Signal Mediated Growth Control by Osmotic Stress

9506987古斯汀这项研究解决了细胞如何感知和应对渗透压力的根本问题。在国家自然科学基金资助的前期工作中，研究表明酿酒酵母具有一种渗透敏感信号转导通路，称为HOG激酶(由HOG1编码)和MAP激酶(由PBS2编码)。缺乏这两个基因的突变体在高渗透压诱导的渗透调节和渗透耐受所需蛋白质的表达方面存在特定的缺陷。该实验室最近发现，暴露在渗透压增加下的酵母细胞暂时将细胞周期停滞在G2期；HOG 1和PBS2突变体对这种渗透应激反应是有缺陷的。导致细胞周期停滞的机制目前尚不清楚，但工作假设是Hog1p负调控细胞周期蛋白依赖的蛋白激酶CDc28p的G2特异性功能。长期的目标是从分子上了解渗透胁迫是如何调节细胞周期的。这个项目的重点是确定HOG通路是如何诱导G2期停滞的。两个主要目标是(1)研究高渗透压诱导的、依赖于Hog1的细胞周期蛋白依赖性蛋白激酶CDc28在G2中的功能调节所需的机制，以及(2)分离和鉴定介导G2细胞停滞的HOG途径的分子靶点。达到上述目标的具体实验是：(1)使用G2期细胞来确定高渗透压和HOG途径突变对G2周期蛋白/CDC28复合体的组蛋白H1激酶活性的影响，G2周期蛋白/CDC28复合体的体内稳定性，以及G2周期蛋白的细胞含量。将确定Cks1或其他已知的CDC28相关蛋白是否介导Hog1对G2特异性CDC28功能的调节。还将确定CDC28磷酸化的变化是否介导了Hog1诱导的G2停滞。通过确定渗透胁迫和hog1突变对细胞周期标志性事件的影响，将研究Hog1抑制G2的生理意义。2)利用亲和层析和免疫共沉淀法分离和鉴定Hog1或G2细胞周期蛋白/CDc28相关蛋白，以介导Hog1诱导的G2细胞停滞。这一渗透调节蛋白激酶级联调控细胞周期的分子分析结果将为研究其他真核系统中活跃的类似渗透反应途径提供重要的实验范式。%这个项目解决了最近在酵母中发现的信号通路的一个新特征，该研究人员在之前的NSF奖中发现了这一点。其信号通路是高渗透压激活基因(HOG)，它明显通过细胞分裂来影响细胞的正常进程。如果细胞暴露在高渗透条件下，如高盐或高糖浓度，它们通常会将细胞周期停滞在被称为G2的时间段，即DNA合成(S期)和有丝分裂(M期)之间的时间间隔。带有不活跃的HOG1基因的细胞不会在G2中停滞，相反，它们会继续分裂，通常是异常的。从G2到M的转变已被证明部分受磷酸化酶或激酶的复合体的循环组装控制，该复合体是CDC28(细胞分裂控制28)基因的产物和被称为细胞周期蛋白的蛋白质，大部分是在细胞周期的进展过程中循环地产生的。细胞周期蛋白是在G2期形成的，可以在G2/M转换过程中进行，我们称之为G2细胞周期蛋白。允许通过G1(有丝分裂和脱氧核糖核酸合成之间的时间间隔)/S过渡的周期蛋白称为G1周期蛋白。这个项目的重点是G2周期蛋白和CDC28激酶形成的复合体的性质如何受到Hog1的影响。活性、稳定性和与其他蛋白质的结合都是Hog 1作用的候选者。为了确定G2Cyclin/CDc28复合体中是否存在介导Hog1诱导的细胞周期停滞的其他调节成分，将根据它们与复合体成分的关联来鉴定和分离其他成分。这项工作很重要，因为它可以作为真菌和植物细胞对不断变化的环境的反应的模型，例如部分脱水或盐度增加，这两者都会改变细胞的渗透条件。***

9506987 Gustin The research addresses the fundamental problem of how cells sense and respond to osmotic stress. In previous work funded by NSF, it was shown that the yeast Saccharomyces cerevisiae has an osmosensing signal transduction pathway called the HOG kinase (encoded by HOG1) and a MAP kinase kinase (encoded by PBS2). Mutants lacking either gene have specific defects in the high osmolarity-induced expression of proteins need for osmoregulation and osmotolerance. This laboratory recently made the exciting discovery that yeast cells exposed to an increase in osmolarity temporarily arrest their cell cycle in the G2 phase; hog 1 and pbs2 mutants are defective for this osmotic stress response. The mechanism responsible for this cell cycle arrest is currently unknown, but the working hypothesis is that Hog1p negatively regulates a G2-specific function of the cyclin- dependent protein kinase Cdc28p. The long-range goal is a molecular understanding of how the cell cycle is regulated by osmotic stress. The specific focus of this project is to determine how the HOG pathway induces a G2 phase arrest. Two major objectives are (1) to investigate mechanisms required for high osmolarity-induced, Hog1-dependent regulation of the function of the cyclin-dependent kinase Cdc28 in G2, and (2) to isolate and characterize the molecular targets of the HOG pathway that mediate G2 arrest. Specific experiments to meet the stated objectives are: (1) Use G2-phase cells to determine the effect of high