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Characterization of new substrates targeted by Yersinia tyrosine phosphatase YopH.

de la Puerta ML, Trinidad AG, del Carmen Rodríguez M, Bogetz J, Sánchez Crespo M, Mustelin T, Alonso A, Bayón Y - PLoS ONE (2009)

Bottom Line: In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions.Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase.These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, Valladolid, Spain.

ABSTRACT
YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

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Pull-down assays with different deletion mutants of YopH.A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.
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pone-0004431-g003: Pull-down assays with different deletion mutants of YopH.A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.

Mentions: YopH binds tyrosine phosphorylated proteins through two domains, the N-terminal domain (amino acids 1–129) and the catalytic domain (amino acids 193–468), which presents two interaction sites, the catalytic pocket and a second site on the opposite side of this domain [30]. Between these sites, there is a Pro-rich region (PRR) that may bind to SH3 domains. To analyze the interaction of YopH with the proteins studied here and based on this domain structure and on the biochemical data related to YopH substrate interaction, we generated four deletion mutants of YopH: N129 (amino acids 1–129), N220 (amino acids 1–220), C339 (amino acids 129–468), and C277 (amino acids 193–468) (Fig. 3 A). These peptides were used as GST-fusion proteins in pull-down assays using lysates from pervanadate stimulated cells. Using this approach, we observed that Fyb, a well-known YopH substrate, binds YopH through the N129 and the catalytic domain. Gab1 and Gab2 also bind through both domains but they do not bind to C227, indicating that, for binding, they required additional amino acids present in the PRR. Taking into account that Gab adaptors lack SH3 domain, association would imply another mechanism not determined yet. In the case of Vav, p85, and Lck, the stronger association was observed with the deletion mutant C339, which contains the catalytic domain and the Pro-rich region. All these proteins contain SH3 domains that might interact with the YopH PRR, thus explaining why removal of this region abrogates the interaction and why the N220 construct binds slightly to these proteins while the N129 domain shows no binding at all. According to the data shown in Fig. 3B, the proteins that bind to YopH can be divided into three groups: i) proteins that interact with both the catalytic domain and the N129 domain (Fyb), ii) proteins that interact with the extended catalytic domain, which includes PRR domain and the N129 domain (Gab1 and Gab2), and iii) proteins that only bind to the extended catalytic domain C339 (Lck, p85, and Vav).


Characterization of new substrates targeted by Yersinia tyrosine phosphatase YopH.

de la Puerta ML, Trinidad AG, del Carmen Rodríguez M, Bogetz J, Sánchez Crespo M, Mustelin T, Alonso A, Bayón Y - PLoS ONE (2009)

Pull-down assays with different deletion mutants of YopH.A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637541&req=5

pone-0004431-g003: Pull-down assays with different deletion mutants of YopH.A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.
Mentions: YopH binds tyrosine phosphorylated proteins through two domains, the N-terminal domain (amino acids 1–129) and the catalytic domain (amino acids 193–468), which presents two interaction sites, the catalytic pocket and a second site on the opposite side of this domain [30]. Between these sites, there is a Pro-rich region (PRR) that may bind to SH3 domains. To analyze the interaction of YopH with the proteins studied here and based on this domain structure and on the biochemical data related to YopH substrate interaction, we generated four deletion mutants of YopH: N129 (amino acids 1–129), N220 (amino acids 1–220), C339 (amino acids 129–468), and C277 (amino acids 193–468) (Fig. 3 A). These peptides were used as GST-fusion proteins in pull-down assays using lysates from pervanadate stimulated cells. Using this approach, we observed that Fyb, a well-known YopH substrate, binds YopH through the N129 and the catalytic domain. Gab1 and Gab2 also bind through both domains but they do not bind to C227, indicating that, for binding, they required additional amino acids present in the PRR. Taking into account that Gab adaptors lack SH3 domain, association would imply another mechanism not determined yet. In the case of Vav, p85, and Lck, the stronger association was observed with the deletion mutant C339, which contains the catalytic domain and the Pro-rich region. All these proteins contain SH3 domains that might interact with the YopH PRR, thus explaining why removal of this region abrogates the interaction and why the N220 construct binds slightly to these proteins while the N129 domain shows no binding at all. According to the data shown in Fig. 3B, the proteins that bind to YopH can be divided into three groups: i) proteins that interact with both the catalytic domain and the N129 domain (Fyb), ii) proteins that interact with the extended catalytic domain, which includes PRR domain and the N129 domain (Gab1 and Gab2), and iii) proteins that only bind to the extended catalytic domain C339 (Lck, p85, and Vav).

Bottom Line: In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions.Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase.These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, Valladolid, Spain.

ABSTRACT
YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

Show MeSH
Related in: MedlinePlus