Study on the hyperosmotic stress-responsive signal transduction pathway

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We found that the Ssk1 response regulator of the His-Asp phosphorelay in the yeast osmosensing pathway is degraded through the ubiquitin-proteasome system only when dephosphorylated. This degradation was shown to contribute to repression of unwanted activation of the pathway.By a co-precipitation experiment using chemical cross-linker, we detected transient interaction between Sho1, a component of the yeast osmosensing machinery, and Las 17, a major actin regulatory protein, upon hyperosmotic stress. The interaction occurred prior to the Sho1-Pbs2 MAPKKK interaction. When the Sho1-Las17 interaction was prevented, the Kss1 MAPK for the filamentous growth pathway was spuriously activated upon hyperosmotic stress. We propose that the Sho1-Las17 interaction sequesters Sho1 from activating the downstream MAPK pathways to establish the signaling specificity.We also found that Las17 loses its localization upon hyperosmotic stress. Under this condition, Las17 is phosphorylated via GSK-3. The GSK-3-mediated phosphorylation and consequent delocalization of Las17 is suggested to contribute to the actin delocalization induced by hyperosmotic stress.The Rim101 pathway, which also contributes to halo-tolerance in yeast, was shown to be activated via the endosomal accumulation of the ESCRT complex, the sorting machinery for membrane proteins destined to vacuolar degradation. Based on this observation, we proposed the activation mechanism of the pathway.We also found that the PP2CbX protein phosphatase, which represses the mammalian stress-activated MAPK pathway, forms a ternary complex of PP2CbX-ANKRA2-MAP3K. Our data suggest that ANKRA2 acts as a scaffold protein to facilitate dephosphorylation of MAP3K by PP2CbX.