A Two-Component Signal Transduction Pathway in DictyosteliumDevelopment

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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. ***