Stepwise multi-gate control of the HOG MAPK pathway under hyperosmotic stress

, adaptation to hyperosmotic stress is mediated by the Hog1 mitogen-activated protein kinase (MAPK) via the high-osmolarity glycerol (HOG) pathway, which comprises a MAPK cascade and two upstream branches, SHO1 and SLN1. In the SHO1 branch, hyperosmotic stress is detected by transmembrane proteins such as Sho1, Opy2, Hkr1, and Msb2; however, the signaling steps directly controlled by the stress have remained unclear. Here, we show that hyperosmotic stress regulates three distinct steps within the SHO1 branch. It promotes the phosphorylation of the MAP2K Pbs2 by the MAP3K Ste11 through Sho1-dependent protein interactions and requires Hkr1, and it also regulates an upstream step required for Ste11 activation that depends on Hkr1 and Opy2. Together with a previously described step in Pbs2-mediated Hog1 phosphorylation, these findings show that osmotic stress regulates the pathway at three levels, defining stepwise multi-gate control of HOG pathway activation.