Tacie Hall and Dr. Julianne Grose, Department of Microbiology and Molecular Biology
When PAS kinase is knocked out in mice placed on high-fat diets, these mice show such symptoms as decreased weight gain, hypermetabolic phenotype, decreased liver triglyceride accumulation, and retained insulin sensitivity when compared with their wild type littermates.1 These symptoms are highly associated with chronic diseases commonly seen in humans today, such as diabetes, obesity, and heart disease. PAS kinase is a highly conserved protein from yeast to man which allows for the use of yeast as model organism to help discover the molecular mechanisms behind PAS kinase function. Since little is known about how PAS kinase functions, it is important to identify interactions in human PAS kinase since metabolic pathways are not identical in humans and yeast. Thus, my research was intended to discover putative protein-protein interactions of human PAS kinase in order to determine the pathways that human PAS kinase regulates.
Several yeast-two-hybrid screens were conducted using human PAS kinase, however, no interactions occurred. Since we were unable to elucidate protein-protein interactions of human PAS kinase, we then hypothesized that the human PAS kinase construct was active in yeast. To prove this hypothesis we ran tests to see if human PAS kinase might suppress a deficiency in yeast PAS kinase and take over the role of the yeast PAS kinase. These tests were conducted by over expressing Ugp1 which is lethal to the cell. Human PAS kinase was then over expressed to see if it could rescue lethality of Ugp1 over expression. The results of these tests were negative. An over expression of yeast PAS kinase will rescue the cell from the lethality of over expression of Ugp1, but human PAS kinase will not.
Unable to express human PAS kinase using the yeast-two-hybrid system, I was able to continue research on yeast PAS kinase 1 in elucidating protein-protein substrates.i Yeast-two-hybrid screens were performed to discover putative protein interactions (including sequencing). These interactions were then confirmed through PAS kinase dependence tests in which plasmids are purified from yeast, transformed into E-coli to increase copy number, and finally transformed back into two yeast strains. One strain containing an empty vector, the other containing PAS kinase. Growth occurring on the empty vector showed that the protein did not interact with PAS kinase.
Some of the proteins that have been elucidated from these screens and determined dependent upon PAS kinase are involved in such pathways as glucose deprivation (Pbp1), glycolysis and gluconeogenesis (Fba1), spindle integrity to prevent the premature exit from mitosis (Ibd2), and transcription activation and regulation of genes that regulate glucose metabolism (IES6 and Yap6).
The libraries that have been used for screens are nearly saturated, thus, few screens remain to be completed. Dependency tests for existing screens are nearing completion as well, thus, we anticipate publication by February 2013.
Following publication of these results, research will continue concerning yeast PAS kinase including the characterization of the proteins that have been determined as dependant. Characterization of these proteins will be done using co-immunoprecipitation studies, kinase assays using 32P radioactive phosphate to see if the substrate is phosphorylated in the presence and absence of PAS kinase, and phenotypic assays in which the substrate’s function will be tested with respect to PAS kinase levels and activity. This will be done for each protein to determine how PAS kinase affects function. Findings from our continued research may prove to result in therapeutic targets for treating such diseases as heart disease, diabetes, and obesity.
References
- Hao HX, Rutter J. 2008. The role of PAS kinase in regulating energy metabolism. IUBMB Life; 60(4): 204-9.