From the Department of Biological Chemistry,
University of Michigan Medical School, Life Sciences Institute,
Ann Arbor, Michigan 48109 and the § Department of
Molecular Genetics and Microbiology, Center for Infectious Diseases,
State University of New York, Stony Brook, New York 11794
Received for publication, February 4, 2003
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ABSTRACT |
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Pathogenic Yersinia contain a
virulence plasmid that encodes genes for intracellular effectors, which
neutralize the host immune response. One effector, YopM, is necessary
for Yersinia virulence, but its function in host cells is
unknown. To identify potential cellular pathways affected by YopM,
proteins that co-immunoprecipitate with YopM in mammalian cells were
isolated and identified by mass spectrometry. Results demonstrate that
two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein
kinase 1 (RSK1), interact directly with YopM. These two kinases
associate only when YopM is present, and expression of YopM in cells
stimulates the activity of both kinases. RSK1 is activated directly by
interaction with YopM, and RSK1 kinase activity is required for
YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs
independently of the actions of YopJ on the MAPK pathway. YopM is also
required for Yersinia-induced changes in RSK1 mobility in
infected macrophage cells. These results identify the first
intracellular targets of YopM and suggest YopM acts to stimulate the
activity of PRK2 and RSK1.
The bacterium Yersinia has three species that are
pathogenic for humans. Yersinia pestis causes plague and is
transmitted by fleas from rodent reservoirs to humans, frequently
resulting in fatal infections. This bacterium is responsible for three
plague pandemics and has been involved in two plague epidemics in
western India as recently as 1944 (1). Two other species,
Yersinia enterocolitica and Yersinia
pseudotuberculosis, are also human pathogens, and infection with
these species results in gastrointestinal disorders such as
appendicitis, ileocolitis, and mesenteric adenitis. Contaminated food
or water is the most common route of infection by these species, with
swine serving as a major reservoir for these bacteria (2). Although the
severity of the disease and the mode of transmission of pathogenic
Yersinia species differs, they all target lymphoid tissues
and are able to block innate immune responses to allow extracellular
replication of the bacterium.
Yersinia spp. harbor a virulence plasmid of ~70 kb that
encodes proteins termed Yops1
(for Yersinia outer
proteins) that are either intracellular effectors or
membrane proteins that create a delivery system for these effectors (3,
4). Six effector proteins from Yersinia (YopE, YopH, YopJ/P,
YopM, YopT, and YpkA/YopO) are delivered into eukaryotic cells to
inactivate the host immune response. YopE, YopH, YopT, and YpkA target
the actin cytoskeleton through different mechanisms to prevent
phagocytosis of the bacterium. YopJ inhibits the production of certain
inflammatory cytokines by blocking the mitogen-activated protein kinase
(MAPK) and NF The effects of YopM on host response to Yersinia infection
are largely unknown. However, YopM is required for full virulence of
Y. pestis and Y. enterocolitica, as demonstrated
in mouse infection models (5, 6). The YopM protein is an acidic,
~42-kDa protein composed almost entirely of leucine-rich repeat (LRR)
motifs. The LRR motif is thought to be a protein-protein interaction
motif and is present in multiple signaling molecules found both
intracellularly and extracellularly (7). Initial studies suggested that
YopM is secreted from the bacteria and interacts with thrombin to
inhibit platelet aggregation (8). Subsequent studies demonstrated that YopM is translocated into target cells via the type III secretion apparatus (9). Once inside the target cell, YopM moves from the
cytoplasm to the nucleus via a vesicle-associated pathway (10, 11). A
gene chip microarray analysis of Y. enterocolitica-infected macrophage cell lines suggests that YopM affects the expression of
genes involved in cell cycle and cell growth (12). However, the
mechanism by which YopM produces these effects on gene expression is
unknown. This paper describes the identification of two intracellular targets of YopM, protein kinase C-like 2 (PRK2) and ribosomal S6
protein kinase 1 (RSK1).
Cell Culture and Transfection--
Human embryonic kidney 293 cells were maintained in DMEM (Invitrogen) + 10% fetal bovine serum
(FBS, Invitrogen) + penicillin/streptomycin (Invitrogen). Cells were
transfected in 100-mm plates with 5 µg of DNA using FuGENE 6 (Roche
Applied Science) at a ratio of 3:1 (FuGENE 6:DNA) according to the
manufacturer's instructions. Mouse macrophage J774A.1 cells were
maintained in DMEM + 10% FBS (Hyclone) + 1 mM sodium
pyruvate (Invitrogen).
Antibodies--
The following antibodies were used in the
experiments described: anti-FLAG M2 (Sigma), anti-PRK2 (Cell Signaling
Technology and Transduction Laboratories), anti-RSK1 (C-21, Santa Cruz
Biotechnology), anti-HA (Y-11, Santa Cruz Biotechnology and clone 3F10,
Roche Applied Science), anti-GST (B-14, Santa Cruz Biotechnology),
anti-GFP (FL, Santa Cruz Biotechnology), anti-CREB (Cell Signaling
Technology), and anti-Akt (Cell Signaling Technology). YopM polyclonal
rabbit antisera was a gift of O. Schneewind, University of Chicago
(13).
Plasmid Constructs--
FLAG-YopM was constructed by amplifying
by PCR nucleotides 7-1265 of YopM from the Y. enterocolitica virulence plasmid, pYVe8081, and cloning into
pFLAG-CMV-2 (Eastman Kodak Co.), resulting in the incorporation of an
amino-terminal FLAG tag. EGFP-YopM was constructed by PCR amplifying
YopM (nucleotides 7-1265) from FLAG-YopM and cloned into pEGFP-C1
(Clontech) resulting in a fusion protein with an
amino-terminal EGFP tag. The p67N-YopM plasmid was constructed by PCR
amplifying nucleotides 1-1265 of Y. enterocolitica YopM and
cloning into pMMB67HE (14). YopM-6xHis was a gift of Don Huddler
(University of Michigan). FLAG-PRK2 wild type and FLAG-PRK2 kinase-deficient were gifts of Gary Johnson, University of
Colorado Health Sciences Center (15). PRK2 deletion constructs were
produced by PCR amplification of regions of human PRK2 and cloning into the mammalian expression vector pEBG-3X, resulting in the incorporation of a GST tag on the amino terminus. Mammalian expression constructs of
avian RSK1 (HA-RSK1 and HA-RSK1 K112/464R) were gifts of John Blenis,
Harvard Medical School (16). The mammalian expression construct of rat
RSK1 (pMT2-HA-RSK1) was a gift of Valerie Castle, University of
Michigan (17). RSK1 deletion constructs were produced by PCR
amplification of regions of rat RSK1 and cloning into pcDNA3-HA. Subcloning an EcoRI fragment of pMT2-HA-RSK1 into pcDNA3
created the RSK1 template for in vitro
transcription/translation. pSFFV-YopJ-FLAG was a gift of Kim Orth
(18).
