 |
INTRODUCTION |
The extracellular microbial pathogen, Yersinia,
disrupts critical signaling pathways in the infected host by injecting,
via a type III secretion system, a number of virulence factors, or Yersinia outer proteins
(Yops)1 into a target host
cell (1). Before translocation into the target host, Yops remain in a
quiescent state inside the pathogen due to lack of substrate or
activator or due to the presence of a chaperone (2). Each effector
(Yop) appears to mimic the activity of an eukaryotic protein, which is
then used to alter the signaling machinery in the target cell, yielding
an advantage for the survival of the pathogen during infection.
Identification of the activity of each Yop has uncovered an essential
regulatory mechanism utilized by eukaryotic cells. Three of the Yops,
YopT (a papain-like protease), YopE (a GTPase-activating
protein), and YpkA (a serine kinase), each contribute to the
disruption of the actin cytoskeleton, thereby preventing phagocytosis
of this extracellular pathogen (3-5). A fourth effector, YopH, is a
tyrosine phosphatase that enhances the inhibition of phagocytosis of
the pathogen by dephosphorylating proteins in the focal adhesion
complex, resulting in the disassembly of this essential complex
(6-8).
Another Yersinia virulence factor, YopJ, blocks the innate
immune response by blocking cytokine production and inducing apoptosis in the infected macrophage (2). The infected host cell cannot respond
to a Yersinia infection because YopJ inhibits the MAP kinase
and NF
B pathways by preventing the activation (via phosphorylation) of the superfamily of mitogen-activated protein kinase kinases (including the MKKs (MAP kinase kinases) and IKK
(I
B kinase
)
(9). Because the derived secondary structure of YopJ is highly similar
to that of the known secondary structure of a cysteine protease from
adenovirus and the eukaryotic ubiquitin-like protein protease (Ulp1),
YopJ is thought to mimic this type of hydrolase activity. Inactivating
mutations in the catalytic triad of YopJ are unable to inhibit the
aforementioned signaling pathways, further supporting the proposal that
YopJ acts as a protease (10).
Homologues of YopJ, collectively referred to as YopJ proteases, are
found in plant and animal pathogens as well as in a plant symbiont. A
homologue expressed by the animal pathogen Salmonella, AvrA,
inhibits only the NF
B pathway but not the MAP kinase pathways and
requires an intact catalytic triad for this activity (11). Similarly, a
homologue from Xanthomonas, AvrBst, requires a functional catalytic site for induction of the host defense response in plant cells (10). Members of the YopJ family of proteases from both the plant
and animal pathogens disrupt/modulate signaling pathways in the
eukaryotic host by a mechanism that involves some type of hydrolase
activity, resulting in disruption of homeostasis in the infected cell.
To date, all of the effectors expressed by Yersinia disrupt
signaling in the target cells by disrupting the homeostasis of posttranslational modifications. For example, the extremely active tyrosine phosphatase YopH dephosphorylates cellular targets so efficiently that the complementary kinases are unable to maintain a
regulated state (6-8). Similar scenarios are observed with the serine
kinase YpkA and with the GTPase-activating protein YopE (3, 5).
The effector YopJ is proposed to disrupt a reversible posttranslational
modification in the form of an ubiquitin-like protein. The secondary
structure of YopJ is similar to that of the proteases AVP and Ulp1,
which cleave ubiquitin conjugates and ubiquitin-like protein
conjugates, respectively (10, 12-14). Therefore, YopJ is thought to
disrupt an essential posttranslational modification that is required
for activation of mammalian MAP kinase and NF
B pathways.
Yeast (Saccharomyces cerevisiae) MAP kinase signaling
pathways have an architecture similar to the signaling pathways
utilized by higher eukaryotes, which consist of regulated kinase
cascades and cell surface receptors that receive and transmit
extracellular signals (15). Genetic studies on ubiquitin-like protein
conjugation in yeast demonstrate that this system is evolutionarily
conserved and essential for survival (13). Because YopJ is a member of a family of virulence factors that are conserved in both animal and
plant pathogens, it is appealing to propose that the targets of YopJ in
these signaling pathways are evolutionarily conserved and utilized in a
similar manner in yeast (2, 16). Herein, we utilize yeast genetics to
demonstrate that the MAP kinase pathways used for pheromone (mating
pathway) and high osmolarity growth (HOG pathway) can be inhibited by
YopJ but not by the catalytically inactive form of YopJ, demonstrating
that the mechanism of regulation disrupted by YopJ is evolutionarily conserved.
