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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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Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 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. The p-values from one-way ANOVA analysis are shown.
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pone-0071015-g003: Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 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. The p-values from one-way ANOVA analysis are shown.

Mentions: Class II PI3-kinases are less sensitive to PI3-kinase inhibitors [31] and Class II-mediated Akt activation has been shown to be required for glucose-stimulated insulin secretion [32]. Therefore, we targeted expression of all Class II PI3-kinase isoforms using siRNA and analyzed Akt activation by Salmonella. Knockdown of PI3K-C2β led to a significant decrease in SopB-dependent Akt activation, while that of other Class II PI3-kinase isoforms was without effect (Figure 3). Though silencing PI3K-C2β reduced Akt phosphorylation, this kinase seemed to only have a partial role in Akt activation in HeLa cells, indicating that other kinases are also involved.


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)

Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 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. The p-values from one-way ANOVA analysis are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750030&req=5

pone-0071015-g003: Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type S. Typhimurium for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 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. The p-values from one-way ANOVA analysis are shown.
Mentions: Class II PI3-kinases are less sensitive to PI3-kinase inhibitors [31] and Class II-mediated Akt activation has been shown to be required for glucose-stimulated insulin secretion [32]. Therefore, we targeted expression of all Class II PI3-kinase isoforms using siRNA and analyzed Akt activation by Salmonella. Knockdown of PI3K-C2β led to a significant decrease in SopB-dependent Akt activation, while that of other Class II PI3-kinase isoforms was without effect (Figure 3). Though silencing PI3K-C2β reduced Akt phosphorylation, this kinase seemed to only have a partial role in Akt activation in HeLa cells, indicating that other kinases are also involved.

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