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Multiple host kinases contribute to Akt activation during Salmonella infection.

Roppenser B, Kwon H, Canadien V, Xu R, Devreotes PN, Grinstein S, Brumell JH - PLoS ONE (2013)

Bottom Line: SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche.In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell.Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

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SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔsopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).
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pone-0071015-g002: SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔsopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).

Mentions: PI3-kinases, which phosphorylate PIs at the 3-position of the inositol ring, can be divided into three main classes depending on their substrate specificity [30]. We tested all 3 classes of PI3-kinases for their ability to influence SopB-mediated Akt activation. Class I PI3-kinases can be effectively inhibited by the PI3-kinase inhibitors LY294002 and Wortmannin. HeLa cells were treated with varying concentrations of LY294002 (Figure 2) or Wortmannin (Figure S2), then infected with wild type Salmonella. Akt activation was measured in cell lysates with anti-pAkt-Ser473 antibodies. Addition of low concentrations of LY294002 did not significantly impair SopB-mediated Akt activation during infection. However, higher concentrations, e.g. 50-100 µM, LY294002 significantly reduced the Akt activation by approximately 25% compared to the control. Wortmannin showed a very similar impact on Akt activation. In contrast, Akt activation by EGF was abolished by low concentrations of either inhibitor. This indicates that Class I PI3-kinases, which are normally inhibited by LY294002 or Wortmannin, play a minor role in the SopB-mediated Akt activation in HeLa cells.


Multiple host kinases contribute to Akt activation during Salmonella infection.

Roppenser B, Kwon H, Canadien V, Xu R, Devreotes PN, Grinstein S, Brumell JH - PLoS ONE (2013)

SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔsopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750030&req=5

pone-0071015-g002: SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type S. Typhimurium for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with ΔsopB mutant S. Typhimurium for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type S. Typhimurium or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).
Mentions: PI3-kinases, which phosphorylate PIs at the 3-position of the inositol ring, can be divided into three main classes depending on their substrate specificity [30]. We tested all 3 classes of PI3-kinases for their ability to influence SopB-mediated Akt activation. Class I PI3-kinases can be effectively inhibited by the PI3-kinase inhibitors LY294002 and Wortmannin. HeLa cells were treated with varying concentrations of LY294002 (Figure 2) or Wortmannin (Figure S2), then infected with wild type Salmonella. Akt activation was measured in cell lysates with anti-pAkt-Ser473 antibodies. Addition of low concentrations of LY294002 did not significantly impair SopB-mediated Akt activation during infection. However, higher concentrations, e.g. 50-100 µM, LY294002 significantly reduced the Akt activation by approximately 25% compared to the control. Wortmannin showed a very similar impact on Akt activation. In contrast, Akt activation by EGF was abolished by low concentrations of either inhibitor. This indicates that Class I PI3-kinases, which are normally inhibited by LY294002 or Wortmannin, play a minor role in the SopB-mediated Akt activation in HeLa cells.

Bottom Line: SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche.In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell.Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Show MeSH
Related in: MedlinePlus