Ted in the transgenic line under a range of osmotic stress at various time points (Figure 5D). Unexpectedly, the cells expressing the mutant ssk2D (1,240) lost the sensitivity to the mild osmotic stress (0.2 M sorbitol) (Figure 5D). Furthermore, the response is significantly attenuated under osmotic stress compared with that of the wild type Ssk2p. The strain ste11Dssk1D showed a quick response with a high amplitude.DiscussionIt is well known that dephosphorylated Ssk1p can activate the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. Though some studies indicated an additional input for Pbs2 [24,25], there is no specific research on it. In this paper, we showed that Ssk2p can be activated and it then activates the HOG pathway independent of Ssk1p under osmotic stress. We propose that there is another regulator that can bind to the Ssk2p and activate the Ssk2p. The region which is essential for Ssk1p-independent activation of the HOG pathway is identified from aa 177 to aa 240 in Ssk2p. The findings can explain previous reports that STL1 and GRE2 are induced 8- to 38-fold in ste11Dssk22D cells but exhibit little 298690-60-5 supplier induction (,1.7- fold) in hog1D or pbs2D strains [24]. We observed that Z-360 biological activity deletion of the binding domain for the X factor significantly attenuates the activation of Hog1p under osmotic stress. It is possible that the deletion might change the conformation of Ssk2p, making it less accessible for Ssk1p. The observation also raised the possibility that binding of the unknown X factor increases the affinity of Ssk1p to Ssk2p in the presence of osmotic stress. Budding yeast keeps three MAPKKKs, Ste11p, Ssk2p and Ssk22p, to activate one MAPKK Pbs2p to activate the HOG pathway upon hyperosmotic stress.At a crude level, they appear to be functionally redundant. However, 18055761 as our study shows, they have distinct activation patterns. The Ste11 branch is less sensitive than the Sln1-Ssk1-Ssk2 cascade under mild osmotic shock, but it alone enables osmoresistance of the host cell almost as good as the wild type strain. The Sln1-Ssk1-Ssk2 cascade exhibit both sensitivity and tolerance to the various levels of osmotic stress. The X-Ssk2 branch only responds to the severe osmotic stress (i.e. at concentrations higher than 0.5 M sorbitol, KCL or NaCL). Its duration of activation is also much shorter. In comparison, the Sln1-Ssk1-Ssk22 cascade displays less sensitivity, slower activation, and low level of activation capacity even though Ssk22p is highly homologous to Ssk2p. The wild type cells employ a combination of these activation patterns in their osmostress response. Besides the activation pattern, the three MAPKKKs in the HOG pathway have different roles in salt tolerance. Our study shows that Ste11p and Ssk2p cope with salt stress 
caused by sodium equally well, but Ssk22p displays a poorer capacity, implicating the role of Ste11p and Ssk2p in the activation of parallel processes when the cell is under toxic cation stress. Our results also show that the salt-resistance requires high level activation of Ssk2p, which could be achieved through synergistic activation of Ssk1p and the X factor. In conclusion, we uncovered another input into Ssk2p in the HOG pathway and identified the receiver domain (amino acids 177,240) in Ssk2p which is essential for the alternative activation pathway. Ssk2p is essential in salt tolerance besides its role in the activation of the HOG pathway. It would be very interesting if the experimental observations reported here can be foll.Ted in the transgenic line under a range of osmotic stress at various time points (Figure 5D). Unexpectedly, the cells expressing the mutant ssk2D (1,240) lost the sensitivity to the mild osmotic stress (0.2 M sorbitol) (Figure 5D). Furthermore, the response is significantly attenuated under osmotic stress compared with that of the wild type Ssk2p. The strain ste11Dssk1D showed a quick response with a high amplitude.DiscussionIt is well known that dephosphorylated Ssk1p can activate the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. Though some studies indicated an additional input for Pbs2 [24,25], there is no specific research on it. In this paper, we showed that Ssk2p can be activated and it then activates the HOG pathway independent of Ssk1p under osmotic stress. We propose that there is another regulator that can bind to the Ssk2p and activate the Ssk2p. The region which is essential for Ssk1p-independent activation of the HOG pathway is identified from aa 177 to aa 240 in Ssk2p. The findings can explain previous reports that STL1 and GRE2 are induced 8- to 38-fold in ste11Dssk22D cells but exhibit little induction (,1.7- fold) in hog1D or pbs2D strains [24]. We observed that deletion of the binding domain for the X factor significantly attenuates the activation of Hog1p under osmotic stress. It is possible that the deletion might change the conformation of Ssk2p, making it less accessible for Ssk1p. The observation also raised the possibility that binding of the unknown X factor increases the affinity of Ssk1p to Ssk2p in the presence of osmotic stress. Budding yeast keeps three MAPKKKs, Ste11p, Ssk2p and Ssk22p, to activate one MAPKK Pbs2p to activate the HOG pathway upon hyperosmotic stress.At a crude 
level, they appear to be functionally redundant. However, 18055761 as our study shows, they have distinct activation patterns. The Ste11 branch is less sensitive than the Sln1-Ssk1-Ssk2 cascade under mild osmotic shock, but it alone enables osmoresistance of the host cell almost as good as the wild type strain. The Sln1-Ssk1-Ssk2 cascade exhibit both sensitivity and tolerance to the various levels of osmotic stress. The X-Ssk2 branch only responds to the severe osmotic stress (i.e. at concentrations higher than 0.5 M sorbitol, KCL or NaCL). Its duration of activation is also much shorter. In comparison, the Sln1-Ssk1-Ssk22 cascade displays less sensitivity, slower activation, and low level of activation capacity even though Ssk22p is highly homologous to Ssk2p. The wild type cells employ a combination of these activation patterns in their osmostress response. Besides the activation pattern, the three MAPKKKs in the HOG pathway have different roles in salt tolerance. Our study shows that Ste11p and Ssk2p cope with salt stress caused by sodium equally well, but Ssk22p displays a poorer capacity, implicating the role of Ste11p and Ssk2p in the activation of parallel processes when the cell is under toxic cation stress. Our results also show that the salt-resistance requires high level activation of Ssk2p, which could be achieved through synergistic activation of Ssk1p and the X factor. In conclusion, we uncovered another input into Ssk2p in the HOG pathway and identified the receiver domain (amino acids 177,240) in Ssk2p which is essential for the alternative activation pathway. Ssk2p is essential in salt tolerance besides its role in the activation of the HOG pathway. It would be very interesting if the experimental observations reported here can be foll.