Immunoprecipitation--
293 cells were transfected in 100-mm
plates with 5 µg of expression plasmid for a total of 40-45 h. Prior
to harvest, cells were starved in DMEM for 16-18 h. Cells were washed
with PBS and lysed in RIPA Mass Spectrometry--
Immunoprecipitates from 12 to 16 100-mm
plates of transfected 293 cells were separated by SDS-PAGE followed by
staining in 0.25% Coomassie Blue, 10% glacial acetic acid, 40% MeOH.
The gel was destained in 10% glacial acetic acid, 40% MeOH and dried
onto Whatman paper. Protein bands of interest were excised from the dried gel and digested with trypsin as described previously (21). MALDI-TOF mass spectrometry was performed on a Voyager DE-STR instrument (Applied Biosystems) in linear positive mode or reflector positive mode (for PSD analysis) using In Vitro Kinase Assay--
Immunoprecipitated proteins were
incubated for 30 min at room temperature in 20 mM Hepes, pH
7.4, 10 mM MgAc, 1 mM dithiothreitol, 50 µM ATP, 20 µCi of [ In Vitro Pull-down Assay--
The PRK2 and RSK1 kinases were
in vitro transcribed and translated in the presence of
[35S]methionine (PerkinElmer Life Sciences) using the
TNT T7 Quick-coupled Transcription/Translation System
(Promega) from linearized PRK2-V5 (AgeI) or
pcDNA3-HA-RSK1 (NcoI) plasmid templates. Radiolabeled proteins were incubated with bacterially produced recombinant YopM-6xHis protein bound to nickel-agarose beads (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40.
Beads were subsequently washed with 50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 10 mM imidazole and
run out on SDS-PAGE. The gel was fixed in 40% MeOH, 10% glacial
acetic acid, treated with Amplify (Amersham Biosciences), and dried
onto Whatman paper. The gel was then exposed to film at Yersinia Strains and Infection--
The Y. pseudotuberculosis strains used in this study were derived from
YP126 (serogroup O:3). YP22 has been described previously (22) and is
deficient for the expression of YopE, YopH, and YopK proteins. The YP33
strain was derived from YP22 and contains a frameshift in
yopM, disrupting the expression of the YopM protein. The
YP33/yopM strain was constructed by introducing the
IPTG-inducible YopM expression plasmid, p67N-YopM into YP33 through
conjugation. Yersinia strains were grown in Luria broth at
26 °C, diluted 1:40 in Luria broth supplemented with 20 mM sodium oxalate and 20 mM MgCl2
(A600 0.1), and grown for 1 h at
26 °C. These cultures were then shifted to 37 °C and grown for an
additional 2 h. For infections using YP33/yopM, YopM
expression was induced with 0.1 mM IPTG for the
2 h at 37 °C. Yersinia were washed in Hanks'
balanced salt solution (Invitrogen) and resuspended at
A600 0.5 in Hanks' balanced salt solution.
J774A.1 cells were grown to 80-90% confluency in DMEM + 10% FBS + 1 mM sodium pyruvate and infected for 2 h at 37 °C at
a multiplicity of infection of 50.
YopM Associates with Two Protein Kinases in Mammalian
Cells--
The crystal structure of YopM has been solved and reveals
that the YopM monomer has an amino-terminal
To identify potential cellular targets affected by this
Yersinia effector, a biochemical approach was used to
identify proteins that interact with YopM in mammalian cells. In this
approach, proteins that associate with YopM in transiently transfected
cells were isolated by immunoprecipitation and identified by mass
spectrometry. Lysates from 293 cells transfected with a FLAG-YopM
expression vector or the empty FLAG vector were immunoprecipitated with
anti-FLAG antibody, and the associated
proteins were visualized by silver stain of SDS-PAGE gels (Fig.
1A). Three proteins with approximate molecular masses
of 120, 80, and 78 kDa were found to specifically co-immunoprecipitate
with YopM.
The YopM-associated proteins were excised from a Coomassie-stained gel,
trypsin-digested, and the resulting peptides analyzed by
matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)
mass spectrometry or nanospray tandem mass spectrometry (LC/MS/MS). The
pool of p120 tryptic peptides was analyzed by MALDI-TOF (Fig.
1B). The peptide values from the peptide mass fingerprint
were used to search the NCBI non-redundant data base using two web site
tools, ProFound (prowl.rockefeller.edu/cgi-bin/ProFound) (24), and
MS-Fit (prospector.ucsf.edu). Both programs predicted the identity of
p120 to be protein kinase C-like 2 (PRK2), a novel serine/threonine
protein kinase of the protein kinase C family (25, 26). In addition,
MALDI post-source decay (PSD) was employed to obtain partial sequence
information on two prominent peptides in the mass fingerprint and were
found to match the PRK2 sequence.
The identity of the p80 and p78 proteins was determined by LC/MS/MS
analysis (Fig. 1C). The MS/MS fragmentation spectra was used
to search the NCBI non-redundant data base for protein identification. Three MS/MS spectra from the p80 and p78 samples unambiguously identified these proteins as p90 ribosomal protein S6 kinase 1 (RSK1/p90rsk/MAPKAP-K1), a member of the growth
factor-regulated S6 serine/threonine kinase family (27, 28).
PRK2, RSK1, and YopM Form a Novel Protein Complex in
Cells--
The mass spectral identification of p120, p80, and p78 was
confirmed by Western blot analysis of YopM-associated proteins (Fig.
2A). Lysates from 293 cells
transfected with a FLAG-YopM plasmid or vector alone were
immunoprecipitated with anti-FLAG antibody, and proteins were
visualized by sequential Western blots using anti-FLAG, anti-PRK2, and
anti-RSK1 antibodies. A single ~45-kDa protein corresponding to
FLAG-YopM was detected in anti-FLAG Western blots. Anti-PRK2 Western
blots reacted with a single band of 120 kDa co-immunoprecipitating with
FLAG-YopM. Anti-RSK1 Western blots detected a YopM
co-immunoprecipitating doublet of ~80 kDa. These results confirm the
mass spectral identification of p120 as PRK2 and p80/p78 as RSK1.