 |
EXPERIMENTAL PROCEDURES |
Strains, Media, Plasmids, and Antibodies--
All yeast strains
used were obtained from the BY4741/2/3 series from Research Genetics
(Huntsville, AL). All yeast media containing 2% dextrose (Glu) or 2%
raffinose, 1% galactose (Gal) were prepared as described (17). YopJ
(J) and YopJ(C/A) (J(C/A)) were amplified from wild type and mutant
templates, respectively (10), using a 5' oligonucleotide encoding an
EcoRI site and a 3' oligonucleotide encoding a FLAG epitope
tag followed by a termination codon and a XhoI site. PCR
products were cloned into p413Gal1 (18). Yeast transformations were
carried out by the lithium acetate procedure (19). Tetrad analysis was
carried out as described (17). Antibodies used in this study include
anti-FLAG M2 (Sigma #A2220), anti-phospho-p44/42 MAP kinase antibody
(Cell Signaling #9101), anti-Fus3p (Santa Cruz #sc-6722),
anti-phospho-P38 antibody (Cell Signaling #9211), anti-Hog1p (Santa
Cruz #sc-6815), and anti-porin (Molecular Probes, #A-6449).
Immunoreactive species were detected by immunoblot analysis using
horseradish peroxidase-conjugated anti-rabbit (Amersham Biosciences),
anti-mouse (Amersham Biosciences), or anti-goat (Amersham Biosciences)
followed by visualization with enhanced chemiluminescence detection
reagents (Amersham Biosciences, ECL+).
Pheromone Induction Assays--
Halo assays were performed as
described in Hoffman et al. (20). Liquid cultures (2%
raffinose, 1% galactose) were induced with 50 nM
factor (Sigma A-3917) for 15 min and terminated by snap-freezing in dry
ice/isopropanol followed by extraction of total protein as described by
Ooi et al. (21).
Hyperosmotic Stress Induction Assays--
Yeast cultures were
grown on plates containing either 2% dextrose or 2% raffinose, 1%
galactose in the presence or absence of 1 M sorbitol.
Liquid cultures were stimulated for the indicated periods of time with
0.7 M sorbitol and terminated by snap-freezing in dry
ice/isopropanol followed by extraction of total protein as described by
Ooi et al. (21).
 |
RESULTS |
Yeast Expressing YopJ Respond Inefficiently to
Factor--
To
determine whether YopJ would affect the yeast MAP kinase pathways, the
expression of YopJ is put under the control of a GAL1
promoter resulting in the induction of YopJ expression only when
induced by growth on galactose. When the parental strain BY4741
(Research Genetic, Inc.) is transformed with either an empty vector
(V), wild type YopJ (J), or the catalytically inactive form of YopJ
(J(C/A)), immunoblot analysis confirms that wild type YopJ and the
mutant YopJ(C172A) are expressed on media containing galactose (Gal)
but are not detectable on media containing glucose (Glu) (Fig.
1A). To analyze whether YopJ
has any effect on the ability of yeast cells to respond to the
pheromone-regulated MAP kinase pathway, we assayed whether cells could
respond to
factor in the presence of wild type YopJ but not in the
presence of the catalytically inactive form of YopJ. The growth
inhibition by pheromone on the MATa cells is assessed by
formation of halos in the presence of
factor (20). Yeast cells with
either an empty vector or expressing the catalytically inactive form of YopJ (J(C/A)) plasmid are able to produce halos of similar size in
response to
factor (Fig. 1B). However, almost
undetectable halos are formed with yeast cells expressing wild type
YopJ, indicative of a disruption in the pheromone signaling pathway
(Fig. 1B).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
YopJ inhibits the pheromone-induced MAP
kinase pathway. A, expression of YopJ (J)
and mutant YopJ(C172A) (J(C/A)) is induced by growth in
galactose (Gal) but not in dextrose medium. Total protein isolated from
2 × 107 cells that were grown overnight on Glu or Gal
medium (as described under "Experimental Procedures") were
immunoblotted with anti-FLAG antibody followed by immunoblotting with
anti-porin antibody to confirm equal loading. B, growth
inhibition by factor is disrupted in yeast expressing wild type
YopJ. Log phase cells were plated in soft agar and exposed to a filter
disc containing 20 µg of factor. Halo formation, indicative of
growth inhibition, is observed in cells containing an empty control
vector (V) or mutant YopJ (J(C/A)) but not with
cells expressing wild type YopJ (J). C,
expression of YopJ compromises the ability of yeast to mate. Mating
efficiency between BY4741-MATa cells containing an empty
control vector and BY4742-MAT cells is calculated at
100% (actual mating efficiency is 95%). The efficiency of mating
BY4741-MATa cells containing catalytically inactive YopJ
(C/A) mutant with BY4742-MAT cells is ~100%, whereas
mating BY4741-MATa cells containing wild type YopJ with
BY4742-MAT cells is reduced to ~10%.
