Ase activity of Ssk2p [26,31]. Osmotic Mirin site stress induces a rapid disassembly of the actin cytoskeleton [31,33]. Actin cytoskeleton disassembly induces Ssk2p to translocate from the cytosol to the septin cytoskeleton of the bud neck [26,31,32]. Therefore, we asked whether actin disassembly would activate the Ssk2p to activate the HOG pathway. Lat B was used to induce rapid and complete disassembly of the actin cytoskeleton in strains BY4741 and ste11Dssk1D [34]. Within 20 min of Lat B treatment, neither strain displayed activation of Hog1p (Figure 2C). After 20 min incubation of both cells in 200 uM lat B, samples were fixed for Rd-phalloidin staining of actin structures. No actin structures were observed in the cells (Figure 2D). The results were in accordance with previous JW-74 web observation that activity of Hog1p activity is affected neither by actin-destabilizing drug latrunculin A, nor by actin-stabilizing drug jasplakinolide [21]. These results indicate that X factor may not be the actin disassembly.A Receiver Domain (Amino Acids 177,240) Near the Nterminus of SSK2 is Needed for the Activation of SSK2 Independent of SSKAs observed above, Ssk2p can be activated without Ssk1p under osmotic stress, whereas the Ssk22p cannot. We carried out a sequence alignment analysis of the two proteins Ssk2p and Ssk22p. As shown in Figure 3, the sequence comparison shows that Ssk2p and Ssk22p are quite similar. The similarity of the kinase domains of these two MAPKKKs is higher than that of the N-terminal noncatalytical domains. Ssk2p is larger than Ssk22p, mainly due to an extra N-terminal segment (1,176). There isSsk2p can be Activated Independent of Ssk1p under Severe Osmotic StressAs described above, the HOG pathway was activated in the ssk1Dste11D mutant under osmotic stress but not in the ste11Dssk2Dssk22D mutant, which indicated Ssk2p and Ssk22p may be activated independent of Ssk1p under osmotic stress. It hasAlternative 15755315 Activation of Ssk2p in Osmotic StressFigure 1. Hog1p phosphorylation level and growth phenotypes for the wild type (WT) and mutant yeast cells under 
various osmotic and salt stress conditions. A. Hog1p MAPK phosphorylation (P-Hog1p) was detected in the ssk1Dste11D mutant under hyperosmotic stress. Cells were exposed to different level of osmotic stress induced by sorbitol (concentration shown) in YPD medium for the time indicated. B. Same experiment as in A but for the wild type strain which shows higher sensitivity and a longer duration of the response. C. Hog1p phosphorylation was not detected in the ste11Dssk2Dssk22D mutant. D. Hog1p phosphorylation assay under ionic osmotic stress in the ssk1Dste11D double mutant. Cells were exposed to a different levels of salt stress induced by NaCl (concentration shown) in YPD medium for the time indicated. E. Same as in D but for the wild type cells. F. The ssk1Dste11D mutant exhibited better growth than hog1D mutant under osmotic stress. Serial dilutions (from left to right in each panel) 
of indicated strains were spotted onto YPD and salt plates and growth was scored after 3 days. doi:10.1371/journal.pone.0054867.ga high variable N-terminal segment (177,240) in Ssk22p. Previous study has identified the Ssk1p binding domain (294,413) in Ssk2p [7]. We assume that the binding site for the X factor is located in the near N-terminal region.To determine the region in Ssk2p that is essential for its activation in the absence of Ssk1p, various segments near the Nterminus were deleted in Ssk2p. Thes.Ase activity of Ssk2p [26,31]. Osmotic stress induces a rapid disassembly of the actin cytoskeleton [31,33]. Actin cytoskeleton disassembly induces Ssk2p to translocate from the cytosol to the septin cytoskeleton of the bud neck [26,31,32]. Therefore, we asked whether actin disassembly would activate the Ssk2p to activate the HOG pathway. Lat B was used to induce rapid and complete disassembly of the actin cytoskeleton in strains BY4741 and ste11Dssk1D [34]. Within 20 min of Lat B treatment, neither strain displayed activation of Hog1p (Figure 2C). After 20 min incubation of both cells in 200 uM lat B, samples were fixed for Rd-phalloidin staining of actin structures. No actin structures were observed in the cells (Figure 2D). The results were in accordance with previous observation that activity of Hog1p activity is affected neither by actin-destabilizing drug latrunculin A, nor by actin-stabilizing drug jasplakinolide [21]. These results indicate that X factor may not be the actin disassembly.A Receiver Domain (Amino Acids 177,240) Near the Nterminus of SSK2 is Needed for the Activation of SSK2 Independent of SSKAs observed above, Ssk2p can be activated without Ssk1p under osmotic stress, whereas the Ssk22p cannot. We carried out a sequence alignment analysis of the two proteins Ssk2p and Ssk22p. As shown in Figure 3, the sequence comparison shows that Ssk2p and Ssk22p are quite similar. The similarity of the kinase domains of these two MAPKKKs is higher than that of the N-terminal noncatalytical domains. Ssk2p is larger than Ssk22p, mainly due to an extra N-terminal segment (1,176). There isSsk2p can be Activated Independent of Ssk1p under Severe Osmotic StressAs described above, the HOG pathway was activated in the ssk1Dste11D mutant under osmotic stress but not in the ste11Dssk2Dssk22D mutant, which indicated Ssk2p and Ssk22p may be activated independent of Ssk1p under osmotic stress. It hasAlternative 15755315 Activation of Ssk2p in Osmotic StressFigure 1. Hog1p phosphorylation level and growth phenotypes for the wild type (WT) and mutant yeast cells under various osmotic and salt stress conditions. A. Hog1p MAPK phosphorylation (P-Hog1p) was detected in the ssk1Dste11D mutant under hyperosmotic stress. Cells were exposed to different level of osmotic stress induced by sorbitol (concentration shown) in YPD medium for the time indicated. B. Same experiment as in A but for the wild type strain which shows higher sensitivity and a longer duration of the response. C. Hog1p phosphorylation was not detected in the ste11Dssk2Dssk22D mutant. D. Hog1p phosphorylation assay under ionic osmotic stress in the ssk1Dste11D double mutant. Cells were exposed to a different levels of salt stress induced by NaCl (concentration shown) in YPD medium for the time indicated. E. Same as in D but for the wild type cells. F. The ssk1Dste11D mutant exhibited better growth than hog1D mutant under osmotic stress. Serial dilutions (from left to right in each panel) of indicated strains were spotted onto YPD and salt plates and growth was scored after 3 days. doi:10.1371/journal.pone.0054867.ga high variable N-terminal segment (177,240) in Ssk22p. Previous study has identified the Ssk1p binding domain (294,413) in Ssk2p [7]. We assume that the binding site for the X factor is located in the near N-terminal region.To determine the region in Ssk2p that is essential for its activation in the absence of Ssk1p, various segments near the Nterminus were deleted in Ssk2p. Thes.