The ability of YopM to interact with these cellular proteins was
examined in Yersinia-infected cells to determine whether the
interactions detected in transfected cells are also seen in the context
of an infection. The macrophage cell line J774A.1 was infected with
yopM+ (YP22) or yopM
The interaction of YopM with PRK2 and RSK1 was also assessed in
vitro to determine whether these kinases bind to YopM directly. Recombinant YopM with a His6 tag was bound to nickel beads
and incubated with radiolabeled in vitro
transcribed/translated kinases (Fig. 2C). Proteins bound to
the YopM beads were separated by SDS-PAGE, and the association of the
kinases was visualized by fluorography. Both PRK2 and RSK1 bound to
recombinant YopM in vitro. Deletion of the carboxyl terminus
of these kinases abolished interaction with YopM in this assay (data
not shown). These results, as well as results described in experiments
to follow (Fig. 3), indicate that a
region in the carboxyl-terminal half of these kinases is required for
interaction with YopM. The in vitro binding data suggest a
direct interaction between YopM and PRK2, as well as YopM and RSK1.
Further experiments were performed to determine whether YopM forms a
single complex with these two kinases or two separate kinase complexes.
Lysates from vector-transfected cells or cells transfected with
FLAG-YopM were immunoprecipitated with anti-PRK2, anti-RSK1, or
anti-FLAG antibodies, and proteins were visualized by Western blot
(Fig. 2D). In vector-transfected cells, PRK2 and RSK1 do not
co-immunoprecipitate, indicating that these two kinases normally do not
associate in cells (lanes 2 and 3). However, in cells that express YopM, PRK2 co-immunoprecipitated both YopM and RSK1
(lane 4). RSK1 immunoprecipitates also showed interaction with both PRK2 and YopM (lane 5). These results demonstrate
that YopM forms a single complex with PRK2 and RSK1 in transfected cells and that YopM induces the association of PRK2 and RSK1.
The subcellular localization of YopM is distinct from the other Yop
effectors. YopM has been demonstrated to be injected into the cytoplasm
of cells and translocate to the nucleus via a vesicle-associated pathway (10, 11). PRK2 has been described as a cytoplasmic protein,
whereas RSK1 has been demonstrated to shuttle between the cytoplasm and
the nucleus of cells. To determine whether the subcellular localization
of PRK2 and RSK1 is altered through association with YopM, the
localization of the kinases was examined and compared with their
localization in cells expressing YopM (Fig. 2E). Nuclear and
cytoplasmic extracts were made from cells transfected with vector or
FLAG-YopM, immunoprecipitated with anti-PRK2, anti-RSK1, or anti-FLAG
antibodies, and visualized by Western blot. As a control for proper
cell fractionation, the localization of a nuclear transcription factor,
CREB, was also assessed in these extracts by Western blot. In
vector-transfected as well as FLAG-YopM-transfected cells, PRK2 and
RSK1 were localized primarily to the cytoplasm, with some nuclear
localization as well. YopM was also detected in both cellular
compartments, with the majority of the YopM protein localized in the
cytoplasm. These results demonstrate that PRK2, RSK1, and YopM localize
to similar cellular locations, and association of the kinases with YopM
does not redirect their localization.
Different Regions of PRK2 and RSK1 Are Required for YopM
Association--
In order to characterize further the protein
interactions in the YopM complex, truncated kinase constructs were
tested for their ability to co-immunoprecipitate with YopM (Fig. 3).
The PRK2 domain structure contains three homologous repeat (HR1)
regions in the amino terminus, followed by a central repeat region
similar to the pseudosubstrate site of protein kinase C kinases (HR2), an SH3 domain-binding PXXP motif, and a carboxyl-terminal
serine/threonine kinase domain (26). PRK2 deletion constructs were
constructed in-frame with an amino-terminal GST tag and transfected
into cells with FLAG-YopM. YopM complexes were immunoprecipitated with
anti-FLAG antibody and Western-blotted with anti-GST antibody (Fig.
3A). YopM co-immunoprecipitated GST-PRK2 constructs
containing the carboxyl-terminal amino acids 512-985 (
The RSK1 domain structure consists of two distinct kinase domains
separated by a linker region. Deletion constructs of RSK1 were
constructed in-frame with an amino-terminal HA tag. The HA-RSK1 expression constructs were co-transfected into cells with FLAG-YopM, followed by anti-FLAG immunoprecipitation and Western blot analysis with anti-HA antibody (Fig. 3B). FLAG-YopM was able to
co-immunoprecipitate full-length HA-RSK1 (FL) and the HA-RSK1 construct
composed of amino acids 1-423 ( Association of PRK2 and RSK1 with YopM Enhances Their Kinase
Activities--
We have demonstrated that YopM forms a complex with
two cellular kinases, PRK2 and RSK1. In vitro kinase assays
were performed to determine the effect of complex formation on the
kinase activity of PRK2 and RSK1 (Fig.
4). Endogenous kinases were
immunoprecipitated from vector-transfected or cells transfected with
FLAG-YopM and used in an in vitro kinase assay with
[
The relative amounts of YopM expression required to activate PRK2 and
RSK1 were investigated by in vitro kinase assays from cells
transfected with increasing amounts of FLAG-YopM plasmid. Cells used
for assessing PRK2 kinase activity were transfected with 0-5 µg of
FLAG-YopM, whereas cells used in the RSK1 assays were transfected with
0-1 µg of FLAG-YopM, with the total amount of transfected plasmid
maintained at 5 µg with the addition of vector plasmid. Cells were
starved for 16 h and the endogenous PRK2 and RSK1
immunoprecipitated, and activity was assessed by in vitro
kinase assay (Fig. 4, B and C). Results showed a
dose-dependent increase in both PRK2 and RSK1 kinase
activity with increasing amounts of YopM expressed in cells. The
amounts required for maximal activity of the two kinases are different,
with RSK1 activated by much lower amounts of YopM expressed. These
results demonstrate that the endogenous kinase activity of PRK2 and
RSK1 is increased in a dose-dependent manner by YopM
expressed in cells and that this activation of kinase activity is
specific to two kinases that interact with YopM.
Mechanism of PRK2 and RSK1 Activation by YopM--
The previous
experiments do not differentiate whether the increase of PRK2 and RSK1
kinase activity is due to direct actions of YopM on the kinases or
through stimulation of cellular pathways that lead to the activation of
these kinases. To address this, recombinant YopM protein was added to
in vitro kinase assays with endogenous PRK2 or RSK1
immunoprecipitated from serum-starved cells (Fig.
5A). Addition of recombinant
YopM increased the kinase activity of RSK1 in vitro but did
not significantly affect PRK2 kinase activity. The results suggest that
YopM has a direct effect on RSK1 kinase activity and that the
stimulation of PRK2 activity is due to an indirect mechanism.
The role of PRK2 and RSK1 kinase activities in the stimulation of the
kinases by YopM was assessed using kinase-deficient mutants of PRK2 and
RSK1 that contain mutations in critical lysines of their kinase domains
that render them inactive (Fig. 5, B-D). In these
experiments, cells were co-transfected with an EGFP-tagged YopM
(EGFP-YopM), an HA-tagged RSK1 (HA-RSK, either wild-type or
kinase-deficient), and a FLAG-tagged PRK2 (FLAG-PRK2, either wild-type
or kinase-deficient). These cells were then serum-starved and
components of the YopM complex immunoprecipitated, and kinase activity
was analyzed by in vitro kinase assays.
