CAREER: Tyrosine Phosphorylation and Kinase Activity as a Regulator of Sodium-Glucose Transporters

钠-葡萄糖连接转运体（SGLTs）是细胞摄取葡萄糖所必需的蛋白质，葡萄糖是一种必需的营养物质。这些蛋白质活性的改变会导致葡萄糖吸收不良和营养缺乏。不幸的是，调控SGLT活性的细胞事件知之甚少。酪氨酸磷酸化尤其如此，酪氨酸磷酸化是一种可以改变蛋白质结构和功能的修饰形式，但尚未研究其与SGLT活性的关系。因此，这项NSF CAREER研究项目的长期目标是提高对SGLT酪氨酸磷酸化或激活这种修饰形式的酪氨酸激酶蛋白如何调节葡萄糖进入细胞的变化的理解。该项目还包括若干改进教育的战略，其中包括强调sglt的生物学重要性，以及培训和招募将领导未来科学、技术、工程和数学工作的年轻科学家。总的来说，这个研究项目有望通过涉及葡萄糖转运体的教育见解和新的科学发现，影响许多科学学科，包括分子、细胞和系统生物学。这个NSF CAREER项目的研究目标是确定一个新的翻译后事件，调节SGLT活性和葡萄糖吸收的可变性。溶质载体转运体，包括sglt，被认为是被动的孔，可以单独或通过电化学梯度将底物移动到细胞膜上。然而，作为当前项目的一部分，需要验证的假设是sglt是在细胞表面被特定激酶酪氨酸磷酸化的，并且这些磷酸化事件与运输功能有关。为了解决这一假设，该项目将1)利用细胞分离和质谱法评估不同细胞区室中SGLTs的磷酸化状态；2)通过位点定向诱变、激酶抑制剂和SGLT活性测定，确定酪氨酸磷酸化位点在调节SGLT活性中的作用；3)利用磷酸化蛋白质组学分析和激酶相互作用分析鉴定介导SGLT活性的酪氨酸激酶。此外，拟议研究目标的战略和成果将用于提高基础科学知识，产生研究热情，并提高生物科学领域的领导能力。为此，将为贫困的高中生设立一个年度暑期研究职位，与中学教育工作者合作，提高对可复制和高质量科学的认识，并开发在线内容，以提高对转运蛋白的熟悉程度。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The sodium-glucose-linked transporters (SGLTs) are proteins that are necessary for cellular uptake of glucose, which is an essential nutrient. Altered activity of these proteins can lead to poor glucose absorption and nutrient deficiency. Unfortunately, cellular events that regulate SGLT activity are poorly understood. This is particularly true for tyrosine phosphorylation, a form of modification that can change protein structure and function, that has not yet been studied in relation to SGLT activity. Therefore, the long-term goal of this NSF CAREER research project is to improve the understanding of how SGLT tyrosine phosphorylation, or tyrosine kinase proteins that activate this form of modification, regulate changes in glucose movement into cells. The project also includes several strategies for educational improvements that include highlighting the biological importance of SGLTs, as well as the training and recruitment of junior scientists who will lead future science, technology, engineering, and mathematic efforts. Collectively, this research project is expected to impact many scientific disciplines, including molecular, cellular, and systems biology, through educational insight and novel scientific discoveries involving glucose transporters.The research objective of this NSF CAREER project is to identify a novel post-translational event that regulates variability in SGLT activity and glucose absorption. Solute carrier transporters, including SGLTs, have been associated as passive pores that move substrates across cell membranes alone or with electrochemical gradients. However, the hypothesis to be tested as part of the current project is that SGLTs are tyrosine phosphorylated by specific kinases at the cell surface, and that these phosphorylation events are associated with transport function. To address this hypothesis, the project will 1) evaluate the phosphorylation status of SGLTs in different cellular compartments using cell fractionation and mass spectrometry; 2) identify the role of tyrosine phosphorylation sites in regulating SGLT activity using site-directed mutagenesis, kinase inhibitors, and SGLT activity assays; and 3) identify tyrosine kinases that mediate SGLT activity using phosphoproteomic analysis and kinase interaction assays. Additionally, the strategies and outcomes from the proposed research objectives will be used to improve basic scientific knowledge, generate a passion for research, and improve leadership capabilities in the field of biological sciences. This will be done by establishing an annual summer research position for underprivileged high school students, working with middle school educators to increase recognition of reproducible and high-quality science, and developing online content that will increase familiarity with transporter proteins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.