
P-loop to the formation of a stable enzyme-substrate complex.
Substrate-binding activity was scored by the ability of the mutant
proteins to localize to focal adhesions in infected HeLa cells and to
coprecipitate with Cas in detergent lysates of infected HeLa cells.
These results of both assays indicated that the N-terminal domain is
the major determinant of substrate-binding activity in YopH but that
the P-loop also contributes, albeit weakly, to substrate trapping. The
ability of Cas to coprecipitate with YopH was not completely eliminated
by the presence of the V31G and R409A substitutions, suggesting that an
additional substrate-binding interface may be present elsewhere in the
protein. Persson et al. (19) have reported that part of a
surface-exposed loop in the PTP domain (residues 223-226) is involved
in targeting YopH to focal complexes. However, their data argue that
this region functions as a localization sequence and is not involved in
substrate recognition (19).
The V31G substitution was introduced into catalytically active YopH to
determine whether the N-terminal domain is important for efficient
dephosphorylation of substrates by YopH in vivo. The
presence of the V31G substitution resulted in a dramatic decrease in
the efficiency of Cas and paxillin dephosphorylation by YopH in
infected HeLa cells. These results support our original proposal that
the N-terminal domain functions to increase the efficiency of substrate
recognition by YopH in vivo (11). It is also possible that
the N-terminal domain allows YopH to act processively during the
dephosphorylation of multiply phosphorylated substrates such as Cas and paxillin.
It is becoming apparent that translocated proteins of other type III
secretion systems may be arranged in a modular fashion with at least
two, and possible more, distinct effector domains (36-38). In
addition, a single modular domain may in fact be multifunctional, as is
the case for the YopH N-terminal domain. This raises the possibility
that the N-terminal domains of other translocated type III proteins
will perform multiple functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity (27). Colonies that displayed reduced
-galactosidase activity were tested for the production of
BD-YopH1-129M45 fusion protein by immunoblotting. Yeast cell lysates
were prepared from 25 ml of culture grown to an
A600 value of ~0.68 (equivalent to
2.5 × 108 cells) in SD-Trp-Leu media. After
centrifugation the pellet was resuspended in 50 µl of ice-cold
ddH2O, 250 µl of glass beads, and 200 µl of 1× Laemmli
sample buffer supplemented with 10%
-mercaptoethanol. Samples were
vortexed at high speed for 1 min and then chilled on ice for 30 s.
This procedure was repeated twice. Samples were boiled for 3 min and
briefly centrifuged prior to loading. Samples of 10 µl were separated
on 12% SDS polyacrylamide gels and analyzed by immunoblotting using
anti-M45 (see below). The two-hybrid assay was repeated with
reconstructed binding domain vectors to verify that reduced expression
of
-galactosidase was specific to mutations in the YopH1-129M45
coding region. Plasmid DNA was isolated from 53 Y153 colonies that
produced full-length BD-YopH1-129M45 protein. The plasmid DNA was used
to transform Escherichia coli strain MH4 to Leu prototrophy
to select for the LEU2 gene carried by the pBD-YopH1-129M45
plasmid (27). Plasmid DNA was isolated from Leu+ colonies of MH4 and
digested with BamHI and EcoRI to remove the
YopH1-129M45 coding regions. The regions coding for mutant
YopH1-129M45 proteins were inserted between the BamHI and EcoRI sites of pBDMI, and the resulting plasmids were
introduced into Y153 along with the pAD-Cas+Src plasmid. Colonies of
Leu+ Trp+ transformants on filters were tested for expression of
-galactosidase as described above. This procedure was repeated using
pAD-SycH in place of pAD-Cas+Src to identify mutations that selectively interfered with binding of the YopH N-terminal domain to Cas. The
regions coding for YopH1-129M45 in purified pBD-YopH1-129M45 plasmids
were sequenced to identify mutations that resulted in reduced binding
to Cas. The G41S and D106G codon substitutions were separated by a
standard restriction fragment subcloning procedure.
