Histidine to Aspartate Phosphoryl Transfer in Yeast

描述(申请人提供)：在酿酒酵母中，SLN1、YPD1和SSK1蛋白形成一个多步骤的HJS-Asp磷酸传递信号通路，控制对高渗胁迫的适应性反应。YPD1是一种典型的组氨酸磷酸转移(HPT)蛋白，需要将磷酸基从膜结合的传感器组氨酸激酶SLN1转移到反应调节蛋白SSK1。这一建议的重点是通过磷酸化和去磷酸化来调节SSK1的功能。YPD1在酵母渗透压调节途径中具有双重功能，一是将磷酸基穿梭到SSK1上，二是在非渗透胁迫条件下稳定SSK1的磷酸化状态。与大多数双组分反应调节蛋白相反，SSK1在磷酸化后变得不活跃。高渗应激通过一种鲜为人知的机制导致SSK1的快速去磷酸化，从而使SSK1与下游的MAPK级联反应并激活。关于环境条件如何影响调节蛋白复合体的结合或解离，我们知之甚少。因此，本文提出的研究将解决YPD1/SSK1相互作用如何受到环境条件和反应调节因子磷酸化状态的影响。此外，还将探讨SSK1在高渗胁迫下迅速去磷酸化的机制(S)。这项应用的具体目的是：1)获得YPD1-反应调节分子复合体的共晶结构；2)检测在体内和体外影响YPD1/SSK1复合体形成的环境条件；3)研究SSK1通过去磷酸化激活的可能机制(S)。这项工作对基础科学和生物医学的两个主要领域具有重要意义。最重要的是，拟议的研究将有助于更好地理解细胞信号通路中受调控的蛋白质-蛋白质相互作用。在更广泛的背景下，这些研究可能为开发新型抗菌和抗真菌药物提供基础。

DESCRIPTION (provided by applicant): In Saccharomyces cerevisiae, the SLN1, YPD1, and SSK1 proteins form a multi-step Hjs-Asp phosphorelay signaling pathway that controls adaptive responses to hyperosmotic stress. YPD1, a prototypical histidine-containing phosphotransfer (HPt) protein, is required for phosphoryl group transfer from the membrane-bound sensor histidine kinase SLN1 to the response regulator protein SSK1. The focus of this proposal is on the regulation of SSK1 function by phosphorylation and dephosphorylation. YPD1 serves a dual function in the yeast osmoregulatory pathway by shuttling phosphoryl groups to SSK1 and also by stabilizing the phosphorylated state of SSK1 under non-osmotic stress conditions. Contrary to most two-component response regulator proteins, SSK1 is rendered inactive when phosphorylated. Hyperosmotic stress, through a mechanism that is poorly understood, results in rapid dephosphorylation of SSK1, which allows SSK1 to interact with and activate a downstream MAP kinase cascade. Very little is known about how environmental conditions influence the association or dissociation of regulatory protein complexes. Hence, the studies proposed herein will address how YPD1/SSK1 interactions are influenced by both environmental conditions and the response regulator phosphorylation state. Mechanism(s) by which SSK1 is rapidly dephosphorylated in response to hyperosmotic stress will also be examined. The specific aims of this application are to i) obtain co-crystal structures of YPD1-response regulator complexes, ii) examine environmental conditions that affect YPD1/SSK1 complex formation both in vivo and in vitro, and iii) examine possible mechanism(s) of activation of SSK1 via dephosphorylation. This work has significance to two major areas of basic scientific and biomedical importance. Foremost, the proposed studies will lead to a better understanding of regulated protein-protein interactions in the context of cell signaling pathways. In a broader context, these studies may provide a basis for development of novel antibacterial and antifungal drugs.