A Two-Component Signal Transduction Pathway in DictyosteliumDevelopment

9728463卢米斯信号在细胞表面被接收并转导到细胞内的生理反应。本研究分析了土壤变形虫盘根菌的双组分系统，该系统由一个传感器激酶和一个响应调节因子组成。当信号激活感应激酶时，它会将其中一种组氨酸自磷酸化，然后将磷酸盐传递给反应调节器。Dictyostelium的感应激酶由dhkA基因编码，反应调节因子由regA基因编码。使dhkA失活的突变在任何条件下都能阻断产孢，而使regA失活的突变即使在没有前柄衍生信号的情况下也能导致快速产孢。在这些实验中，DhkA与前体细胞释放的小肽SDF-2结合前后将测量DhkA的组氨酸激酶活性。DhkA中被激酶磷酸化的氨基酸也将被确定。这些实验的结果将为进一步了解这种传感器激酶的功能提供坚实的基础。随后，这些研究人员将建立条件来测量磷酸盐从DhkA到反应调节因子RegA的接力。这些研究人员最近发现，RegA是一种水解cAMP的酶，从而通过从其调节亚基中去除cAMP来阻止蛋白激酶A （PKA）的活性。遗传学研究表明，RegA被Dhka磷酸化后不再具有活性，这可能是因为它不再与PKA的调控亚基结合。这里概述的实验将测试磷酸化和未磷酸化形式的RegA与PKA调控亚基的相互作用。这种相互作用所需的特定蛋白质结构域将被定义。两种组分系统已被证明在细菌、真菌和植物的各种反应中起作用，本实验室已从小鼠组织中分离出一种组氨酸激酶cDNA，可能在信号转导中起作用。这些研究将提供关于这两个组成系统如何运作的进一步细节。重要的信号转导途径之一是双组分系统，其中信号由蛋白激酶接收，然后将组氨酸残基自磷酸化，然后将该磷酸盐转移到另一种称为反应调节因子的蛋白质上，该蛋白质反过来激活其他蛋白质，最终导致对信号的反应。由于这两个组成系统在许多细菌和高等植物以及可能的脊椎动物中都起作用，因此了解这些系统的生化和遗传机制是很重要的。＊＊＊

9728463 Loomis Signals are received on the surface of cells and transduced to physiological responses within the cells. This research is analyzing a two-component system in the soil amoeba Dictyostelium, which consists of a sensor kinase and a response regulator. When the signal activates the sensor kinase, it autophosphorylates one of its histidines and then relays the phosphate to the response regulator. In Dictyostelium the sensor kinase is encoded by the dhkA gene and the response regulator is encoded by the regA gene. Mutations that inactivate dhkA block sporulation under all conditions while those that inactivate regA result in rapid sporulation even in the absence of prestalk derived signals. In these experiments the histidine kinase activity of DhkA will be measured before and after it binds the small peptide, SDF-2, that is released by prestalk cells. The amino acids in DhkA that are phosphorylated by the kinase will also be determined. The results of these experiments will provide a solid foundation for further understanding how this sensor kinase functions. Conditions will then be established by these investigators to measure the relay of the phosphate from DhkA to the response regulator, Reg A. These investigators have recently found that RegA is an enzyme that hydrolyzes cAMP and thereby prevents protein kinase A (PKA) from being active by removing cAMP from its regulatory subunit. Genetic studies indicate that RegA is not active when it is phosphorylated by Dhka, possibly because it can no longer associate with the regulatory subunit of PKA. Experiments outlined here will test for the interactions of the phosphorylated and unphosphorylated forms of RegA with the regulatory subunit of PKA. The specific protein domains necessary for this interaction will be defined. Two component systems have been shown to function in various responses of bacteria, fungi and plants, and this laboratory has isolated a cDNA from mouse tissue that is a histidine kinase and may func tion in signal transduction. These studies will provide further details on how these two component systems operate. One of the important signal transduction pathways is the two component system in which signals are received by a protein kinase, which then autophosphorylates a histidine residue, and then transfers this phosphate to another protein known as the response regulator, which in turn activates other proteins ultimately resulting in a response to the signal. The research described here is looking at the detailed mechanisms by which this process occurs in the soil amoeba Dictyostelium Since these two component systems operate in many bacteria and higher plants and possibly vertebrates, it is important to understand the biochemical and genetic mechanisms of these systems. ***