9506987 Gustin The research addresses the fundamental problem of how cells sense and respond to osmotic stress. In previous work funded by NSF, it was shown that the yeast Saccharomyces cerevisiae has an osmosensing signal transduction pathway called the HOG kinase (encoded by HOG1) and a MAP kinase kinase (encoded by PBS2). Mutants lacking either gene have specific defects in the high osmolarity-induced expression of proteins need for osmoregulation and osmotolerance. This laboratory recently made the exciting discovery that yeast cells exposed to an increase in osmolarity temporarily arrest their cell cycle in the G2 phase; hog 1 and pbs2 mutants are defective for this osmotic stress response. The mechanism responsible for this cell cycle arrest is currently unknown, but the working hypothesis is that Hog1p negatively regulates a G2-specific function of the cyclin- dependent protein kinase Cdc28p. The long-range goal is a molecular understanding of how the cell cycle is regulated by osmotic stress. The specific focus of this project is to determine how the HOG pathway induces a G2 phase arrest. Two major objectives are (1) to investigate mechanisms required for high osmolarity-induced, Hog1-dependent regulation of the function of the cyclin-dependent kinase Cdc28 in G2, and (2) to isolate and characterize the molecular targets of the HOG pathway that mediate G2 arrest. Specific experiments to meet the stated objectives are: (1) Use G2-phase cells to determine the effect of high osmolarity and HOG pathway mutants on the histone H1 kinase activity of the G2 cyclin/Cdc28 complex, the in vivo stability of the G2 cyclin/Cdc28 complex, and the cellular content of G2 cyclins. It will be determined whether Cks1 or other known Cdc28-associated proteins mediate Hog1 regulation of G2-specific Cdc28 functions. It will also be determined whether changes in Cdc28 phosphorylation mediate Hog1-induced G2 arrest. The physiological significance of G2 arrest by Hog1 will be investigated by determining effects of osmotic stress and the hog1 mutation on cell cycle landmark events. 2) Use affinity chromatography and co-immunoprecipitation to isolate and characterize Hog1- or G2 cyclin/Cdc28-associated proteins mediating Hog1-induced G2 arrest. Results of this molecular analysis of cell cycle regulation by an osmotically-regulated protein kinase cascade will provide an important experimental paradigm for studies on similar osmotic response pathways active in other eukaryotic systems. %%% This project addresses a new feature of a signaling pathway recently discovered in yeast by this investigator in a prior NSF award. The signaling pathway is the High Osmotic-stress activated Genes (HOG) which apparently have influence over the normal progression through cell division. If the cells are exposed to high osmotic conditions, such as high salt or sugar concentrations, they normally arrest the cell cycle in the period of time called "G2", the Gap of time between DNA synthesis (S phase) and mitosis (M phase). Cells with an inactive HOG1 gene, do not arrest in G2, rather they continue on to divide, often abnormally. The transition from G2 into M has been shown to be controlled, in part, by the cyclical assembly of complexes of a phosphorylating enzyme, or kinase, which is the product of the Cdc28 (Cell Division Control 28) gene and proteins called cyclins, which are, in most part, cyclically made during the progression through the cell cycle. The cyclins which are made during G2 and allow progression through the G2/M transition are called the G2 cyclins. Cyclins which allow progression through the G1 (the Gap of time between mitosis and DNA synthesis)/S transistion are called G1 cyclins. The focus of this project is how the nature of the complex formed by G2 cyclins and the Cdc28 kinase is influenced by the Hog1. The activity, stability, and associations with other proteins are all candidates for Hog 1 action. To determine if other regulatory components are present in the G2cyclin/Cdc28 complex which mediate Hog1-induced cell cycle arrest, the other components will be identified and isolated by virtue of their association with components of the complex. This work is important because it may serve as a model for fungal and plant cellular response to a changing environment, such as partial desication or increase in salinity, both of which change the osmotic conditions of the cell. ***
