Yeast Osmosensors Hkr1 and Msb2 Activate the Hog1 MAPK Cascade by Different Mechanisms

Abstract

To cope with environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the mitogen-activated protein kinase (MAPK) Hog1, which controls an array of osmoadaptive responses. Two independent, but functionally redundant, osmosensing systems involving the transmembrane sensor histidine kinase Sln1 or the tetraspanning membrane protein Sho1 stimulate the Hog1 MAPK cascade. Furthermore, the Sho1 signaling branch itself also involves the two functionally redundant osmosensors Hkr1 and Msb2. However, any single osmosensor (Sln1, Hkr1, or Msb2) is sufficient for osmoadaptation. We found that the signaling mechanism by which Hkr1 or Msb2 stimulated the Hog1 cascade was specific to each osmosensor. Specifically, activation of Hog1 by Msb2 required the scaffold protein Bem1 and the actin cytoskeleton. Bem1 bound to the cytoplasmic domain of Msb2 and thus recruited the kinases Ste20 and Cla4 to the membrane, where either of them can activate the kinase Ste11. The cytoplasmic domain of Hkr1 also contributed to the activation of Ste11 by Ste20, but through a mechanism that involved neither Bem1 nor the actin cytoskeleton. Furthermore, we found a PXXP motif in Ste20 that specifically bound to the Sho1 SH3 (Src homology 3) domain. This interaction between Ste20 and Sho1 contributed to the activation of Hog1 by Hkr1, but not by Msb2. These differences between Hkr1 and Msb2 may enable differential regulation of these two proteins and provide a mechanism through Msb2 to connect regulation of the cytoskeleton with the response to osmotic stress.

Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway

“…We later found that disruption of HKR1 together with MSB2 in the ssk2/ 22⌬ background completely inhibited Hog1 activation, indicating that Hkr1 and Msb2 are functionally redundant (11). Because the signaling mechanisms employed by Hkr1 and Msb2 are significantly different, they were termed the HKR1 and MSB2 subbranches, respectively (12). Msb2, but not Hkr1, also acts at the head of the filamentous growth Kss1 MAPK signaling pathway (15,16).…”

“…The cytoplasmic regions of Msb2 functionally interact with the scaffold protein Bem1 (12). Bem1 binds, among other proteins, Ste20 and its activator, Cdc42.…”

“…Thus, Pbs2 and Hog1 are common to both branches. The SHO1 branch can be further divided into the MSB2 and HKR1 subbranches (11,12). The namesake component of the MSB2 subbranch, Msb2, was originally identified to be a multicopy suppressor of the cdc24 budding defect, suggesting that its function is related to signaling by the Cdc42 small G protein (13).…”

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

“…In contrast, disruption of both HKR1 and MSB2 together, in an ssk2/22⌬ background, completely inhibits Hog1 activation, indicating that Hkr1 and Msb2 are functionally redundant (30). Because the signaling mechanisms employed by Hkr1 and Msb2 appear to be significantly different, we have further divided the SHO1 branch into the HKR1 and MSB2 subbranches (29,31). Hkr1 and Msb2 also differentially regulate the filamentous growth Kss1 MAPK signaling pathway (32,33).…”

“…The cytoplasmic region of Msb2 functionally interacts with the scaffold protein Bem1 (31), which binds, among other proteins, Ste20 and the Ste20-activating protein Cdc42. Thus, Msb2 and Bem1 help activate Ste20 on the plasma membrane.…”