HISTIDINE TO ASPARTATE PHOSPHORYL TRANSFER IN YEAST

酿酒酵母中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，涉及三个蛋白质，SLN1，YPD1和SSK1，它们与细菌的“双组分”信号转导相关。这个磷酸传递系统反过来调节下游的丝裂原活化蛋白(MAP)激酶级联反应。这里提出的研究重点是了解结构/功能关系，这些关系决定了分子相互作用，从而决定了磷酰基转移。X射线结晶学研究结合含有组氨酸的磷酸传递蛋白YPD1的诱变实验，将获得关于YPD1表面(S)与SLN1和SSK1相关同源结构域相互作用的见解。反应调节蛋白SSK1代表了MAP激酶级联反应的一种新的调节蛋白。因此，通过启动SSK1的结构研究，我们有望发现关于磷酸化在调节SSK1功能中的作用以及SSK1的分子表面(S)的独特而重要的信息，这些信息可用于与MAP激酶级联的下游蛋白相互作用。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依赖于激酶的信号通路具有广泛的基础意义。此外，由于相关的MAP激酶在控制细胞生长和分化的通路中发挥作用，而且组氨酸到天冬氨酸的磷酸化转移对于人类Nm23蛋白的肿瘤抑制活性是必不可少的，因此这里提出的研究可能会为癌症的病因提供见解。总体而言，这项研究的基本目标是更好地理解蛋白质磷酸化作为细胞调节的一种普遍形式。

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.