The relative contribution of PRK2 and RSK1 kinase activity to the YopM
complex was assessed by in vitro kinase assays with immunoprecipitated EGFP-YopM from transfected cells (Fig.
5B). In this assay, no kinase activity over control levels
(lane 1) was seen in immunoprecipitates from cells
transfected with both kinase-deficient PRK2 and RSK1 (lane
5), indicating that these are the only two kinases responsible for
YopM complex phosphorylation of MBP. The kinase activity of the YopM
complex was decreased by expression of kinase-deficient PRK2
(lane 3) and completely abolished by expression of
kinase-deficient RSK1 (lane 4). These results demonstrate
that both PRK2 and RSK1 kinase activity contribute to YopM-associated
phosphorylation, and RSK1 activity is required for complex kinase activity.
The effect of RSK1 kinase activity on the activation of PRK2 by YopM
was assessed through in vitro kinase assays of
immunoprecipitated FLAG-PRK2 from the cells described above (Fig.
5C). As these assays were performed from serum-starved
cells, PRK2 kinase activity is only significantly stimulated in cells
expressing EGFP-YopM (lane 3). Immunoprecipitation of a
kinase-deficient FLAG-PRK2 from cells expressing wild-type HA-RSK1 and
EGFP-YopM still showed the ability (albeit a decreased ability when
compared with wild-type) to phosphorylate MBP, probably due to
co-immunoprecipitation of active RSK1 (lane 4).
Immunoprecipitates of wild-type FLAG-PRK2 from cells expressing
kinase-deficient HA-RSK1 and EGFP-YopM demonstrated weak kinase
activity (lane 5), indicating that RSK1 activity affects the
kinase activity of PRK2.
The activity of RSK1 was also assessed in cells expressing
kinase-deficient FLAG-PRK2 and EGFP-YopM (Fig. 5D).
Serum-starved transfected cells were immunoprecipitated with anti-HA
antibody and immunoprecipitates used for in vitro kinase
assays. The activity of HA-RSK1 from serum-starved cells was
undetectable in this assay (lane 2), and expression of
EGFP-YopM increased the kinase activity of HA-RSK1 (lane 3).
HA-RSK1 activity was unaffected by expression of kinase-deficient
FLAG-PRK2 (lane 4). Kinase reactions that included
kinase-deficient HA-RSK1 demonstrated no significant kinase activity
(lanes 5 and 6). These results indicate that
FLAG-PRK2 kinase activity does not affect the EGFP-YopM stimulated
activity of HA-RSK1. Taken together, these data support the model that YopM stimulates the kinase activity of RSK1, and the kinase activity of
RSK1 stimulates the kinase activity of PRK2.
YopM Is Required for an Increase of RSK1 Phosphorylation during
Yersinia Infection, Independent of the Effects of YopJ--
In a
Yersinia infection, multiple Yop effector proteins are
injected into the cell cytosol. One of these effector proteins, YopJ,
is a cysteine protease that has been shown to target the MAPK pathway
by inactivating MAPK kinases (18) (Fig.
6A). The Ras/MAPK pathway
activates RSK1, through the phosphorylation of RSK1 by Erk1/2 (17). The
Erk1/2 pathway is effectively blocked by the actions of YopJ, leading
us to investigate whether YopM can activate RSK1 in cells that also
express YopJ. In vitro kinase assays were performed with
endogenous RSK1 immunoprecipitated from serum-starved cells transfected
with vector, FLAG-YopM, FLAG-YopJ, or both FLAG-YopM and FLAG-YopJ
(Fig. 6B). The ability of RSK1 to phosphorylate MBP
increased with the expression of FLAG-YopM (lane 3). This
increased activity was not affected by co-expression of FLAG-YopJ
(lane 5). These results demonstrate that YopM is able to
activate RSK1 kinase activity independent of the effects of YopJ in
cells.
We also examined the role of YopM in the activation of RSK1 in
macrophage cells infected with yopM+ (YP22) or
yopM
To determine whether this change in mobility of RSK1 is due
specifically to the actions of YopM, YopM expression was complemented in the YP33 strain by introduction of an IPTG-inducible YopM expression plasmid (YP33/yopM). The YP33/yopM strain
produced a low level of YopM protein without IPTG addition, and a
significant amount of YopM protein was produced with IPTG stimulation
(YP33/yopM+) (Fig. 6C, lower panel).
The molecular weight of Y. enterocolitica YopM produced by
the expression plasmid (~42 kDa) is different from YopM produced by
Y. pseudotuberculosis YP22 (~48 kDa). This heterogeneity
of YopM protein size has been reported previously (29) with no known
effect on YopM function. Expression of low levels of YopM modestly
increased the amount of slower migrating forms of RSK1 (lane
4), and expression of high levels of YopM caused a significant
decrease in RSK1 mobility, similar to infection with YP22 (compare
lanes 2 and 5). These results indicate that infection of macrophage cells with Y. pseudotuberculosis
causes a decrease in the mobility of RSK1 and that YopM protein
expression is required for this change in RSK1 mobility.
In this study, we describe the identification of the first
intracellular targets of the Yersinia virulence factor YopM.
YopM is unique in comparison to the other Yop effectors, as it does not
contain any obvious catalytic domains. The crystal structure of
tetrameric YopM demonstrates a large surface for protein-protein interactions, suggesting a role as a protein scaffold (23). There are
many examples of scaffolding proteins that regulate the activity of
protein kinase pathways, including both the MAPK and protein kinase C
pathways (30, 31). These scaffolding proteins are predicted to regulate
not only the efficiency of kinase activation but also the specificity
of substrates and the amplitude of the kinase cascade response (32).
Our data suggest that non-catalytic YopM acts as a protein scaffold to
recruit and activate proteins that have catalytic function, the
serine/threonine kinases PRK2 and RSK1.
Functional studies of PRK2 suggest that it is a component of several
signaling pathways involving the cytoskeleton, receptor tyrosine
kinases, regulation of translation, and cell survival (Fig.
7). The kinase activity of PRK2 is
stimulated by the binding of RhoA and has been demonstrated to alter
the actin cytoskeleton (33, 34). Lipids have also been demonstrated to
activate PRK2 and specifically negative phospholipids, such as
cardiolipin (35). PRK2 is involved in signaling pathways activated by
receptor tyrosine kinases through interaction with the adaptor proteins
Nck and Grb4 (36, 37). PRK2 is also activated by interactions with MAPK
kinase kinase 2, a kinase activated in response to several stimuli,
including growth factors and cross-linking of antigen receptors on T
cells (15). In addition, PRK2 plays a role in initiating translation
through phosphorylation of the mRNA cap binding factor, eIF4E (38).
