A Hog1 Inhibitor for Analyzing Kinase Signaling Figure 7. 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 hog1D 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 mM) 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 mM) 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 b-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 a-factor (10 mM) and assayed for b-galactosidase activity as described above. Induced expression of the FUS1 gene in response to a-factor required Fus3 and Kss1 but was independent of Hog1 [38]. doi:10.1371/journal.pone.0020012.g007 used); a significant overlap between the inhibitor-sensitive and negative genetic interaction gene-sets would indicate whether the inhibitor targets a particular MAPK. Importantly, we found a statistically significant overlap between those gene-sets for Hog1 (p = 0.00127; Table S1) but not for any of the other MAPKs (p.0.05), suggesting that 4a is selectively inhibiting Hog1. To test selectivity in a different way, we exposed cells to a- factor, a condition that activates the Fus3 and Kss1 MAPKs, and monitored expression of the FUS1-lacZ reporter gene as a read-out for Fus3 and Kss1 kinase activities (Figure 6D). Exposing cells to a- factor resulted in strong activation of FUS1-lacZ expression. Pre- treating cells with 4a did not reduce FUS1-lacZ expression in response to a-factor. These data suggest that 4a does not inhibit the Fus3 and Kss1 MAPKs, at least not at concentrations that fully inhibit Hog1, as judged by the lack of osmotic stress-induced STL1-lacZ expression in the presence of 4a (Figure 7C). Hence, 4a appears to selectively target the Hog1 kinase for inhibition. Use of the novel inhibitors to elucidate the G1 checkpoint function of Hog1 We recently demonstrated that yeast cells lacking Hog1 are highly As(III) sensitive [14] and exhibit a persistent arrest in the G1 phase of the cell cycle due to accumulation of the cell cycle- dependent kinase inhibitor Sic1 [14,20]. In contrast to previous findings showing the involvement of Hog1 in promoting hyperosmotic stress-induced G1 checkpoint arrest (18), our data suggested that Hog1 is not required for cell cycle delay in G1 in the presence of As(III), but instead plays a crucial role in cellular recovery from As(III)-induced G1 cell cycle arrest. However, in response to As(III) stress, Hog1 fulfills also other functions unrelated to cell cycle regulation, like inhibition of As(III) influx by affecting the transport activity of the Fps1 glycerol channel [20]. Thus, by using the HOG1 deletion mutant (hog1D) only, we could not pin-point neither time nor mechanism of Hog1 action during G1 cell cycle delay and recovery. Nevertheless, we showed with an analogue-sensitive mutant of Hog1 (Hog1-as) and 1-NM- PP1 inhibitor, that lack of Hog1 kinase activity is responsible for persistent G1 arrest in the presence of As(III) [20]. However, the results of the cell cycle experiments with the Hog1-as allele were confounded by the fact that in the absence of 1-NM-PP1, the Hog1-as strain displayed much longer As(III)-induced G1 delay than wild-type cells [14,20]. 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 a-factor arrest in the presence of 0.5 mM As(III) by monitoring the level of phosphorylated Hog1 (Figure 8). We found that Hog1 phosphorylation peaked within the first 30 minutes of As(III) exposure as previously shown for asynchro- PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20012