The proposal described herein uses the biochemical and genetic tools of S. cerevisiae to uncover novel molecular mechanisms involved in PAS kinase regulation and function. PAS kinase is a recently discovered sensory protein kinase that may be a key player in the development of diabetes and obesity, however, little is known about the pathways and proteins involved. The first hypothesis under investigation is that phosphorylation regulates PAS kinase activity. In vivo phosphorylation of the two yeast PAS kinase homologs, Psk1 and Psk2, has recently been detected using anti-phosphoserine antibodies. In addition, Mike Tyers'lab has detected multiple Psk1 and Psk2 phosphosites in a high-throughput study of yeast kinases. The role of these phosphosites in PAS kinase regulation will be examined through phosphosite mutation and subsequent in vivo and in vitro kinase assays. Yeast PAS kinase is activated by two growth conditions, conditions that elicit cell integrity stress and growth on respiratory carbon sources. Activation by respiratory carbon sources occurs quickly and is dependent on the protein kinase Snf1. Thus, phosphorylation of PAS kinase could be due to autophosphorylation or transphosphorylation by another protein kinase, such as Snf1. The use of kinase-dead mutants of Psk1 and Psk2 will distinguish between these possibilities. In addition, the role of Snf1 in the phosphorylation of PAS kinase will be explored. The second hypothesis involves the characterization of putative PAS kinase substrates. Five proteins have been targeted as putative substrates based on high-throughput screens for PAS kinase substrates or interacting proteins. The in vivo protein- protein interactions between PAS kinase and most of these proteins have been verified using yeast two-hybrid and copurification techniques. Two of the proteins, Gsy2 and Gph1, are involved in glycogen metabolism, a pathway PAS kinase is known to regulate. Characterization of the other three, eIF1A, Caf20, and Sam1, may uncover novel roles for PAS kinase in the regulation of translation, and cofactor biosynthesis. These substrates will be verified through characterization of their interaction with PAS kinase, in vitro and in vivo phosphorylation studies, localization studies, and enzymatic and phenotypic assays in wild type versus PAS kinase-deficient yeast. Successful completion of this project will not only elucidate the role of yeast PAS kinase in metabolic regulation, but also provide a wealth of tools and methodologies for future study. These tools include PAS kinase phosphosite mutants and mutants that bind substrates more tightly, facilitating identification of novel substrates through further protein-protein interaction studies. Since pathways and proteins are often conserved between yeast and mammalian cells, our studies may uncover novel targets for the treatment of metabolic disease.
PAS kinase is a recently discovered sensory protein kinase that may be a key player in the development of diabetes and obesity. The objective of this proposal is to use the biochemical and genetic tools of S. cerevisiae to characterize the molecular mechanisms of PAS kinase regulation and function. Since pathways and proteins are often conserved between yeast and mammalian cells, our findings may yield valuable insight for the therapeutic treatment of metabolic disease.
|DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D et al. (2014) A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1. Mol Biol Cell 25:2199-215|