Finally, PRK2 affects cell survival pathways through regulation of Akt
kinase activity (39, 40).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B signaling pathways. YopJ has also been implicated in
apoptosis induction in macrophages through caspase-8 activation and
inhibition of NF
B-induced transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
buffer (150 mM
NaCl, 50 mM Tris, pH 8, 0.4 mM EDTA, 10%
glycerol, 1% Nonidet P-40, 1 mM pefabloc (Roche Applied
Science), 10 µM leupeptin (Roche Applied Science), 1 mM benzamidine (Sigma), 10 µM E64 (Sigma))
followed by centrifugation at 15,000 rpm for 30 min at 4 °C. For
cell fractionation experiments, nuclear and cytoplasmic extracts were
prepared as described previously (19). Lysates were precleared with
10-20 µl of protein G-agarose (Invitrogen) for 30 min of rocking at
4 °C. Lysates were then incubated with 15-20 µl of anti-FLAG
M2-Sepharose (Sigma) or 1-2 µg of antibody and 10-20 µl of
protein G-agarose for 1-2 h at 4 °C. Beads were washed 4 times with
1 ml of 1 M RIPA
buffer (1 M
NaCl, 50 mM Tris, pH 8, 0.4 mM EDTA, 10%
glycerol, 1% Nonidet P-40), 2 times with 1 ml of 500 mM
RIPA
buffer (500 mM NaCl, 50 mM
Tris, pH 8, 0.4 mM EDTA, 10% glycerol, 1% Nonidet P-40),
and 2 times with 1 ml of RIPA
buffer. Immunoprecipitates
used in kinase assays were washed an additional 2 times with 1 ml of 20 mM Hepes, pH 7.4, 10 mM MgAc. Silver staining
of SDS-PAGE gels was performed as described previously (20).
-cyano-4-hydroxycinnamic acid
as the UV absorbing matrix. Peptide values from the mass fingerprint
were used to search the NCBI data base using the Profound and MS-Fit
software tools. Nanospray LC/MS/MS was performed on a LCQDECA
quadrupole ion trap mass spectrometer (Thermo Finnigan) equipped with a
picoview nanospray source (New Objective). A 5-µl aliquot of the pool
of tryptic peptides was injected onto a 75-µm inner diameter × 15-cm C18 picofrit column (New Objective) that terminated in a 15-µm
tip spray needle. Peptides were eluted with 60% acetonitrile, 1%
formic acid. Mass spectra was acquired using the Top3 triple play mode,
and MS/MS spectra were used to search the NCBI using SEQUEST software.
-32P]ATP
(PerkinElmer Life Sciences), with 1 µg of MBP (Sigma) as substrate in
a 20-µl reaction volume. One-third of the reaction was separated by
SDS-PAGE, dried onto Whatman paper, and exposed to film at
80 °C
with an intensifying screen.
80 °C with
a screen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical hairpin
followed by a curving repeat structure composed of LRR motifs (23). The LRR motifs in YopM form a structure that has a concave face composed of
-sheets and a polyproline II helical conformation on the convex side. YopM is also shown to form a homo-tetramer that creates a
tube-like structure with a pore of ~35 Å. The structure does not
indicate a catalytic function for YopM but instead suggests that it may
act as a protein scaffold recruiting proteins into a complex.
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Fig. 1.
Identification of YopM-associated
proteins by mass spectrometry. A, silver-stained
SDS-PAGE of anti-FLAG immunoprecipitates from vector or
FLAG-YopM-transfected 293 cells. Molecular mass markers are indicated
on the left in kilodaltons. B, mass
fingerprint of trypsin-digested p120 generated by MALDI-TOF. Unique
peptide masses are shown in red and were used to search the
NCBI non-redundant data base. The indicated peptide sequences were
determined by MALDI-PSD. C, MS/MS spectrum of p80 peptide,
[M + H]2+ = 733.8. The observed y and b fragment ions
corresponding to the indicated peptide sequence are shown.
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Fig. 2.
Analysis of the protein interactions of the
YopM complex. A, anti-FLAG immunoprecipitates from
vector or FLAG-YopM-transfected 293 cells Western-blotted with
anti-PRK2, anti-RSK1, and anti-FLAG antibodies sequentially.
B, J774A.1 cells infected with Y. pseudotuberculosis YP22 (yopM+) or YP33
(yopM ) strains, immunoprecipitated with YopM
antisera, and proteins visualized by Western blot. Immunoglobulin heavy
chain is indicated by an asterisk. C,
in vitro binding of transcribed/translated
[35S]methionine-labeled kinases to recombinant YopM-6xHis
bound to nickel-agarose (YopM) or nickel-agarose
(beads) and visualized by fluorography. D,
Western blot analysis of anti-PRK2, anti-RSK1, or anti-FLAG
(FLAG or c) immunoprecipitates (IP)
from vector or FLAG-YopM-transfected 293 cells. E, subcellular localization of PRK2, RSK1, and YopM in 293 cells.
Nuclear (n) and cytoplasmic (c) extracts were
made from transfected cells, immunoprecipitated, and visualized by
Western blot. Western blots of equivalent amounts of nuclear and
cytoplasmic extracts determined localization of the nuclear
transcription factor CREB. Molecular mass markers are indicated on the
left in kilodaltons.
(YP33) Y. pseudotuberculosis strains, immunoprecipitated
with YopM polyclonal antisera, and the co-immunoprecipitation of RSK1 and PRK2 determined by Western blot. YopM co-immunoprecipitated RSK1
(Fig. 2B), but the co-immunoprecipitation of PRK2 was not detectable in this assay (data not shown). The inability to detect PRK2
may be due to a number of factors but is most likely associated with
the quality of the YopM antisera. For example, when
FLAG-YopM-transfected 293 cells were immunoprecipitated with the YopM
antiserum, the interaction of PRK2 and YopM was much weaker and
difficult to detect (data not shown), indicating that the antiserum
does not effectively immunoprecipitate the YopM-PRK2 complex. However, our result firmly establishes that YopM forms a complex with RSK1 in
Y. pseudotuberculosis-infected macrophage cells.
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Fig. 3.
Analysis of the regions of PRK2 and RSK1
required for interaction with YopM. A, anti-FLAG
immunoprecipitates (IP) from 293 cells transfected with
FLAG-YopM and vector (GST) or GST-PRK2 fusion plasmids.