|
|
To quantitatively analyze how effectively YopJ blocks the pheromone
signaling pathway, the efficiency of mating of the various yeast
strains was determined. Mating of the BY4741-MATa cells with
haploid BY4742-MAT
cells results in the predicted number of diploid cells based on the number of BY4741-MATa cells
mated to a 10-fold excess of BY4742-MAT
cells (Fig.
1C). Likewise, BY4741-MATa cells expressing the
catalytically inactive form of YopJ (J(C/A)) mated with excess
BY4742-MAT
cells produce diploid cells similar in number
to the vector control (Fig. 1C). In contrast, when
BY4741-MATa cells expressing the wild type YopJ are mated with excess BY4742-MAT
cells, mating efficiency is only
~10% that of cells containing empty vector (Fig. 1C).
These results support the proposal that expression of wild type YopJ
but not the catalytically inactive mutant YopJ, compromises the ability of yeast cells to respond to pheromone stimulus by disrupting an
evolutionarily conserved signaling mechanism in a MAP kinase pathway.
YopJ Blocks the Pheromone MAP Kinase Pathway Upstream of Fus3p
Activation--
Yeast cells treated with pheromone trigger the mating
response and also activate an adaptation response by inducing the
expression of proteins that allow the yeast to recover from growth
inhibition induced by pheromone. One of these proteins is a secreted
protease (Sst1p) that degrades
factor, thereby limiting the
external zone of stimulus (22). Adaptation is also regulated by a
GTPase-activating protein (Sst2p) that stimulates the hydrolysis of GTP
by the active
G-protein subunit, Gpa1p, thereby accelerating the
rate of recovery to basal state in the pheromone-stimulated yeast cells
(23). Deletion of either SST1 or SST2 results in
MATa yeast strains that are hypersensitive to
factor
and, thus, form superhalos in the presence of pheromone. To test
whether YopJ blocks the pheromone-sensing MAP kinase pathway upstream
or downstream of pheromone-induced transcription, wild type and mutant
YopJ were transformed into the sst1
and
sst2
strains and assayed for their ability to form halos
in response to
factor. The sst2
cells containing
either an empty vector or overexpressing the catalytically inactive
mutant YopJ (C172A) under the control of the GAL promoter formed superhalos in the presence of
factor (Fig.
2A). In contrast, sst2
cells expressing wild type YopJ failed to form
superhalos (Fig. 2A). Similar results were observed with the
sst1
strain (data not shown). These results indicate that
the block by the Yersinia effector YopJ was upstream of the
transcriptionally induced adaptive response.

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 2.
YopJ inhibits the pheromone-induced MAP
kinase pathway upstream of Fus3p phosphorylation. A,
sst2 cells expressing YopJ do not form superhalos. The
formation of superhalos is observed in sst2 cells
containing a control vector (V) and with mutant YopJ plasmid
(J(C/A)) cells but not with wild type YopJ (J)
plasmid. B, YopJ inhibits the phosphorylation of Fus3p.
BY4741-MATa cells containing empty control vector
(V), wild type YopJ plasmid, or mutant YopJ
(J(C/A)) plasmid were grown to log phase in Gal media and
induced for 5 min with factor (200 nM). Representative
immune blots are shown that are used to assay for induction of Fus3p
activation by phosphorylation (anti-phospho-p44/42 MAP kinase
antibody). The asterisk indicates cross-reacting MAPK Kss1p.