-galactosidase activity in Y153 strains containing mutant
pBD-YopH1-129M45 plasmids was quantitated using a colorimetric liquid
assay as described previously (27). Cultures of Y153 strains containing
mutant pBDMI-YopH1-129M45 plasmids and pAD-Cas+Src were grown in
selective media (SD-Trp-Leu) to an A600 value of ~1.0. Yeast cells in 5 ml of culture were assayed for
-galactosidase activity using chlorophenol
red-
-D-galactopyranoside as described (27). Reactions
were incubated for 35 min. Measurement of
-galactosidase activity in
yeast cells containing mutant pBDMI-YopH1-129M45 plasmids and pAD-SycH
was performed using cells derived from 0.1 ml of culture and the
substrate O-nitrophenyl-
-D-galactopyranoside (27). Reactions were incubated for 15 min. Units of
-galactosidase activity were calculated as described (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity was detected when
both fusion proteins and Src were produced in the yeast reporter strain
(Fig. 1A). In contrast,
-galactosidase activity was not detected when both fusion proteins
were expressed in yeast cells in the absence of Src (data not shown).
Thus, a specific two-hybrid interaction between the N-terminal domain of YopH and tyrosine-phosphorylated Cas was detected.
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Fig. 1.
Identification of amino acid substitutions in
the YopH N-terminal domain that interfere with binding to Cas but not
SycH in the yeast two-hybrid system. Units of -galactosidase
activity expressed by cultures of a yeast two-hybrid reporter strain
producing the indicated wild-type or mutant BD-YopH1-129M45 fusion
proteins was quantitated using a colorimetric assay. A,
yeast cultures producing AD-Cas+Src fusion protein as the interaction
target of BD-YopH1-129M45. B, yeast cultures producing
AD-SycH fusion protein as the interaction target of BD-YopH1-129M45.
Values shown are the mean ± S.D. for reactions performed in
triplicate on a single culture of each strain.
-galactosidase activity. 43 colonies
that showed reduced
-galactosidase activity were chosen for further characterization (see "Experimental Procedures"). Five of these colonies produced intact BD-YopH1-129M45 protein that interacted weakly with tyrosine-phosphorylated Cas in the two-hybrid system. The
binding domain vectors were isolated from these colonies, and the
regions coding for residues 1-129 of YopH were sequenced. Four
plasmids contained single codon changes in the YopH coding region
(corresponding to Q11R, V31G, A33D, and N34D). One plasmid contained
two codon changes (G41S and D106G) that were subsequently separated by
restriction fragment subcloning. As shown in Fig. 1A, five
of the six single amino acid substitutions (the exception being D106G)
resulted in reduced levels of
-galactosidase activity in the
two-hybrid assay.
-galactosidase activity
as compared with the wild-type (Fig. 1B). Lysates of the
yeast strains producing the mutant BD-YopH1-129M45 proteins were
analyzed by immunoblotting with the M45 antibody to examine levels of
protein expression. As shown in Fig. 2,
all of the mutant fusion proteins were produced at approximately the
same level as the wild-type fusion protein. These results suggested
that the V31G, A33D, N34D, G41S, and D106G substitutions actually
resulted in tighter binding of the YopH N-terminal domain to SycH.
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Fig. 2.
Levels of wild-type and mutant
BD-YopH1-129M45 fusion proteins produced by cultures of the yeast
reporter strain. Total protein was extracted from yeast cultures
producing BD-YopH1-129M45 fusion proteins containing the indicated
amino acid substitutions. Protein samples were separated by SDS
polyacrylamide gel electrophoresis and analyzed by immunoblotting with
anti-M45.
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Fig. 3.
Amino acid substitutions in the N-terminal
domain that interfere with binding to Cas do not interfere with YopH
translocation. HeLa cells were infected for 2 h with YP19
(pPYopHM45) (lane 1), YP17 (pPROH) (lane 2), YP17
(pPYopHM45) (lane 3), YP17 (pPYopHQ11RM45) (lane
4), YP17 (pPYopHV31GM45) (lane 5), YP17
(pPYopHA33DM45) (lane 6), or YP17 (pPYopHN34DM45)
(lane 7). The infected cells were lysed in 1% Nonidet P-40,
and insoluble protein was removed by centrifugation. Equivalent amounts
of total soluble protein were separated by SDS polyacrylamide gel
electrophoresis and analyzed by immunoblotting with anti-M45.
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Fig. 4.
The N-terminal domain is required for
efficient localization of inactive YopH to focal adhesions.
HeLa cells were infected for 2 h with the following: YP17
(pPYopHM45) (wt; A), YP17 (pPYopHV31GM45)
(V31G; B), YP17 (pPYopHC403SM45)
(C403S; C), YP17 (pPYopHR409AM45)
(R409A; D), YP17 (pPYopHV31GC403SM45)
(VGCS; E and G), or YP17
(pPYopHV31GR409AM45) (VGRA; F and H).
