The long-term objective of this application is to understand in molecular terms how intracellular signal transduction is mediated by protein phosphorylation and dephosphorylation. This proposal intends to study the genetic and biochemical bases of osmoregulatory signal transduction in budding yeast. The yeast response to high external osmolarity is governed by two independent osmosensors, SLN1 and SHO1. Although these osmosensors are structurally distinct, they ultimately regulate the same downstream element, the HOG1 MAP kinase. Yeast is an ideal model system to study the general principles governing intracellular signal transduction, not only because it is genetically tractable, but also because its signaling pathways have many features in common with human cells. SLN1 and SHO1 are the only eukaryotic models currently available for the mammalian osmosensors, which have not been molecularly identified. Mammalian osmoregulation is important for the maintenance of homeostasis as well as for kidney function. The regulatory mechanism of a MAPK cascade is particularly important to mammalian oncogenesis, because many oncogenes exert their effects through hyperactivation of MAP kinases.
The specific aims of this proposal are: 1) Studies on the SLN1 osmosensing mechanism, and the regulation of the SLN1-YPD1-SSK1 phosphorelay. SLN1 mutants that are defective in signal sensing or transmission, and their intragenic suppressor mutants, will be isolated and characterized. Regulators of the SLN1-YPD1-SSK1 phsophorelay, such as a histidine phosphatase or an aspartate phosphatase, will be identified and studied. Studies on the SSK2/SSK22 ativation mechanism by SSK1. The domains that are essential for the interaction of SSK1 and the SSK2/SSK22 MAPKKKs will be identified. Three specific SSK2 activation mechanisms will then be addressed: dimerization of SSK2; involvement of an SSK2 autoinhibitory site; and phosphorylation of SSK2 either by an autophosphorylation mechanism or by another protein kinase. 3) Studies on the regulation of the STE11 MAPKKK and the PBS2 MAPKK by the SHO1 osmosensor. Additional components on the SHO1 branch of the yeast osmoregulatory signal pathway will be identified, and the mechanism that determines the specificity of the PBS2 and STE11 kinases will be investigated. Finally, the SHO1 osmosensing mechanism will be genetically analyzed.