Total Fus3p was detected using an anti-Fus3p antibody. Immunoblotting
with porin and FLAG antibodies is used to confirm protein loads and
YopJ expression, respectively. The relative fold change in
phosphorylation of Fus3p was determined by dividing the intensity of
the signal of phospho-Fus3p by that of total Fus3p as detected on an
Alpha ImagerTM 2200.
|
|
Previous studies demonstrate that YopJ is able to block the activation
of the MAP kinase pathway in mammalian cells upstream of the MAP kinase
activation (9). To assess whether YopJ blocks the mating pathway at a
similar point, cells containing empty vector, overexpressing wild type
YopJ or the catalytically inactive YopJ under the control of the
GAL promoter were tested for their ability to affect
phosphorylation levels of the pheromone-induced MAPK Fus3p. When the
cells containing empty vector or overexpressing the catalytically
inactive YopJ (C/A) are treated with pheromone, the MAPK Fus3p is
activated by phosphorylation as shown in Fig. 2B. When cells
overexpressing wild type YopJ are treated with
factor, a very low
level of Fus3p phosphorylation is detected (Fig. 2B). The
low level of phosphorylated Fus3p observed in YopJ-expressing cells is
consistent with inability of YopJ-expressing cells to mate efficiently.
Therefore, similar to what is observed with mammalian MAP kinase
signaling pathways, YopJ blocks the pheromone MAPK pathway upstream of
the activation of the MAP kinase.
Yeast Cells Expressing YopJ Lead to Growth Arrest in Response to
High Osmolarity--
YopJ blocks mammalian MAP kinase signaling
pathways and the yeast pheromone-sensing pathway, suggesting a
conserved regulatory mechanism underlying the MAP kinase pathways. To
further support this hypothesis we tested whether YopJ had any effect
on the HOG MAP kinase pathway by growing the various yeast strains on
media containing 1 M sorbitol (Sorb). When these strains
are grown on dextrose medium (no induction of the GAL
promoter) and transferred to dextrose plates containing 1 M
sorbitol, all strains adapt to media containing 1 M
sorbitol by activating the HOG MAP kinase pathway (Fig.
3A). When these strains are
grown on galactose plates (induction of YopJ expression) and
transferred to galactose media containing 1 M sorbitol,
cells containing empty vector or the catalytically inactive YopJ(C/A),
but not cells overexpressing wild type YopJ, are able to adapt and grow
on media containing 1 M sorbitol (Fig. 3B).
Therefore, the expression of the active form of YopJ has an adverse
effect on the ability of yeast to adapt to hyperosmotic stress as
indicated by the lack of growth. To determine whether the YopJ effect
was reversible, we tested whether cells overexpressing wild type YopJ
from the 1 M Sorb-Gal plate that are growth-inhibited could
recover and grow on a 1 M Sorb-Glu plate. As observed in
Fig. 3C, the "growth-inhibited" cells overexpressing
wild type YopJ from the 1 M Sorb-Gal plate are able to
recover and grow in a manner similar to that of cells grown on glucose
and 1 M sorbitol. Turning off YopJ expression allows cells
to adapt and grow in media containing 1 M sorbitol, thereby
demonstrating that the effect caused by YopJ expression is reversible.
These observations are consistent with the hypothesis that YopJ affects
the equilibrium of a reversible posttranslational modification.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of YopJ inhibits the ability of
yeast to adapt to growth on high salt. Yeast containing empty
vector (V) or Gal-inducible YopJ (J) or mutant
YopJ(C/A) (J(C/A)) vector are able to grow on dextrose
(Glu) plates (A), 1 M sorbitol
(Sorb), and dextrose (Glu) plates (B)
but not on 1 M sorbitol (Sorb), galactose
(Gal) plates (C). D, yeast cells
expressing wild type YopJ that are transferred from the 1 M
Sorb, Gal, plates to 1 M Sorb, Glu plates are able to
recover and grow.