Proteins were visualized by Western blot. Immunoglobulin heavy chain is
marked with an asterisk. GST-PRK2 fusion proteins are
diagrammed at the bottom, with ovals indicating
HR1 domains, hexagons representing the HR2 domain, the
box indicating the PXXP motif, and the
rectangle representing the kinase domain. B,
anti-FLAG immunoprecipitates from 293 cells transfected with FLAG-YopM
and vector or HA-RSK1 fusion plasmids. Proteins were visualized by
Western blot. HA-RSK1 fusion proteins are diagrammed at the
bottom with the rectangles representing the
kinase domains. FL indicates full-length HA-RSK1. Molecular
mass markers are indicated on the left in kilodaltons.
512N) and
648-985 (
648N). A weak interaction was also detected between YopM
and GST-PRK2 amino acids 331-651 with long exposures. These results
indicate that the major region of PRK2 required for interaction with
YopM is between amino acids 648 and 985 which encompasses the kinase domain of PRK2.
423C). Further truncation of HA-RSK1
abolished co-immunoprecipitation with YopM. These results suggest that
interaction of RSK1 and YopM requires the presence of the linker region
between the dual kinase domains of RSK1.
-32P]ATP and myelin basic protein (MBP) as substrate.
As both RSK1 and PRK2 kinase activity is increased in cells grown in
serum, transfected cells were serum-starved for 16 h prior to
immunoprecipitation with rabbit IgG (c), anti-PRK2, anti-RSK1, or
anti-Akt antibodies (Fig. 4A). The ability of both PRK2 and
RSK1 to phosphorylate MBP was increased when YopM was expressed in
cells. The kinase activity of Akt, a kinase not found in the YopM
complex, was not affected by the expression of YopM. These results
suggest that expression of YopM specifically activates PRK2 and
RSK1.
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Fig. 4.
YopM increases the in vitro
kinase activity of endogenous PRK2 and RSK1.
A, in vitro kinase assays performed with
immunoprecipitated (IP) kinases or rabbit IgG
immunoprecipitates (c) from transfected, serum-starved 293 cells. The phosphorylation of MBP was assessed by autoradiography of
half the reaction. The other half of the reaction was analyzed by
Western blot to assess the amount of kinases immunoprecipitated in the
assay. Molecular mass markers are indicated on the left in
kilodaltons. B, in vitro kinase assays
performed with endogenous PRK2 immunoprecipitated from serum-starved
293 cells transfected with increasing amounts of FLAG-YopM plasmid
(0-5 µg). Assays were performed as in A. C, in
vitro kinase assays performed with endogenous RSK1
immunoprecipitated from serum-starved 293 cells transfected with
increasing amounts of FLAG-YopM plasmid (0-1 µg). Assays were
performed as in A.
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Fig. 5.
Activation mechanism of YopM-associated
kinases. A, in vitro kinase assays
performed using rabbit IgG (c), anti-PRK1, or anti-RSK1
immunoprecipitates (IP) from serum-starved 293 cells with or
without the addition of recombinant YopM-6xHis protein. The
phosphorylation of the MBP substrate was assessed by autoradiography of
half the reaction. The other half of the reaction was analyzed by
Western blot to assess the amount of kinases immunoprecipitated.
B, in vitro kinase assays performed with
anti-GFP immunoprecipitates from serum-starved 293 cells transfected
with expression plasmids for EGFP-YopM and wild-type (WT) or
kinase-deficient (KD) HA-RSK1 and FLAG-PRK2 kinases.
Expression of the transfected constructs was determined by Western blot
of whole cell lysates. C, in vitro kinase
assay performed as in B, except anti-FLAG antibody was used
to immunoprecipitate FLAG-PRK2 proteins. Half of the reaction was
analyzed by Western blot to assess the amount of FLAG-PRK2
immunoprecipitated in this assay. D, in
vitro kinase assay performed as in C, except HA-RSK1
was immunoprecipitated from cells using anti-HA antibody. Molecular
mass markers are indicated on the left in kilodaltons.
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Fig. 6.
YopM is required for an increase of RSK1
phosphorylation during Yersinia infection, independent
of the effects of YopJ. A, schematic diagram of
the Ras/MAPK pathway leading to the activation of RSK1. Shown are the
kinases that YopJ inhibits and YopM activates. B,
in vitro kinase assays were performed with rabbit IgG
(c) or anti-RSK1 immunoprecipitates (IP) from
serum-starved, transfected 293 cells with MBP included as a substrate.
Amounts of RSK1 immunoprecipitated were detected by anti-RSK1 Western
blot, and expression of FLAG-YopM and FLAG-YopJ was confirmed in
anti-FLAG Western blot of cell lysates. Molecular mass markers are
indicated on the left in kilodaltons. C,
analysis of RSK1 migration from J774A.1 cells infected with Y. pseudotuberculosis strains by anti-RSK1 Western blot (upper
panel). Expression of YopM was detected by anti-YopM Western blot
of cell lysates (lower panel). Yersinia strains
used were YP22 (yopM+), YP33
(yopM ), YP33/yopM
(yopM
+ yopM plasmid), and
YP33/yopM+ (yopM
+ yopM
plasmid + IPTG).
(YP33) Y. pseudotuberculosis
strains (Fig. 6C). The macrophage cell line J774A.1 was left
uninfected or infected with Y. pseudotuberculosis; whole cell lysates were made, and the mobility of the RSK1 protein was
visualized by Western blot. In uninfected cells, RSK1 protein appears
as multiple bands due to differences in migration of multiply phosphorylated forms of RSK1 (lane 1). Upon infection of the
cells with YP22, RSK1 decreases in mobility, presumably due to an
increase in phosphorylation of the kinase (lane 2). The
apparent amount of RSK1 protein is less in lysates from YP22-infected
cells due to unknown reasons but may be due to decreased reactivity of
the antibody to the highly phosphorylated form of RSK1. Infection with
YP33 results in the appearance of faster migrating (less phosphorylated) forms of RSK1 (lane 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Conceptual model of YopM complex
formation. In cells, RSK1 and PRK2 form separate signaling
pathways with distinct activators and substrates. With the introduction
of YopM into the cell, RSK1 and PRK2 are recruited into a new complex.
Interaction of RSK1 with YopM activates its kinase activity, which in
turn increases the kinase activity of PRK2. The activated YopM complex
is now able to phosphorylate unidentified, possibly novel
substrates.
