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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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Hog1 kinase activity is required to relieve As(III)-induced                                G1 checkpoint arrest.(A) Kinetics of Hog1 activation during G1 checkpoint                            adaptation in response to As(III) stress. Hog1 phosphorylation was                            monitored as in Figure                                6. (B) HOG1 deletion or the addition of                                4b resulted in persistent G1 arrest in the                            presence of As(III). (C) As(III)-induced G1 checkpoint delay                            can be prolonged by addition of 4b until just before onset                            of the S phase. (D) Removal of 4b quickly relieves                                G1 arrest. Wild-type (W303-1A) and the isogenic                                HOG1 deletion mutant (hog1Δ)                            were synchronized in G1 with 5 µM α-factor and                            released in fresh medium in the presence or absence of 0.5 mM sodium                            arsenite [As(III)]. 4b (1 µM) was added as                            indicated. After washing out the inhibitor (in 7D), the cells were                            resuspended in fresh medium containing 0.5 mM As(III). The percentage of                            cells that remained in G1 was determined by the                            α-factor-nocodazole trap assay.
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pone-0020012-g008: Hog1 kinase activity is required to relieve As(III)-induced G1 checkpoint arrest.(A) Kinetics of Hog1 activation during G1 checkpoint adaptation in response to As(III) stress. Hog1 phosphorylation was monitored as in Figure 6. (B) HOG1 deletion or the addition of 4b resulted in persistent G1 arrest in the presence of As(III). (C) As(III)-induced G1 checkpoint delay can be prolonged by addition of 4b until just before onset of the S phase. (D) Removal of 4b quickly relieves G1 arrest. Wild-type (W303-1A) and the isogenic HOG1 deletion mutant (hog1Δ) were synchronized in G1 with 5 µM α-factor and released in fresh medium in the presence or absence of 0.5 mM sodium arsenite [As(III)]. 4b (1 µM) was added as indicated. After washing out the inhibitor (in 7D), the cells were resuspended in fresh medium containing 0.5 mM As(III). The percentage of cells that remained in G1 was determined by the α-factor-nocodazole trap assay.

Mentions: Having a novel and potent inhibitor of wild-type Hog1 at hand, we wanted to identify the execution point of Hog1 function in regulating the G1/S checkpoint in the presence of As(III). First, we determined the kinetics of Hog1 activation in G1-synchronized wild-type cells released from α-factor arrest in the presence of 0.5 mM As(III) by monitoring the level of phosphorylated Hog1 (Figure 8).


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)

Hog1 kinase activity is required to relieve As(III)-induced                                G1 checkpoint arrest.(A) Kinetics of Hog1 activation during G1 checkpoint                            adaptation in response to As(III) stress. Hog1 phosphorylation was                            monitored as in Figure                                6. (B) HOG1 deletion or the addition of                                4b resulted in persistent G1 arrest in the                            presence of As(III). (C) As(III)-induced G1 checkpoint delay                            can be prolonged by addition of 4b until just before onset                            of the S phase. (D) Removal of 4b quickly relieves                                G1 arrest. Wild-type (W303-1A) and the isogenic                                HOG1 deletion mutant (hog1Δ)                            were synchronized in G1 with 5 µM α-factor and                            released in fresh medium in the presence or absence of 0.5 mM sodium                            arsenite [As(III)]. 4b (1 µM) was added as                            indicated. After washing out the inhibitor (in 7D), the cells were                            resuspended in fresh medium containing 0.5 mM As(III). The percentage of                            cells that remained in G1 was determined by the                            α-factor-nocodazole trap assay.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104989&req=5

pone-0020012-g008: Hog1 kinase activity is required to relieve As(III)-induced G1 checkpoint arrest.(A) Kinetics of Hog1 activation during G1 checkpoint adaptation in response to As(III) stress. Hog1 phosphorylation was monitored as in Figure 6. (B) HOG1 deletion or the addition of 4b resulted in persistent G1 arrest in the presence of As(III). (C) As(III)-induced G1 checkpoint delay can be prolonged by addition of 4b until just before onset of the S phase. (D) Removal of 4b quickly relieves G1 arrest. Wild-type (W303-1A) and the isogenic HOG1 deletion mutant (hog1Δ) were synchronized in G1 with 5 µM α-factor and released in fresh medium in the presence or absence of 0.5 mM sodium arsenite [As(III)]. 4b (1 µM) was added as indicated. After washing out the inhibitor (in 7D), the cells were resuspended in fresh medium containing 0.5 mM As(III). The percentage of cells that remained in G1 was determined by the α-factor-nocodazole trap assay.
Mentions: Having a novel and potent inhibitor of wild-type Hog1 at hand, we wanted to identify the execution point of Hog1 function in regulating the G1/S checkpoint in the presence of As(III). First, we determined the kinetics of Hog1 activation in G1-synchronized wild-type cells released from α-factor arrest in the presence of 0.5 mM As(III) by monitoring the level of phosphorylated Hog1 (Figure 8).

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