A Hog1 Inhibitor for Analyzing Kinase Signaling Figure 8. 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 (hog1D) were synchronized in G1 with 5 mM a-factor and released in fresh medium in the presence or absence of 0.5 mM sodium arsenite [As(III)]. 4b (1 mM) 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 a-factor-nocodazole trap assay. doi:10.1371/journal.pone.0020012.g008 nously growing cells [14]. Interestingly, Hog1 phosphorylation and activation was maintained for up to 180 minutes from a-factor arrest release in the presence of 0.5 mM As(III), though it gradually decreased from 90 minutes (Figure 8A). More impor- tantly, the a -factor-nocodazole trap assay (to determine the number of G1 cells versus post-G1 (S/G2/M) cells) performed on the same culture as above (Figure 8A), revealed that in the presence of As(III), wild-type cells started to enter S phase 80– 100 min from a-factor release (Figure 8B). Thus, when cells are exposed to As(III) in G1, Hog1 is phosphorylated and hence activated not only immediately after As(III) addition but also at much later time points during the recovery from G1 arrest. Next, we determined the effect of 4a or 4b on Hog1-dependent cell cycle progression during As(III) exposure. Wild-type cells were synchronized in G1 by a-factor, released in fresh medium containing 0.5 mM As(III) in the presence of 1 mM inhibitor, and analyzed by the a-factor-nocodazole trap assay (Figures 8B and S5). The addition of 4a or 4b resulted in a persistent G1 delay, which was indistinguishable from that of hog1D cells. Importantly, neither of the inhibitors had any effect on the G1/S cell cycle transition in the absence of As(III) (Figures 8B and S5). Knowing that Hog1 also has non-cell-cycle related functions during As(III) stress [14] and having established the kinetics of Hog1 activation in G1 upon As(III) addition (Figure 8A), we took advantage of our inhibitors to show the execution role of Hog1 in timely entry into the S phase. G1-synchronized cells were resuspended in fresh medium in the presence of As(III), and inhibitor was added 0, 40, 80, or 100 minutes after release (Figures 8C and S5). Inhibition of Hog1 kinase activity prevented entry into the S phase of any cell that still remained in G1 at the time of inhibitor addition. In a reciprocal experiment, G1-synchronized cells were released in the presence of As(III) and inhibitor to execute a persistent G1 arrest followed by washing out the inhibitor at the 120 minute time point (Figures 8D and S5). After removal of inhibitor from the medium, cells gradually entered the S phase despite the presence of As(III). Taken to- gether, our new data obtained with inhibitors 4a and 4b strongly support the notion that Hog1 kinase activity is specifically required for the execution of G1 arrest release when the cell adapts to the presence of As(III) and is ready to resume the cell cycle. In conclusion, potent and fast acting inhibitors targeting the wild-type version of the yeast MAPK Hog1 have been developed. Our novel inhibitors (4a and 4b) are structurally related to SB203580 (Figures 1 and 2), a commercially available inhibitor of p38a, p38b, and AKT/PKB. Importantly, in contrast to SB203580, both of our inhibitors enter yeast cells efficiently (Figures 6, S1, S2, S6 and S7), allowing exploration of rapid signal transduction events in living cells. Indeed, 4a and 4b inhibit Hog1 activity to similar levels, both in vitro and in vivo (Figures 5, 7, 8, S4 and S5). The yeast HOG pathway is one of the most studied MAPK pathways, and the availability of these novel inhibitors for rapid and selective inactivation of Hog1 will be essential to dissect novel aspects of the signaling process as well as to define novel biological roles for Hog1. Indeed, in this paper we successfully used these inhibitors to pin-point the time of Hog1 action during recovery from G1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during As(III) stress (Figures 8 and S5). Moreover, combination of 4a and 4b together with the as-kinase inhibitor 1-NM-PP1 opens the possibility to simultaneously modulate the activities of two MAPK pathways for studying signaling cross-talk. Finally, these com- pounds may also prove of value for studying Hog1 signaling in PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20012