RSK1 is a member of the growth factor-regulated S6 serine/threonine
kinase family (27, 28). These kinases have two non-identical kinase
domains, and the amino-terminal kinase domain is thought to be
responsible for phosphorylating substrates of RSK containing the
consensus motif of
(Arg/Lys)-X-Arg-X-X-(Ser/Thr) or
Arg-X-(Ser/Thr) (where X is any amino acid). The
Ras/MAPK pathway activates the RSK1 kinase through phosphorylation by
Erk1/2 (Fig. 7). In addition, the carboxyl-terminal kinase domain of
RSK1 and phosphoinositide-dependent protein kinase-1 (PDK1)
also provide activating signals (28, 41). Upon activation, RSK1 has
been demonstrated to phosphorylate substrates such as transcription
factors (c-Fos, CREB, and SRF), transcriptional co-activators
(CREB-binding protein), translational regulators (ribosomal S6
protein), cell cycle regulators (Myt1 and
Na+/H+ exchanger isoform 1), apoptotic factors
(BAD), and kinases (GSK3 and IKK
), suggesting roles for this
kinase in cellular proliferation, translational regulation, cell
survival, and glycogen synthesis.
The kinases PRK2 and RSK1 have not been reported to interact under normal cellular conditions. Immunoprecipitation of PRK2 and RSK1 from transfected cells demonstrated that these kinases are recruited into a novel complex through interaction with YopM. The interaction of RSK1 and YopM was also seen in macrophage cells infected with Y. pseudotuberculosis. However, interaction of PRK2 with YopM was undetectable in infected cells, due to technical limitations of the antibodies used in these experiments and the level of PRK2 protein expression in these cells. The interaction of PRK2 and YopM from transfected cells is able to withstand multiple washes with buffers containing 1 M NaCl and 1% detergent, suggesting that it is a specific component of the YopM complex along with RSK1.
The formation of the YopM-kinase complex results in the activation of both RSK1 and PRK2. A model for activation of these kinases by YopM is diagramed in Fig. 7. In cells, RSK1 and PRK2 form separate signaling pathways with distinct activators and substrates. Cellular kinases RSK1 and PRK2 are recruited to YopM, and the direct interaction of RSK1 with YopM results in the activation of RSK1 kinase activity. The active RSK1 kinase then functions to stimulate the kinase activity of PRK2, resulting in an active YopM complex. PRK2 activation by YopM could be induced by phosphorylation of PRK2 by RSK1 or through phosphorylation-dependent changes of YopM. We have observed in transfected cells metabolically labeled with [32P]orthophosphate that YopM is phosphorylated (data not shown). Identification of the kinases responsible for this modification, sites of phosphorylation, and the effects of YopM phosphorylation will provide further insight into YopM function.
The cellular targets of PRK2 and RSK1 have been shown to be involved in
several cellular functions, some of which overlap between the two
kinases. Both of these kinases have been implicated in the regulation
of proliferation, apoptosis, and translation (15, 27, 28, 38-40). We
were unable to detect a change in cellular proliferation, translation,
or the activity of some characterized targets of either PRK2 (Akt) or
RSK1 (Bad, Jun, and CREB) in cells transfected with YopM (data not
shown). We propose that the recruitment of PRK2 and RSK1 to YopM may
alter their substrate specificity and/or recruit new substrates to
these kinases, resulting in a novel signaling pathway (Fig. 7). This
hypothesis will need to be tested by an analysis of the
phospho-proteome of yopM+ and
yopM Yersinia-infected cells. We
anticipate that identification of the targets of the YopM complex
described in this paper will provide further insight into the function
of YopM.
In this study, we identify that YopM functions to stimulate endogenous
RSK1 kinase activity. We observed increased activity of RSK1 in
YopM-transfected cells by in vitro kinase assays and by a
change in RSK1 mobility in Y. pseudotuberculosis-infected macrophage cells. Furthermore, the change of RSK1 mobility in infected
cells was shown to be dependent on YopM, as complementation of a
yopM Yersinia strain with a YopM
expression plasmid restored the slower mobility of RSK1. This apparent
activation of RSK1 appears to be independent of upstream activators, as
the strains used also translocate YopJ, a known inhibitor of the
MAPK/Erk1 pathway (18, 22, 42). YopJ-independent activation of RSK1 was
also demonstrated in vitro, as YopM is able to stimulate
RSK1 kinase activity in cells expressing YopJ.
Activation of RSK1 by YopM is conserved function between species of
Yersinia with heterogeneous YopM proteins. It was noted previously (29) that different strains of Y. pseudotuberculosis and Y. enterocolitica produce YopM
proteins that vary from ~41 to ~55 kDa. The difference in size of
these proteins is due to a triplication of a 60-amino acid region near
the carboxyl terminus of YopM. No obvious effects on virulence have
been noted between strains producing different sized YopM proteins;
however, the effect these differences have on YopM function has been
difficult to determine without a defined function for YopM. Infection
with Y. pseudotuberculosis strains secreting an ~42-kDa
Y. enterocolitica YopM protein or the endogenous ~48-kDa
Y. pseudotuberculosis YopM protein both affected the
mobility of RSK1 in macrophage cells. These data suggest a novel and
conserved function for YopM, the activation of RSK1.
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ACKNOWLEDGEMENTS |
---|
We thank members of the Dixon and Bliska laboratories for their advice and assistance, particularly members of the Yop group, Yue Zhang, and Donna Veine. We thank Jeanne Stuckey for assistance with the YopM crystal structure. Critical reading of the manuscript by Carolyn Worby was greatly appreciated. We thank John Blenis, Gary Johnson, Kim Orth, Don Huddler, and Valerie Castle for their gifts of plasmid constructs. Olaf Schneewind provided a generous gift of YopM antisera.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 AI43389 (to J. B. B.), R37 DK18024, and R01 DK18849 (to J. E. D.), and funds from the Ellison Medical Foundation (to J. E. D.).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.
¶ To whom correspondence should be addressed. Tel.: 858-822-3529; Fax: 858-534-6573; E-mail: jedixon@umich.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M301226200
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ABBREVIATIONS |
---|
The abbreviations used are:
Yop, Yersinia outer protein;
MAPK, mitogen-activated protein
kinase;
LRR, leucine-rich repeat;
PRK2, protein kinase C-like 2;
RSK1, p90 ribosomal protein S6 kinase 1;
FBS, fetal bovine serum;
MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight;
PSD, post-source decay;
LC, liquid chromatography;
MS, mass spectrum;
MBP, myelin basic protein;
EGFP, enhanced green fluorescent protein;
DMEM, Dulbecco's modified Eagle's medium;
HA, hemagglutinin;
IPTG, isopropyl-1-thio--D-galactopyranoside;
GST, glutathione
S-transferase;
CREB, cAMP-response element-binding
protein.
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REFERENCES |
---|
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---|
1. | Perry, R. D., and Fetherston, J. D. (1997) Clin. Microbiol. Rev. 10, 35-66[Abstract] |
2. | Bottone, E. J. (1997) Clin. Microbiol. Rev. 10, 257-276[Abstract] |
3. | Juris, S. J., Shao, F., and Dixon, J. E. (2002) Cell Microbiol. 4, 201-211[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Cornelis, G. R.,
Boland, A.,
Boyd, A. P.,
Geuijen, C.,
Iriarte, M.,
Neyt, C.,
Sory, M. P.,
and Stainier, I.
