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Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

Dinér P, Veide Vilg J, Kjellén J, Migdal I, Andersson T, Gebbia M, Giaever G, Nislow C, Hohmann S, Wysocki R, Tamás MJ, Grøtli M - PLoS ONE (2011)

Bottom Line: These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo.Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress.Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

View Article: PubMed Central - PubMed

Affiliation: Medicinal Chemistry, Department of Chemistry, University of Gothenburg, Göteborg, Sweden.

ABSTRACT
The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

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In vivo activity and selectivity of inhibitor                            4a.(A) Nuclear accumulation of Hog1 is prevented in the presence of                                4a. A plasmid encoding a Hog1-GFP fusion protein was                            transformed into the hog1Δ mutant, and living cells                            were analyzed by fluorescence microscopy for Hog1 localization. Cells                            were either untreated or exposed to osmotic stress (0.8 M sorbitol).                            Inhibitor (5 µM) was added to cells 15 minutes before osmotic                            stress was applied. (B) Hog1 dephosphorylation is prevented in the                            presence of 4a. Hog1 phosphorylation was monitored in cells                            exposed to osmotic stress (0.8 M sorbitol) by Western blot analysis                            using an antibody specific to dually phosphorylated p38 MAPK, and an                            anti-Hog1 antibody was used as a control. Inhibitor (5 µM) was                            added to cells 15 minutes before osmotic stress was applied. (C)                            Inhibition of Hog1-dependent gene expression. Exponentially growing                            cells harboring the STL1-lacZ reporter                            were exposed to osmotic stress (0.8 M sorbitol) and assayed for                            β-galactosidase activity as described in the Experimental section.                            Induced expression of the STL1 gene by osmotic stress                            required Hog1 but no other signal transduction pathways. Inhibitor was                            added to cells at the indicated concentrations 10 minutes before osmotic                            stress was applied. The results are the average of three independent                            experiments and the error bars represent standard deviation (s.d.). (D)                                4a is selective for Hog1 inhibition since it does not                            affect the Fus3/Kss1 MAPKs. Exponentially growing cells harboring the                                FUS1-lacZ reporter were exposed to                            α-factor (10 µM) and assayed for β-galactosidase activity                            as described above. Induced expression of the FUS1 gene                            in response to α-factor required Fus3 and Kss1 but was independent                            of Hog1 [38].
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pone-0020012-g007: In vivo activity and selectivity of inhibitor 4a.(A) Nuclear accumulation of Hog1 is prevented in the presence of 4a. A plasmid encoding a Hog1-GFP fusion protein was transformed into the hog1Δ mutant, and living cells were analyzed by fluorescence microscopy for Hog1 localization. Cells were either untreated or exposed to osmotic stress (0.8 M sorbitol). Inhibitor (5 µM) was added to cells 15 minutes before osmotic stress was applied. (B) Hog1 dephosphorylation is prevented in the presence of 4a. Hog1 phosphorylation was monitored in cells exposed to osmotic stress (0.8 M sorbitol) by Western blot analysis using an antibody specific to dually phosphorylated p38 MAPK, and an anti-Hog1 antibody was used as a control. Inhibitor (5 µM) was added to cells 15 minutes before osmotic stress was applied. (C) Inhibition of Hog1-dependent gene expression. Exponentially growing cells harboring the STL1-lacZ reporter were exposed to osmotic stress (0.8 M sorbitol) and assayed for β-galactosidase activity as described in the Experimental section. Induced expression of the STL1 gene by osmotic stress required Hog1 but no other signal transduction pathways. Inhibitor was added to cells at the indicated concentrations 10 minutes before osmotic stress was applied. The results are the average of three independent experiments and the error bars represent standard deviation (s.d.). (D) 4a is selective for Hog1 inhibition since it does not affect the Fus3/Kss1 MAPKs. Exponentially growing cells harboring the FUS1-lacZ reporter were exposed to α-factor (10 µM) and assayed for β-galactosidase activity as described above. Induced expression of the FUS1 gene in response to α-factor required Fus3 and Kss1 but was independent of Hog1 [38].

Mentions: To characterize the action of these inhibitors in vivo, we first monitored how 4a affects Hog1 translocation into the nucleus upon osmotic stress, a process that requires Hog1 kinase activity [9]. To do this, we transformed hog1Δ cells with a plasmid expressing Hog1 fused to GFP (green fluorescent protein) under the control of the endogenous HOG1 promoter and monitored the Hog1-GFP fusion protein by fluorescence microscopy. Exposing cells to 0.8 M sorbitol triggered a rapid accumulation of Hog1-GFP in the nucleus (Figure 7A). In contrast, pre-treating cells with 5 µM of 4a prior to osmotic stress exposure prevented nuclear accumulation of Hog1-GFP in the majority of the cells, suggesting that 4a inhibits Hog1 kinase activity.


Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

Dinér P, Veide Vilg J, Kjellén J, Migdal I, Andersson T, Gebbia M, Giaever G, Nislow C, Hohmann S, Wysocki R, Tamás MJ, Grøtli M - PLoS ONE (2011)

In vivo activity and selectivity of inhibitor                            4a.(A) Nuclear accumulation of Hog1 is prevented in the presence of                                4a. A plasmid encoding a Hog1-GFP fusion protein was                            transformed into the hog1Δ mutant, and living cells                            were analyzed by fluorescence microscopy for Hog1 localization. Cells                            were either untreated or exposed to osmotic stress (0.8 M sorbitol).                            Inhibitor (5 µM) was added to cells 15 minutes before osmotic                            stress was applied. (B) Hog1 dephosphorylation is prevented in the                            presence of 4a. Hog1 phosphorylation was monitored in cells                            exposed to osmotic stress (0.8 M sorbitol) by Western blot analysis                            using an antibody specific to dually phosphorylated p38 MAPK, and an                            anti-Hog1 antibody was used as a control. Inhibitor (5 µM) was                            added to cells 15 minutes before osmotic stress was applied. (C)                            Inhibition of Hog1-dependent gene expression. Exponentially growing                            cells harboring the STL1-lacZ reporter                            were exposed to osmotic stress (0.8 M sorbitol) and assayed for                            β-galactosidase activity as described in the Experimental section.                            Induced expression of the STL1 gene by osmotic stress                            required Hog1 but no other signal transduction pathways. Inhibitor was                            added to cells at the indicated concentrations 10 minutes before osmotic                            stress was applied. The results are the average of three independent                            experiments and the error bars represent standard deviation (s.d.). (D)                                4a is selective for Hog1 inhibition since it does not                            affect the Fus3/Kss1 MAPKs. Exponentially growing cells harboring the                                FUS1-lacZ reporter were exposed to                            α-factor (10 µM) and assayed for β-galactosidase activity                            as described above. Induced expression of the FUS1 gene                            in response to α-factor required Fus3 and Kss1 but was independent                            of Hog1 [38].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104989&req=5

pone-0020012-g007: In vivo activity and selectivity of inhibitor 4a.(A) Nuclear accumulation of Hog1 is prevented in the presence of 4a. A plasmid encoding a Hog1-GFP fusion protein was transformed into the hog1Δ mutant, and living cells were analyzed by fluorescence microscopy for Hog1 localization. Cells were either untreated or exposed to osmotic stress (0.8 M sorbitol). Inhibitor (5 µM) was added to cells 15 minutes before osmotic stress was applied. (B) Hog1 dephosphorylation is prevented in the presence of 4a. Hog1 phosphorylation was monitored in cells exposed to osmotic stress (0.8 M sorbitol) by Western blot analysis using an antibody specific to dually phosphorylated p38 MAPK, and an anti-Hog1 antibody was used as a control. Inhibitor (5 µM) was added to cells 15 minutes before osmotic stress was applied. (C) Inhibition of Hog1-dependent gene expression. Exponentially growing cells harboring the STL1-lacZ reporter were exposed to osmotic stress (0.8 M sorbitol) and assayed for β-galactosidase activity as described in the Experimental section. Induced expression of the STL1 gene by osmotic stress required Hog1 but no other signal transduction pathways. Inhibitor was added to cells at the indicated concentrations 10 minutes before osmotic stress was applied. The results are the average of three independent experiments and the error bars represent standard deviation (s.d.). (D) 4a is selective for Hog1 inhibition since it does not affect the Fus3/Kss1 MAPKs. Exponentially growing cells harboring the FUS1-lacZ reporter were exposed to α-factor (10 µM) and assayed for β-galactosidase activity as described above. Induced expression of the FUS1 gene in response to α-factor required Fus3 and Kss1 but was independent of Hog1 [38].
Mentions: To characterize the action of these inhibitors in vivo, we first monitored how 4a affects Hog1 translocation into the nucleus upon osmotic stress, a process that requires Hog1 kinase activity [9]. To do this, we transformed hog1Δ cells with a plasmid expressing Hog1 fused to GFP (green fluorescent protein) under the control of the endogenous HOG1 promoter and monitored the Hog1-GFP fusion protein by fluorescence microscopy. Exposing cells to 0.8 M sorbitol triggered a rapid accumulation of Hog1-GFP in the nucleus (Figure 7A). In contrast, pre-treating cells with 5 µM of 4a prior to osmotic stress exposure prevented nuclear accumulation of Hog1-GFP in the majority of the cells, suggesting that 4a inhibits Hog1 kinase activity.

Bottom Line: These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo.Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress.Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

View Article: PubMed Central - PubMed

Affiliation: Medicinal Chemistry, Department of Chemistry, University of Gothenburg, Göteborg, Sweden.

ABSTRACT
The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

Show MeSH
Related in: MedlinePlus