The infected cells were processed for immunofluorescence microscopy
using anti-M45 to detect YopH (A-F) or anti-pTyr to detect
tyrosine-phosphorylated proteins (G and H). Bound
antibodies were detected using a secondary antibody conjugated to
TRITC. Representative images captured by epifluorescence microscopy are
shown.
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Fig. 5.
The N-terminal domain of YopH is required for
efficient trapping of Cas in vivo. HeLa cells
were infected for 2 h with YP17 (pPROH) (lane 1), YP17
(pPYopHC403SM45) (lane 2), YP17 (pPYopHV31GC403SM45)
(lane 3), YP17 (pPYopHR409AM45) (lane 4), or YP17
(pPYopHV31GR409AM45) (lane 5). Samples of detergent lysates
prepared from the infected cells were subjected to immunoprecipitation
with anti-M45. Immunoprecipitated proteins were analyzed by
immunoblotting with anti-Cas (A) or anti-pTyr
(B). The filter analyzed in B was stripped of
anti-pTyr and analyzed by immunoblotting with anti-YopH (C)
to control for recovery of YopH protein during the
immunoprecipitation.
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Fig. 6.
The N-terminal domain of YopH is required for
efficient dephosphorylation of substrates in
vivo. HeLa cells were left uninfected (lanes
1 and 6) or were infected for the indicated time (in
min) with YP17 (pPYopHM45) (lanes 2-5) or YP17
(pPYopHV31GM45) (lanes 7-10). Samples of detergent lysates
prepared from the infected cells were separated by SDS polyacrylamide
gel electrophoresis and analyzed by immunoblotting with anti-pTyr
(A). The filter analyzed in A was stripped of
anti-pTyr and analyzed by immunoblotting with anti-YopH (B)
to control for differences in expression levels of YopHM45 and
YopHV31GM45.
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Fig. 7.
The N-terminal domain of YopH is required for
efficient dephosphorylation of Cas in vivo. Cas
was immunoprecipitated from HeLa cell lysates and analyzed by
immunoblotting with anti-pTyr (A). The filter analyzed in
A was stripped of anti-pTyr and analyzed by immunoblotting
with anti-Cas (B) to control for recovery of Cas during the
immunoprecipitation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. YopH
proteins containing amino acid substitutions in the N-terminal domain
were translocated into HeLa cells as efficiently as the wild-type
protein, as determined by cellular fractionation experiments and
immunofluorescence microscopy. Because binding of SycH to YopH is
required for translocation (32), we conclude from these experiments
that it is possible to separate the substrate-binding activity of the
N-terminal domain from its SycH-binding activity.
-helices sandwiched between one two-stranded
-sheet
and one three-stranded
-sheet. The fold of the YopH N-terminal
domain is unlike that of other known phosphotyrosine binding domains
such as the SH2 domain of Src (34) or the phosphotyrosine
binding domain of insulin receptor substrate-1 (35). Q11, V31,
A33, N34, and G41 are located on the surface along a loop connecting
-strand 1 and
-strand 2. This region is rich in positively
charged residues (e.g. K26, R28, K35, and R49)
and is flanked on either side by pockets, which may together form a
shallow phosphotyrosine-binding cleft.
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ACKNOWLEDGEMENTS |
---|
We thank Jonathan Cooper, Stanley Fields, Bruce Mayer, and Patrick Hearing for providing reagents and members of the Bliska laboratory for reviewing the manuscript.
![]() |
FOOTNOTES |
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* This research was supported by National Institutes of Health Grants AI35175 and AI3389 (to J. B. B.).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.
Present address: Nathan S. Kline Inst. for Psychiatric Research,
Orangeburg, NY 10962.
§ To whom correspondence should be addressed. Tel.: 631-632-8782; Fax: 631-632-9797; E-mail: jbliska@ms.cc.sunysb.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M009045200
2 J. Zitzler and J. Bliska, unpublished results.
3 C. Smith and M. Saper, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PTP, protein-tyrosine phosphatase; P-loop, phosphate-binding loop; Cas, p130cas; pTyr, phosphotyrosine; TRITC, tetramethyl rhodamine isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBST, Tris-buffered saline containing 0.05% Tween 20; PCR, polymerase chain reaction; AD, activation domain.
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