osmolarity and HOG pathway mutants on the histone H1 kinase activity of the G2 cyclin/Cdc28 complex, the in vivo stability of the G2 cyclin/Cdc28 complex, and the cellular content of G2 cyclins. It will be determined whether Cks1 or other known Cdc28-associated proteins mediate Hog1 regulation of G2-specific Cdc28 functions. It will also be determined whether changes in Cdc28 phosphorylation mediate Hog1-induced G2 arrest. The physiological significance of G2 arrest by Hog1 will be investigated by determining effects of osmotic stress and the hog1 mutation on cell cycle landmark events. 2) Use affinity chromatography and co-immunoprecipitation to isolate and characterize Hog1- or G2 cyclin/Cdc28-associated proteins mediating Hog1-induced G2 arrest. Results of this molecular analysis of cell cycle regulation by an osmotically-regulated protein kinase cascade will provide an important experimental paradigm for studies on similar osmotic response pathways active in other eukaryotic systems. %%% This project addresses a new feature of a signaling pathway recently discovered in yeast by this investigator in a prior NSF award. The signaling pathway is the High Osmotic-stress activated Genes (HOG) which apparently have influence over the normal progression through cell division. If the cells are exposed to high osmotic conditions, such as high salt or sugar concentrations, they normally arrest the cell cycle in the period of time called "G2", the Gap of time between DNA synthesis (S phase) and mitosis (M phase). Cells with an inactive HOG1 gene, do not arrest in G2, rather they continue on to divide, often abnormally. The transition from G2 into M has been shown to be controlled, in part, by the cyclical assembly of complexes of a phosphorylating enzyme, or kinase, which is the product of the Cdc28 (Cell Division Control 28) gene and proteins called cyclins, which are, in most part, cyclically made during the progression through the cell cycle. The cyclins which are made during G2 and allow progression through the G2/M transition are called the G2 cyclins. Cyclins which allow progression through the G1 (the Gap of time between mitosis and DNA synthesis)/S transistion are called G1 cyclins. The focus of this project is how the nature of the complex formed by G2 cyclins and the Cdc28 kinase is influenced by the Hog1. The activity, stability, and associations with other proteins are all candidates for Hog 1 action. To determine if other regulatory components are present in the G2cyclin/Cdc28 complex which mediate Hog1-induced cell cycle arrest, the other components will be identified and isolated by virtue of their association with components of the complex. This work is important because it may serve as a model for fungal and plant cellular response to a changing environment, such as partial desication or increase in salinity, both of which change the osmotic conditions of the cell. ***