Figure 6. Uptake of inhibitors by yeast cells. (A) A lawn of wild- type yeast cells (BY4741 strain) was spread on solid medium in the absence (control) or presence of osmotic stress (1.5 M sorbitol), and filter discs containing various concentrations of 4a, 4b, and SB203580 were placed on top of the lawn. Inhibition of Hog1 activity could be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress (1.5 M sorbitol). No such halo was visible on control plates in the absence of osmotic stress. Plates were incubated for 48 hours at 30uC. (B) 4a improves growth of PBS2 overexpressing cells. Wild-type and hog1D cells (BY4743 strain) were transformed with an empty plasmid or plasmid overexpressing PBS2. Cells were grown in a microcultivation system in the absence or presence of inhibitor as indicated. doi:10.1371/journal.pone.0020012.g006 allosteric binding site. All these commercially available inhibitors are considered to be important tools for the study of p38 MAP kinase function both in vivo and in vitro, and should be potentially useful for studying Hog1 signaling. However, like SB203580 none of these compounds demonstrated any growth inhibition in the halo assay (Figure S2). The results clearly show that kinase inhibitors developed to target human kinases are not automatically useful for experiments in yeast cells although the homology between the human target and the yeast counterpart is high. Possible explanations for this difference between our pyridinyl- triazole-based inhibitors and the commercial available A Hog1 Inhibitor for Analyzing Kinase Signaling pyridinylimidazole-based inhibitors could be that the pyridinyl- triazole-based inhibitors are recognized by transport proteins that allow efficient uptake or/and pyridinylimidazole-based inhibitors are recognized by transport proteins that allow efficient export of these molecules. To sum up, 4a and 4b are efficiently taken up by cells and can be used for inhibiting Hog1 activity in vivo. In vivo activity 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 hog1D 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 mM 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. Next, we assessed Hog1 activation by monitoring its phosphor- ylation state after osmotic stress by using antibodies that specifically recognize phosphorylated Hog1. Hog1 was rapidly and transiently phosphorylated in response to osmotic stress (0.8 M sorbitol); phosphorylated Hog1 was visible at the 15 minute time point while Hog1 was effectively dephosphorylated after 30 minutes (Figure 7B). Pre-treating cells with 1 mM or 5 mM of 4a prior to osmotic stress exposure resulted in sustained Hog1 phosphorylation (Figure 6B and data not shown). Previous studies have demonstrated that Hog1 kinase activity is required to promote its own dephosphorylation [8,9]. Consistently, a kinase- dead version of Hog1 showed sustained phosphorylation in response to osmotic stress ([9,35] and Figure S3). Hence, sustained Hog1 phosphorylation in the presence of 4a indicates that this compound inhibits Hog1 kinase activity. Finally, we monitored expression of the STL1 gene whose induction during osmotic stress is fully dependent on Hog1 activity [36]. For this, we exposed cells that harbor the STL1 promoter fused to the lacZ reporter gene (STL1-lacZ) to osmotic stress and determined b-galactosidase activity as a read-out for Hog1 activity (Figure 7C). Osmotic stress triggered a strong activation of STL1- lacZ expression. However, pre-treating cells with 1 mM of 4a prior to osmotic stress prevented induction of STL1-lacZ expression, indicating that 4a interferes with Hog1 activity. Treating cells with 4b caused a similar inhibition of STL1-lacZ expression (Figure S4). Collectively, these short-term (minutes to hours) assays (Figuress 7A–C) together with long-term (2–3 days; Figure 6) growth assays clearly show that 4a (and also 4b) effectively inhibits Hog1 activity in vivo. Selectivity Yeast has several MAPK pathways that are activated by various stimuli, and cross-talk between these pathways exists [37,38]. Therefore, inhibitors that act on Hog1 should not target other yeast MAPKs. In order to test selectivity of the novel inhibitors, we did chemical genetic profiling of the yeast deletion mutant collection and scored for mutants with reduced growth in the presence of 500 mM 4a. This screen identified 32 mutants that were at least 2-fold less abundant than the wild-type at the end of the experiment (Table S1), supporting the notion that 4a does not cause a general toxicity to cells. We next compared the set of inhibitor-sensitive mutants to sets of genes/mutants that show a negative genetic interaction with either of the five yeast MAPKs (Hog1, Slt2, Kss1, Fus3, Smk1) (interaction data from [39,40] PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20012