Molecular mechanisms of yeast PAS kinase regulation and function.

描述（由申请人提供）：本文所述的建议使用S.酿酒酵母，以揭示新的分子机制参与PAS激酶的调节和功能。PAS激酶是最近发现的一种感觉蛋白激酶，可能在糖尿病和肥胖的发展中起关键作用，但对所涉及的途径和蛋白知之甚少。研究中的第一个假设是磷酸化调节PAS激酶活性。在体内磷酸化的两个酵母PAS激酶同源物，PSK 1和PSK 2，最近已被检测到使用抗磷酸丝氨酸抗体。此外，Mike Tyers的实验室在酵母激酶的高通量研究中检测到多个Psk 1和Psk 2磷酸化位点。这些磷酸化酶在PAS激酶调节中的作用将通过磷酸化酶突变和随后的体内和体外激酶测定来研究。酵母PAS激酶被两种生长条件激活，即引发细胞完整性应激和在呼吸碳源上生长的条件。呼吸碳源的激活发生迅速，并依赖于蛋白激酶Snf 1。因此，PAS激酶的磷酸化可能是由于另一种蛋白激酶（例如Snf 1）的自磷酸化或转磷酸化引起的。使用Psk 1和Psk 2的激酶死亡突变体将区分这些可能性。此外，Snf 1在PAS激酶磷酸化中的作用也将被探讨。第二个假设涉及假定的PAS激酶底物的表征。基于PAS激酶底物或相互作用蛋白质的高通量筛选，五种蛋白质已被靶向作为推定底物。PAS激酶和这些蛋白质之间的体内蛋白质-蛋白质相互作用已经使用酵母双杂交和共纯化技术进行了验证。其中两种蛋白质Gsy 2和Gph 1参与糖原代谢，已知PAS激酶调节途径。其他三个，eIF 1A，Caf 20和Sam 1的特性，可能会发现PAS激酶在翻译和辅因子生物合成的调节中的新作用。这些底物将通过表征其与PAS激酶的相互作用、体外和体内磷酸化研究、定位研究以及野生型与PAS激酶缺陷型酵母中的酶和表型测定来验证。该项目的成功完成不仅将阐明酵母PAS激酶在代谢调控中的作用，而且为今后的研究提供了丰富的工具和方法。这些工具包括PAS激酶磷酸酶突变体和突变体，结合底物更紧密，促进通过进一步的蛋白质-蛋白质相互作用研究的新底物的识别。由于途径和蛋白质通常在酵母和哺乳动物细胞之间是保守的，我们的研究可能会发现治疗代谢性疾病的新靶点。 公共卫生相关性：PAS激酶是最近发现的一种感觉蛋白激酶，可能是糖尿病和肥胖症发生的关键因素。这项建议的目的是利用S.以表征PAS激酶调节和功能的分子机制。由于途径和蛋白质通常在酵母和哺乳动物细胞之间是保守的，我们的发现可能会为代谢性疾病的治疗提供有价值的见解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.