(1998)
Microbiol. Mol. Biol. Rev.
62,
1315-1352 |
5. | Leung, K. Y., Reisner, B. S., and Straley, S. C. (1990) Infect. Immun. 58, 3262-3271[Medline] [Order article via Infotrieve] |
6. | Mulder, B., Michiels, T., Simonet, M., Sory, M. P., and Cornelis, G. (1989) Infect. Immun. 57, 2534-2541[Medline] [Order article via Infotrieve] |
7. | Kobe, B., and Kajava, A. V. (2001) Curr. Opin. Struct. Biol. 11, 725-732[CrossRef][Medline] [Order article via Infotrieve] |
8. | Reisner, B. S., and Straley, S. C. (1992) Infect. Immun. 60, 5242-5252[Abstract] |
9. | Boland, A., Sory, M. P., Iriarte, M., Kerbourch, C., Wattiau, P., and Cornelis, G. R. (1996) EMBO J. 15, 5191-5201[Abstract] |
10. | Skrzypek, E., Cowan, C., and Straley, S. C. (1998) Mol. Microbiol. 30, 1051-1065[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Skrzypek, E.,
Myers-Morales, T.,
Whiteheart, S. W.,
and Straley, S. C.
(2003)
Infect. Immun.
71,
937-947 |
12. |
Sauvonnet, N.,
Pradet-Balade, B.,
Garcia-Sanz, J. A.,
and Cornelis, G. R.
(2002)
J. Biol. Chem.
277,
25133-25142 |
13. | Lee, V. T., Anderson, D. M., and Schneewind, O. (1998) Mol. Microbiol. 28, 593-601[CrossRef][Medline] [Order article via Infotrieve] |
14. | Furste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M., and Lanka, E. (1986) Gene (Amst.) 48, 119-131[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Sun, W. Y.,
Vincent, S.,
Settleman, J.,
and Johnson, G. L.
(2000)
J. Biol. Chem.
275,
24421-24428 |
16. | Shimamura, A., Ballif, B. A., Richards, S. A., and Blenis, J. (2000) Curr. Biol. 10, 127-135[CrossRef][Medline] [Order article via Infotrieve] |
17. | Grove, J. R., Price, D. J., Banerjee, P., Balasubramanyam, A., Ahmad, M. F., and Avruch, J. (1993) Biochemistry 32, 7727-7738[Medline] [Order article via Infotrieve] |
18. |
Orth, K.,
Palmer, L. E.,
Bao, Z. Q.,
Stewart, S.,
Rudolph, A. E.,
Bliska, J. B.,
and Dixon, J. E.
(1999)
Science
285,
1920-1923 |
19. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
20. | Blum, H., Gross, H. J., and Beier, H. (1989) Virology 169, 51-61[Medline] [Order article via Infotrieve] |
21. | Vacratsis, P. O., Phinney, B. S., Gage, D. A., and Gallo, K. A. (2002) Biochemistry 41, 5613-5624[CrossRef][Medline] [Order article via Infotrieve] |
22. | Palmer, L. E., Hobbie, S., Galan, J. E., and Bliska, J. B. (1998) Mol. Microbiol. 27, 953-965[CrossRef][Medline] [Order article via Infotrieve] |
23. | Evdokimov, A. G., Anderson, D. E., Routzahn, K. M., and Waugh, D. S. (2001) J. Mol. Biol. 312, 807-821[CrossRef][Medline] [Order article via Infotrieve] |
24. | Zhang, W., and Chait, B. T. (2000) Anal. Chem. 72, 2482-2489[CrossRef][Medline] [Order article via Infotrieve] |
25. | Palmer, R. H., Ridden, J., and Parker, P. J. (1994) FEBS Lett. 356, 5-8[CrossRef][Medline] [Order article via Infotrieve] |
26. | Palmer, R. H., Ridden, J., and Parker, P. J. (1995) Eur. J. Biochem. 227, 344-351[Abstract] |
27. | Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892[Abstract] |
28. | Frodin, M., and Gammeltoft, S. (1999) Mol. Cell. Endocrinol. 151, 65-77[CrossRef][Medline] [Order article via Infotrieve] |
29. | Boland, A., Havaux, S., and Cornelis, G. R. (1998) Microb. Pathog. 25, 343-348[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Schaeffer, H. J.,
and Weber, M. J.
(1999)
Mol. Cell. Biol.
19,
2435-2444 |
31. | Bauman, A. L., and Scott, J. D. (2002) Nat. Cell Biol. 4, E203-E206[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Levchenko, A.,
Bruck, J.,
and Sternberg, P. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5818-5823 |
33. | Vincent, S., and Settleman, J. (1997) Mol. Cell. Biol. 17, 2247-2256[Abstract] |
34. |
Zong, H.,
Raman, N.,
Mickelson-Young, L. A.,
Atkinson, S. J.,
and Quilliam, L. A.
(1999)
J. Biol. Chem.
274,
4551-4560 |
35. |
Yu, W. P.,
Liu, J. J.,
Morrice, N. A.,
and Wettenhall, R. E. H.
(1997)
J. Biol. Chem.
272,
10030-10034 |
36. |
Braverman, L. E.,
and Quilliam, L. A.
(1999)
J. Biol. Chem.
274,
5542-5549 |
37. |
Quilliam, L. A.,
Lambert, Q. T.,
MickelsonYoung, L. A.,
Westwick, J. K.,
Sparks, A. B.,
Kay, B. K.,
Jenkins, N. A.,
Gilbert, D. J.,
Copeland, N. G.,
and Der, C. J.
(1996)
J. Biol. Chem.
271,
28772-28776 |
38. | Lee, S. J., Stapleton, G., Greene, J. H., and Hille, M. B. (2000) Dev. Biol. 228, 166-180[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Cryns, V. L.,
Byun, Y.,
Rana, A.,
Mellor, H.,
Lustig, K. D.,
Ghanem, L.,
Parker, P. J.,
Kirschner, M. W.,
and Yuan, J. Y.
(1997)
J. Biol. Chem.
272,
29449-29453 |
40. |
Koh, H.,
Lee, K. H.,
Kim, D.,
Kim, S.,
Kim, J. W.,
and Chung, J.
(2000)
J. Biol. Chem.
275,
34451-34458 |
41. | Richards, S. A., Fu, J., Romanelli, A., Shimamura, A., and Blenis, J. (1999) Curr. Biol. 9, 810-820[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Palmer, L. E.,
Pancetti, A. R.,
Greenberg, S.,
and Bliska, J. B.
(1999)
Infect. Immun.
67,
708-716 |