Kayla Bevard, Brigham Young University
Microbiology and Molecular Biology
Metabolic diseases, such as obesity and diabetes, are endemic in American society. Mutations in PAS kinase, a recently discovered sensory protein kinase, have been shown to cause Maturity Onset Diabetes of the Young (MODY) in humans (Semplici et al., 2011). In addition, PAS kinase deficient mice, when placed on a high fat diet, display phenotypes related to diabetes including resistance to weight gain, insulin insensitivity and triglyceride accumulation (Hao et al., 2007). PAS kinase consists of a sensory PAS domain that binds to and inhibits the kinase domain (Amezcua et al., 2002). Our model for PAS kinase activation involves the generation of a small molecule ligand that binds to the PAS domain and relieves this inhibition. We are currently engaged in several yeast genetic screens which will identify regions in the full length PAS kinase protein that are essential for either PAS kinase activation or for binding of its substrates. The first screen is based on the ability of PAS kinase, when overexpressed, to rescue a temperature-sensitive mutation in Tor2, the tor2(ts). We have isolated both point mutations and truncations in PAS kinase which alleviate the tor2(ts). These mutations solidify our model for PAS domain inhibition and will identify novel regions involved in PAS kinase regulation. Our second screen uses the yeast 2-hybrid to select for both point mutations and truncations that increase the affinity of PAS kinase for its substrate, Pbp1. These mutations will help identify key regions of PAS kinase utilized in substrate recognition. Finally, we will compare the regions affected by our mutations with the regions of PAS kinase that we have found to be well-conserved throughout evolution. Analysis of these specific genetic regions will help elucidate the molecular mechanisms involved in the regulation and function of PAS kinase, a key player in the development of metabolic disease.