Yersinia Effector YopJ Inhibits Yeast MAPK Signaling Pathways by an Evolutionarily Conserved Mechanism*

Sara Yoon, Zhengchang Liu, Yvonne Eyobo, and Kim OrthDagger

From the Department of Molecular Biology, University of Texas Southwestern Medical School, Dallas, Texas 75390-9148

Received for publication, September 26, 2002, and in revised form, November 11, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yersinia effector, YopJ, inhibits the innate immune response by blocking MAP kinase and NFkappa B signaling pathways in mammalian cells. Herein, YopJ is shown to disrupt the MAP kinase signaling pathways in Saccharomyces cerevisiae. Expression of YopJ in yeast blocks the ability of yeast to respond to alpha  factor by disrupting activation of the pheromone signaling pathway upstream of the activation of the MAPK Fus3p. YopJ also blocks the high osmolarity growth (HOG) MAP kinase pathway in yeast upstream of the activation of the MAPK Hog1p. YopJ is proposed to block the MAP kinase pathways in yeast in a similar manner to the way it blocks mammalian signaling pathways, implicating that a novel, evolutionarily conserved mechanism of regulation is utilized for signal transduction by these pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 NFkappa B pathways by preventing the activation (via phosphorylation) of the superfamily of mitogen-activated protein kinase kinases (including the MKKs (MAP kinase kinases) and IKKbeta (Ikappa B kinase beta ) (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 NFkappa 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 NFkappa 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Expressing YopJ Respond Inefficiently to alpha  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 alpha  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 alpha  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 alpha  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).


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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 alpha  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 alpha  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-MATalpha 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-MATalpha cells is ~100%, whereas mating BY4741-MATa cells containing wild type YopJ with BY4742-MATalpha 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-MATalpha 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-MATalpha cells (Fig. 1C). Likewise, BY4741-MATa cells expressing the catalytically inactive form of YopJ (J(C/A)) mated with excess BY4742-MATalpha 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-MATalpha 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 alpha  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 alpha  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 alpha  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 sst1Delta and sst2Delta strains and assayed for their ability to form halos in response to alpha  factor. The sst2Delta 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 alpha  factor (Fig. 2A). In contrast, sst2Delta cells expressing wild type YopJ failed to form superhalos (Fig. 2A). Similar results were observed with the sst1Delta strain (data not shown). These results indicate that the block by the Yersinia effector YopJ was upstream of the transcriptionally induced adaptive response.


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Fig. 2.   YopJ inhibits the pheromone-induced MAP kinase pathway upstream of Fus3p phosphorylation. A, sst2Delta cells expressing YopJ do not form superhalos. The formation of superhalos is observed in sst2Delta 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 alpha  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 alpha  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 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.


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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 sln1Delta 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/sln1Delta diploids, which produce tetrads with a ratio of 2:2 live:dead segregants (26). The lethal phenotype of sln1Delta can be rescued by mutations in the downstream kinases Ssk2p, Pbs2p, or Hog1p (24). SLN1/sln1Delta 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:sln1Delta 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/sln1Delta 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 sln1Delta segregants (Table I). As expected, the four colonies from tetrads of the SLN1:sln1Delta 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:sln1Delta 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 sln1Delta cells. The rescued lethality of sln1Delta 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.

                              
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Table I
YopJ rescues lethality of a sln1Delta mutation
SLN1/sln1Delta 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.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.


    ACKNOWLEDGEMENTS

We thank, for their expert assistance, Janet Thornton with yeast protocols and Alisha Tizenor with figures. We thank Renee Chosed and Sohini Mukherjee for critical reading of the manuscript. We are grateful to Jack Dixon and Ron Butow for their support and encouragement.

    FOOTNOTES

* 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.

Dagger Supported by the Endowed Scholars Program at University of Texas Southwestern Medical Center. To whom correspondence should be addressed: Dept. of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Rm. NA5-320, Dallas, TX 75390-9148. Tel.: 214-648-1685; Fax: 214-648-1488; E-mail: kimorth@hamon.swmed.edu.

Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209905200

    ABBREVIATIONS

The abbreviations used are: Yop, Yersinia outer protein; MAP, mitogen-activated protein; MAPK, MAP kinase; HOG, high osmolarity growth; J, YopJ; J(C/A), YopJ(C/A); Sorb, sorbitol; Gal, galactose.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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