Study on the hyperosmotic stress-responsive signal transduction pathway

我们发现，酵母渗透传感途径中His-Asp磷酸传递途径中的Ssk1反应调节因子只有在去磷酸化时才会通过泛素-蛋白酶体系统降解。这种降解被证明有助于抑制不必要的途径激活。通过使用化学交联剂的共沉淀实验，我们检测到在高渗透胁迫下酵母渗透传感机制的组件Sho1和主要的肌动蛋白调节蛋白LAS 17之间的瞬时相互作用。该相互作用发生在Sho1-PBS2 MAPKKK相互作用之前。当Sho1-Las17相互作用被阻止时，丝状生长途径的Kss1 MAPK在高渗胁迫下被错误地激活。我们认为Sho1-Las17的相互作用隔离了Sho1激活下游的MAPK通路，从而建立了信号特异性。我们还发现Las17在高渗胁迫下失去了它的定位。在此条件下，Las17通过GSK-3被磷酸化。GSK-3介导的Las17的磷酸化和随之而来的去局部化被认为是高渗透胁迫诱导肌动蛋白去局部化的原因。Rim101途径也有助于酵母的晕耐性，被证明是通过ESCRT复合体的内体积累激活的，ESCRT复合体是膜蛋白的分选机制，最终导致空泡降解。在此基础上，我们提出了该通路的激活机制。我们还发现，抑制哺乳动物应激激活MAPK通路的PP2CbX蛋白磷酸酶形成了PP2CbX-ANKRA2-MAP3K三元复合体。我们的数据表明，ANKRA2作为支架蛋白促进PP2CbX对MAP3K的去磷酸化。

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.