A Two-Component Signal Transduction Pathway in DictyosteliumDevelopment

9728463卢米斯 信号在细胞表面被接收，并被转换成细胞内的生理反应。 本研究分析的是土壤阿米巴Dictyosteelium中的一个双组分系统，它由一个传感激酶和一个反应调节剂组成。 当信号激活传感器激酶时，它会使其中一个组氨酸自磷酸化，然后将磷酸盐传递给反应调节剂。 在网柄藻中，传感激酶由dhkA基因编码，而反应调节因子由regA基因编码。 在所有条件下，缺失dhkA的突变阻断孢子形成，而缺失regA的突变导致快速孢子形成，即使在不存在前茎衍生信号的情况下。 在这些实验中，在DhkA结合由前茎细胞释放的小肽SDF-2之前和之后，将测量DhkA的组氨酸激酶活性。还将测定DhkA中被激酶磷酸化的氨基酸。 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。 然后，这些研究人员将建立条件，以测量磷酸盐从DhkA到反应调节剂Reg A的中继。 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在被Dhka磷酸化时不具有活性，这可能是因为它不再与PKA的调节亚基结合。 这里概述的实验将测试RegA的磷酸化和非磷酸化形式与PKA的调节亚基的相互作用。 将定义这种相互作用所需的特定蛋白质结构域。 双组分系统在细菌、真菌和植物的多种反应中起作用，本实验室从小鼠组织中分离到一个组氨酸激酶的cDNA，它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，其中信号被蛋白激酶接收，然后蛋白激酶使组氨酸残基自磷酸化，然后将该磷酸转移到另一种称为反应调节剂的蛋白质，该蛋白质又激活其他蛋白质，最终导致对信号的反应。 这里描述的研究是看这个过程发生在土壤阿米巴Dictyosteoba中的详细机制。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. ***