Molecular mechanisms of yeast PAS kinase regulation and function.

酵母 PAS 激酶调节和功能的分子机制。

基本信息

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
项目类别：
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&apos -kinase-associated serine kinase protein protein interaction respiratory tool yeast protein yeast two hybrid system

项目摘要

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）：本文所述的建议使用酿酒酵母的生化和遗传工具来发现参与PAS激酶调节和功能的新型分子机制。 PAS激酶是最近发现的感觉蛋白激酶，它可能是糖尿病和肥胖症发展的关键参与者，但是，对所涉及的途径和蛋白质知之甚少。所研究的第一个假设是磷酸化调节PAS激酶活性。最近，使用抗磷酸赛抗体检测到了两种酵母PAS激酶同源物PSK1和PSK2的体内磷酸化。此外，Mike Tyers的实验室在酵母激酶的高通量研究中检测到了多个PSK1和PSK2磷脂。这些磷酸材料在PAS激酶调节中的作用将通过磷酸材料突变以及随后的体内和体外激酶分析进行检查。酵母PAS激酶被两种生长条件激活，这些生长条件会引起细胞完整性应激和呼吸碳源的生长。呼吸碳源激活很快就会发生，并取决于蛋白激酶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）自磷酸化或磷酸化引起的。使用PSK1和PSK2的激酶死亡突变体的使用将区分这些可能性。另外，将探索SNF1在PAS激酶磷酸化中的作用。第二个假设涉及推定的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，五种蛋白已作为推定底物作为推定底物。 PAS激酶与大多数这些蛋白质之间的体内蛋白质相互作用已使用酵母两杂交和共晶技术进行了验证。其中两个蛋白GSY2和GPH1参与糖原代谢，已知途径PAS激酶可以调节。其他三个EIF1A，CAF20和SAM1的表征可能会发现PAS激酶在翻译调节和辅因子生物合成中的新作用。这些底物将通过表征它们与PAS激酶，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的相互作用来验证。该项目的成功完成不仅将阐明酵母菌激酶在代谢调节中的作用，而且还为未来的研究提供了大量的工具和方法。这些工具包括PAS激酶磷酸材料突变体和突变体，这些突变体更紧密地结合底物，从而通过进一步的蛋白质 - 蛋白质相互作用研究促进了对新底物的鉴定。由于途径和蛋白质通常在酵母菌和哺乳动物细胞之间保守，因此我们的研究可能会发现用于治疗代谢疾病的新靶标。公共卫生相关性：PAS激酶是最近发现的感觉蛋白激酶，它可能是糖尿病和肥胖症发展的关键人物。该提案的目的是使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。由于途径和蛋白质通常在酵母菌和哺乳动物细胞之间保守，因此我们的发现可能会对代谢疾病的治疗治疗产生有价值的见解。