HISTIDINE TO ASPARTATE PHOSPHORYL TRANSFER IN YEAST

酿酒酵母的渗透调节系统为探索原核和真核细胞中普遍存在的信号转导途径的关键特征提供了一个独特的机会。该信号通路的初始步骤涉及多个组氨酸到天冬氨酸的磷酸化转移事件，涉及与细菌“双组分”信号转导相关的三种蛋白，SLN1， YPD1和SSK1。这个磷接力系统反过来调节下游的有丝分裂原活化蛋白激酶级联反应。本文提出的研究重点是理解决定分子相互作用和磷酸化转移的结构/功能关系。x射线晶体学研究结合含组氨酸磷接力蛋白YPD1的诱变实验将深入了解YPD1表面与SLN1和SSK1相关的同源结构域相互作用。反应调节蛋白SSK1代表了MAP激酶级联的一种新的调节因子。因此，通过启动SSK1的结构研究，预计我们将发现关于磷酸化在调节SSK1功能中的作用以及SSK1可与MAP激酶级联下游蛋白相互作用的分子表面的独特而重要的信息。这项研究将对细菌中的许多双组分信号转导系统和高等真核生物中MAP激酶依赖的信号通路具有广泛的意义。此外，由于相关的MAP激酶级联在控制细胞生长和分化的途径中起作用，并且由于组氨酸到天冬氨酸磷酸化转移对人类Nm23蛋白的肿瘤抑制活性至关重要，因此本文提出的研究可能会为癌症的病因提供见解。总的来说，本研究的基本目的是更好地理解蛋白质磷酸化作为细胞调节的普遍形式。

The osmoregulation system in Saccharomyces cerevisiae offers a unique opportunity in which to explore key features of signal transduction pathways that are prevalent in both prokaryotic and eukaryotic cells. The initial steps of this signaling pathway involve multiple histidine-to-aspartate phosphoryl transfer events involving three proteins, SLN1, YPD1, and SSK1, that are related to bacterial "two-component" signal transducers. This phosphorelay system, in turn, regulates a downstream mitogen- activated protein (MAP) kinase cascade. The research proposed here focuses on understanding structure/function relationships that dictate molecular interactions and hence phosphoryl transfer. X-ray crystallographic studies coupled with mutagenesis experiments of the histidine-containing phosphorelay protein YPD1 will yield insight regarding surface(s) of YPD1 that interact with the homologous domains associated with SLN1 and SSK1. The response regulator protein, SSK1, represents a novel regulator of a MAP kinase cascade. Thus, by initiating structural studies of SSK1, it is anticipated that we will uncover unique and important information regarding the role of phosphorylation in regulating SSK1 function and the molecular surface(s) of SSK1 that are available for interaction with the downstream proteins of the MAP kinase cascade. This study will have broad based implications relevant to the many two-component signal transduction systems in bacteria and MAP kinase-dependent signaling pathways in higher eukaryotes. Moreover, because related MAP kinase cascades function in pathways that control cell growth and differentiation and since histidine-to-aspartate phosphoryl transfer is essential for the tumor-suppressive activity of human Nm23 protein, the research proposed here will likely provide insights into the etiology of cancer. Overall, the fundamental objective of this research is to better understand protein phosphorylation as a universal form of cellular regulation.