A Two-Component Signal Transduction Pathway in DictyosteliumDevelopment

9728463个Loomis信号在细胞表面被接收，并被传递到细胞内的生理反应。这项研究正在分析土壤中的一个双组分系统，该系统由一个传感器激酶和一个反应调节器组成。当信号激活感应器激酶时，它会自动磷酸化其中一个组氨酸，然后将磷酸盐传递给反应调节器。在网柄苔藓中，传感器激酶由dhkA基因编码，反应调节因子由regA基因编码。失活dhkA的突变在所有条件下都会阻止产孢子，而失活RegA的突变会导致快速产孢量，即使在没有前柄来源的信号的情况下也是如此。在这些实验中，将测量DhkA与由前茎细胞释放的小肽SDF-2结合前后的组氨酸激酶活性。DhkA中被该激酶磷酸化的氨基酸也将被测定。这些实验的结果将为进一步了解该传感器激酶的功能提供坚实的基础。然后，这些研究人员将建立条件，以测量磷酸从DhkA到反应调节器REG A的传递。这些研究人员最近发现，RegA是一种水解cAMP的酶，从而通过从cAMP调节亚基中移除cAMP来阻止蛋白激酶A(PKA)的活性。遗传学研究表明，当RegA被Dhka磷酸化时，它不再活跃，可能是因为它不再与PKA的调节亚基联系在一起。这里概述的实验将测试磷酸化和非磷酸化形式的RegA与PKA调节亚单位的相互作用。这种相互作用所需的特定蛋白质结构域将被定义。两个组分系统已被证明在细菌、真菌和植物的不同反应中发挥作用，本实验室从小鼠组织中分离出一种可能在信号转导中发挥作用的组氨酸激酶基因。这些研究将提供关于这两个组成部分系统如何运作的进一步细节。其中一个重要的信号转导途径是两组分系统，在这个系统中，信号被一个蛋白激酶接收，然后它自动磷酸化一个组氨酸残基，然后将这个磷酸盐转移到另一个被称为反应调节器的蛋白质上，后者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有作用，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. ***