|
|
To further explore the possibility that YopJ blocks the HOG MAP kinase
pathway, we tested whether YopJ would rescue the lethality of the
constitutively induced HOG pathway in a sln1
mutant
strain. Sln1p is an osmolarity sensor that negatively regulates the HOG pathway by inhibiting the activation of the downstream kinases (24,
25). In the absence of Sln1p, the MAP kinases are constitutively active, which leads to a lethal phenotype, as is demonstrated by tetrad
analysis of SLN1/sln1
diploids, which produce tetrads with a ratio of 2:2 live:dead segregants (26). The lethal phenotype of
sln1
can be rescued by mutations in the downstream
kinases Ssk2p, Pbs2p, or Hog1p (24). SLN1/sln1
diploids
containing an empty vector (V), wild type YopJ (J), or catalytically
inactive YopJ C172A (J(C/A)) under the control of the GAL
promoter were induced to sporulate, and tetrads were dissected for
spore survival. Dissected tetrads from the
SLN1:sln1
strain containing empty vector (V)
or catalytically inactive YopJ (C/A) plasmid (J(C/A)) results in the
expected live:dead segregant ratio of 2:2 (Table I). In contrast, when tetrads from
SLN1/sln1
strain containing wild type YopJ plasmid (J)
are dissected, 67% of the tetrads produced three or more viable
colonies, supporting the hypothesis that YopJ inhibits HOG MAP kinase
pathway, resulting in survival of the sln1
segregants
(Table I). As expected, the four colonies from tetrads of the
SLN1:sln1
cells containing wild type YopJ plasmid exhibit a live:dead ratio of 2:2 when plated on G418 because the SLN1 was deleted by KanMX4 (Saccharomyces
Deletion Project). All spores from tetrads of the
SLN1:sln1
cells containing wild type YopJ
plasmid with a live:dead ratio of 4:0 also exhibited a 2:2 segregation
for appropriate auxtrophic markers. Extended growth on rich media
resulted in a loss of the YopJ plasmid (HIS3 marker) in strains
encoding wild type SLN1 (G418-sensitive). Loss of the YopJ
plasmid was not observed in the G418-resistant segregants, indicating
that maintenance of this plasmid was required for growth of the
sln1
cells. The rescued lethality of sln1
by YopJ should be due to an extremely low level of expression of YopJ
on dextrose (undetectable by Western blot analysis). The results were
the same regardless of source of carbon (galactose or dextrose) used in
the media for dissections. These genetic observations support the model
that YopJ blocks the HOG MAP kinase pathway downstream of the
osmolarity sensor Sln1p.
View this table:
[in this window]
[in a new window]
|
Table I
YopJ rescues lethality of a sln1 mutation
SLN1/sln1 diploid cells transformed with control vector
(V), wild type YopJ (J), or mutant YopJ (J(C/A)) were grown in
galactose medium and transferred to sporulation medium. Tetrads were
dissected on yeast extract/peptone/dextrose or yeast extract
peptone/galactose medium. After three days of growth, survival of
segregants from each tetrad was determined. Ratio of live:dead
segregants are indicated.
|
|
YopJ Blocks the HOG Pathway Upstream of Hog1p Activation--
As
with all MAP kinase pathways, the distal kinase to be activated in
these pathways is the MAP kinase, and in the case of the HOG MAP kinase
pathway, this kinase is Hog1p (27, 28). In cells containing
empty vector or overexpressing the catalytically inactive YopJ grown on
galactose media containing 0.7 M sorbitol, the Hog1p is
activated, via phosphorylation, as is observed in Fig.
4. In contrast, cells overexpressing the
wild type YopJ induced with the same media are unable to activate the
Hog1p (Fig. 4). Therefore, the Yersinia effector YopJ
appears to block the HOG MAP kinase pathway downstream of Sln1p and
upstream of Hog1p.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4.
Expression of YopJ blocks activation of
Hog1p. Strains containing empty vector (V), wild type
YopJ plasmid (J), or catalytically inactive YopJ(C/A)
plasmid (J(C/A)) were grown to log phase in galactose media
and induced with 0.7 M sorbitol for 2.5 min. Representative
immune blots are shown that are used to assay for induction of Hog1p
activation by phosphorylation (anti-phospho-P38 antibody).
Immunoblotting with porin and FLAG antibodies is used to confirm
protein loads and YopJ expression, respectively.
|
|
 |
DISCUSSION |
Herein, genetic experiments are described that demonstrate MAP
kinase pathways in yeast are susceptible to inhibition by the Yersinia effector YopJ. The point of inhibition is upstream
of the activation of the MAP kinase and downstream of the
receptor-mediated stimulus. The mechanism of inhibition by YopJ is
reversible and, as with mammalian cells, not lethal. These results show
that the mechanism of inhibition mediated by the Yersinia
effector YopJ is evolutionarily conserved from yeast to mammals.
The molecules used to transmit signals via the MAP kinase pathways are
extremely conserved in evolution from yeast to human and even to plants
(15, 29). The conserved proteins utilized in the pathways include, but
are not limited to, G-proteins, kinases, phosphatases, chaperones, and
scaffolds. Regulation of these systems appears to be dictated by the
activation of a cascade of kinases, which results in the activation of
transcription machinery that in turn produces a response. In addition,
eukaryotic cells will also induce adaptation responses so that the
signaling machinery is reset and able to respond to new stimuli. Many
of these regulatory functions are carried out by reversible
posttranslational modifications that can be attenuated by additional
reversible posttranslational modifications.
