Limits...
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.

Show MeSH

Related in: MedlinePlus

IPMK plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. 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) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. 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. (C) Western blot results of ES10 and ES5 from B 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 for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3750030&req=5

pone-0071015-g005: IPMK plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. 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) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. 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. (C) Western blot results of ES10 and ES5 from B 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 for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.

Mentions: In search for the non-canonical host kinase(s) involved in SopB-mediated Akt activation, we performed an esiRNA screen of 778 mammalian kinases. The results of our screen are shown in Table 1. Many of the kinases identified have been previously implicated in regulating Akt activity (highlighted in yellow; see references), supporting the validity of our screen. The top ‘hit’ in our screen was inositol polyphosphate multikinase (IPMK). SopB-mediated Akt activation was impaired by as much as 91% when IPMK expression was targeted by esiRNA (Table 1). We also targeted expression of IPMK using siRNA for comparison. siRNA-mediated silencing of IPMK expression similarly impaired Akt activation, providing additional evidence for its role in HeLa cells (Figure 5A).


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)

IPMK plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. 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) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. 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. (C) Western blot results of ES10 and ES5 from B 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 for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750030&req=5

pone-0071015-g005: IPMK plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type S. Typhimurium for 30 min. As a control, cells were uninfected. 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) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant S. Typhimurium for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. 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. (C) Western blot results of ES10 and ES5 from B 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 for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.
Mentions: In search for the non-canonical host kinase(s) involved in SopB-mediated Akt activation, we performed an esiRNA screen of 778 mammalian kinases. The results of our screen are shown in Table 1. Many of the kinases identified have been previously implicated in regulating Akt activity (highlighted in yellow; see references), supporting the validity of our screen. The top ‘hit’ in our screen was inositol polyphosphate multikinase (IPMK). SopB-mediated Akt activation was impaired by as much as 91% when IPMK expression was targeted by esiRNA (Table 1). We also targeted expression of IPMK using siRNA for comparison. siRNA-mediated silencing of IPMK expression similarly impaired Akt activation, providing additional evidence for its role in HeLa cells (Figure 5A).

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