Molecular mechanisms of yeast PAS kinase regulation and function.

酵母 PAS 激酶调节和功能的分子机制。

• 批准号: 8232500
• 负责人: Julianne H. Grose
• 金额: $ 34.69万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232500
• 关键词: Anabolism; Animal Model; Antibodies; Binding; Biochemical Genetics; Biogenesis; Biological Assay; Calculi; Carbon; Cell Line; Cells; Development; Diabetes Mellitus; Diet; Fatty acid glycerol esters; Future; GLC2 protein; Glycogen (Starch) Synthase; Glycogen Phosphorylase; Growth; Homologous Gene; In Vitro; Insulin Resistance; Investigation; Light; Liver; Mammalian Cell; Mammals; Maps; Metabolic; Metabolic Diseases; Methodology; Mitochondria; Molecular; Mus; Mutagenesis; Mutation; Nutrient; Obesity; Pathway interactions; Peptide Initiation Factors; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphotransferases; Physiological Adaptation; Protein Kinase; Protein phosphatase; Proteins; Regulation; Regulatory Pathway; Reporting; Resistance; Role; Saccharomyces cerevisiae; Sensory; Source; Stress; Structure of beta Cell of islet; Techniques; Therapeutic; Translations; Triglycerides; UTP-Glucose-1-Phosphate Uridylyltransferase; Weight Gain; Work; Yeasts; base; blood glucose regulation; cofactor; glycogen metabolism; high throughput screening; in vivo; insight; interest; methionine adenosyltransferase; mutant; new therapeutic target; novel; phosphatidylinositol 3' -kinase-associated serine kinase; protein protein interaction; respiratory; tool; yeast protein; yeast two hybrid system

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.

描述（由申请人提供）：本文描述的提案使用酿酒酵母的生化和遗传工具来揭示涉及PAS激酶调节和功能的新分子机制。 PAS激酶是一种最近发现的感觉蛋白激酶，可能是糖尿病和肥胖症发展的关键参与者，然而，人们对所涉及的途径和蛋白质知之甚少。正在研究的第一个假设是磷酸化调节 PAS 激酶活性。最近使用抗磷酸丝氨酸抗体检测到两种酵母 PAS 激酶同源物 Psk1 和 Psk2 的体内磷酸化。此外，Mike Tyers 实验室在酵母激酶的高通量研究中检测到了多个 Psk1 和 Psk2 磷酸位点。这些磷酸位点在 PAS 激酶调节中的作用将通过磷酸位点突变和随后的体内和体外激酶测定进行检查。酵母 PAS 激酶由两种生长条件激活，即引发细胞完整性应激的条件和依靠呼吸碳源生长的条件。呼吸碳源的激活发生得很快，并且依赖于蛋白激酶 Snf1。因此，PAS 激酶的磷酸化可能是由于另一种蛋白激酶（例如 Snf1）的自磷酸化或转磷酸化所致。使用 Psk1 和 Psk2 的激酶死亡突变体将区分这些可能性。此外，还将探讨 Snf1 在 PAS 激酶磷酸化中的作用。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的高通量筛选，五种蛋白质已被定位为推定底物。 PAS 激酶与大多数蛋白质之间的体内蛋白质-蛋白质相互作用已使用酵母双杂交和共纯化技术得到验证。其中两种蛋白 Gsy2 和 Gph1 参与糖原代谢，这是已知的 PAS 激酶调节途径。其他三个（eIF1A、Caf20 和 Sam1）的表征可能会揭示 PAS 激酶在翻译调节和辅因子生物合成中的新作用。这些底物将通过其与 PAS 激酶相互作用的表征、体外和体内磷酸化研究、定位研究以及野生型与 PAS 激酶缺陷酵母中的酶促和表型测定来验证。该项目的成功完成不仅将阐明酵母PAS激酶在代谢调节中的作用，而且为未来的研究提供丰富的工具和方法。这些工具包括 PAS 激酶磷酸位点突变体和与底物结合更紧密的突变体，有助于通过进一步的蛋白质-蛋白质相互作用研究识别新的底物。由于酵母和哺乳动物细胞之间的途径和蛋白质通常是保守的，因此我们的研究可能会发现治疗代谢疾病的新靶点。 公共健康相关性：PAS 激酶是一种最近发现的感觉蛋白激酶，可能是糖尿病和肥胖症发展的关键因素。本提案的目的是利用酿酒酵母的生化和遗传工具来表征 PAS 激酶调节和功能的分子机制。由于酵母和哺乳动物细胞之间的途径和蛋白质通常是保守的，因此我们的研究结果可能为代谢疾病的治疗提供有价值的见解。