Previous studies demonstrate that the functions encoded by the
effectors from extracellular pathogen Yersinia mimic
essential activities used in the regulation of these critical
eukaryotic signaling pathways. When injected into the host target cell,
the effectors act to tip the delicate balance of a reversible
posttranslational modification, thereby overriding the host
intracellular signaling machinery in favor of the pathogen during
infection. For example, one of these effectors, YopH, is an extremely
active tyrosine phosphatase that, once inside an infected macrophage,
dephosphorylates focal adhesion signaling machinery at a rate for which
no kinase can compensate. All of the effectors to date have captured
some type of enzymatic activity that deregulates eukaryotic signaling machinery and whose activity is kept quiescent in the prokaryotic pathogen (2, 16).
The Yersinia effector YopJ also has the characteristics of a
typical Yersinia effector in that it encodes an enzymatic
activity that disrupts eukaryotic signaling machinery and tips the
balance of equilibrium to an off state. Previous studies have
demonstrated that YopJ but not the catalytically inactive form of YopJ,
is able to disrupt eukaryotic signaling pathways by preventing the activation of the MAP kinase kinase (MKK) superfamily. The similarity of YopJ to the family of adenoviral proteases, which can cleave ubiquitin conjugates, and to the family of ubiquitin-like protein proteases, which cleave ubiquitin-like protein conjugates, has led to
the hypothesis that YopJ may also target this type of reversible posttranslational modification. The activity of YopJ in mammalian cells, as with yeast, is dependent on the maintenance of a conserved catalytic triad, lending credence to the proposal that YopJ functions as a hydrolase to disrupt the signaling pathways. These studies provide
evidence that a conserved mechanism of regulation, which is susceptible
to inactivation by wild type YopJ, is utilized in the yeast MAP kinase pathways.
Because the yeast MAP kinase pathways and yeast ubiquitin-like systems
are highly conserved with mammalian systems, yeast is a model system to
set up a genetic analysis of the function of the Yersinia
effector YopJ. Our studies have shown that the mechanism of YopJ
inhibition is conserved in yeast, and we have demonstrated that YopJ
can inhibit both the pheromone-induced MAP kinase pathway and the HOG
MAP kinase pathway. Based on the homology of YopJ with other proteases
that attenuate the stability of ubiquitin conjugates and ubiquitin-like
protein conjugates, it is tempting to speculate that YopJ might affect
this type of reversible posttranslational modification. Recent studies
by Firtel and co-workers (30) show that the Dictyostelium
MAP kinase kinase is regulated by an ubiquitin-like protein
modification (sumoylation), supporting the hypothesis that these
signaling pathways can be regulated by yet another reversible
posttranslational modification (30).
Studies by O'Rourke et al. (31) observe cross-talk between
the MAP kinase pathways in yeast, because some of the upstream signaling components are shared between the two pathways (Fig. 5) (31). In our studies with YopJ, we
have utilized a branch of the HOG pathway (Sln1) that shares
no common components with the mating MAP kinase pathway, thereby
leading to our hypothesis that YopJ functions by inhibiting a mechanism
that is conserved in multiple MAP kinase pathways. In mammalian cells,
YopJ prevents the activation of the family of MAP kinase kinases (9),
and we propose in yeast, YopJ prevents the activation of the MAP kinase kinase equivalent; that is, Ste7p for the mating pathway and Pbs2p for
the HOG pathway. The mechanism of regulation would involve a step that
is sensitive to the hydrolase activity of YopJ, which may be mediated
by the proposed sumoylation sites on Ste7 (30). Alternatively, the
mechanism may be mediated by a yet-to-be described modification that is
susceptible to YopJ activity. Future genetic and biochemical studies in
yeast will focus on the identification of the targets of YopJ, which
will help to identify an evolutionarily conserved regulatory mechanism
of eukaryotic MAP kinase signaling pathways.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Schematic representation of the Mating and
HOG MAP kinase pathways. The third branch of the osmo-sensing
pathway and other components of these pathways are omitted for clarity.
YopJ is proposed to act at the level of the MAP kinase kinase
(MKK). MKKK, MKK kinase.
|
|