Studying the ERK phosphorylation of the BCAT protein

研究 BCAT 蛋白的 ERK 磷酸化

• 批准号: 10614553
• 负责人: Ming Dong
• 金额: $ 14.11万
• 依托单位: NORTH CAROLINA AGRI & TECH ST UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10614553
• 关键词: Alzheimer' s Disease; Apoptosis; Binding; Biochemical; Biophysics; Branched-Chain Amino Acids; Breast Cancer Cell; Cancer cell line; Cell Proliferation; Cell Proliferation Regulation; Cell physiology; Cells; Complex; Crystallization; Cytoplasm; Disease; Drops; Event; FRAP1 gene; Family member; Genes; Glutamates; Glutathione Disulfide; Human; Hydrogen Peroxide; In Vitro; Insulin; Investigation; Keto Acids; Kinetics; MAPK1 gene; MAPK3 gene; Mass Spectrum Analysis; Mediating; Metabolic; Mitochondria; Mitogen-Activated Protein Kinases; Modification; Molecular; Molecular Conformation; Neoplasm Metastasis; Neurotransmitters; Oxidants; Oxidation-Reduction; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Process; Proliferating; Protein Biosynthesis; Protein Isoforms; Proteins; Protocols documentation; Publishing; Regulation; Research; Resolution; Role; Signal Transduction; Site; Solvents; Structure; Time; Variant; X-Ray Crystallography; biophysical analysis; branched-chain-amino-acid transaminase; cancer cell; cell motility; crosslink; experimental study; extracellular; insight; malignant breast neoplasm; mutant; overexpression; programs; protein complex; protein expression; protein protein interaction; screening; transamination

The cytosolic and mitochondrial isoforms of human branched chain aminotransferase (hBCATc and hBCATm, respectively) catalyze the transamination of the branched chain amino acids (BCAA) to their respective α-keto acids and glutamate. In addition, we recently found that hBCAT is phosphorylated by the canonical mitogen- activated protein kinase (MAPK) family member, extracellular regulated kinase 1/2 (ERK1/2). Our research team has shown direct evidence that ERK1/2 protein interacts with hBCATc in vitro and in breast cancer cells. More specifically, we found in breast cancer cells the overexpression of BCAT1 (gene encoding hBCATc) resulted in decreased levels of ERK1/2 activation/phosphorylation, and our in vitro experiments showed activated/phosphorylated ERK1/2 phosphorylates hBCATc regulated by the redox center of hBCAT. Together, the physiological implications of these observations are considerable to better understand the cellular and molecular mechanisms of how hBCATc interacts with ERK1/2. In this proposal we will focus on the protein- protein interaction of ERK1/2-mediated phosphorylation of the hBCATc. Based on preliminary studies, we hypothesize that the redox center regulated phosphorylation of hBCATc by ERK1/2 signals this metabolic protein to play additional functional roles in the cell, with the redox center of hBCAT playing an important role in protein-protein interactions between hBCAT and ERK1/2. We hypothesize that hBCAT is differentially phosphorylated by various kinases, which causes a conformational change of hBCAT that potentially confers p-hBCAT with new functions. In the proposed project, we will study the regulatory effects of phosphorylation of hBCAT by ERK1/2 using complementary biochemical and biophysical strategies, including kinetics analysis, mass spectrometry and X-ray crystallography. Likewise, redox regulation of ERK1/2 is also a theme to be developed in this project. Together, these experiments are expected to provide a comprehensive basis for understanding the mechanism of the redox-regulated phosphorylation of BCAT by ERK1/2, and the structural effects of phosphorylation on hBCAT. Moreover, these studies will establish a workflow to examine various hBCAT-kinase interactions and help us further study the phosphorylation-mediated conformational changes of hBCAT by other kinases that will help us to better understand the functional consequences of intracellular phosphorylation of hBCAT. This project will provide for the first-time unique insights into the molecular mechanisms underlying the redox regulation of hBCAT-ERK1/2 interactions, which will fundamentally inform its role in disease conditions such as breast cancer and Alzheimer’s disease.

人类支链氨基转移酶的胞质和线粒体同工型（HBCATC和HBCATM， 催化分支链氨基酸（BCAA）向其各自的α-Keto的跨性别 酸和谷氨酸。此外，我们最近发现，HBCAT被规范有丝分裂原磷酸化 活化的蛋白激酶（MAPK）家族成员，细胞外调节激酶1/2（ERK1/2）。我们的研究 团队已直接证明ERK1/2蛋白在体外和乳腺癌细胞中与HBCATC相互作用。 更具体地说，我们在乳腺癌细胞中发现BCAT1的过表达（编码HBCATC的基因） 导致ERK1/2激活/磷酸化水平降低，我们的体外实验显示 激活/磷酸化的ERK1/2磷酸化的HBCATC由HBCAT的氧化还原中心调节。一起， 这些观察的物理含义相当大，可以更好地理解细胞和 HBCATC如何与ERK1/2相互作用的分子机制。在此提案中，我们将重点放在蛋白质上 HBCATC的ERK1/2介导的磷酸化的蛋白质相互作用。根据初步研究，我们 假设氧化还原中心通过ERK1/2对HBCATC的光谱化表示了这种代谢 蛋白质可以在细胞中发挥其他功能作用，HBCAT的氧化还原中心在 HBCAT与ERK1/2之间的蛋白质蛋白质相互作用。我们假设HBCAT是差异的 由各种激酶磷酸化，这会导致HBCAT的构象变化，这可能会赋予 具有新功能的P-HBCAT。在拟议的项目中，我们将研究摄影的调节作用 ERK1/2的HBCAT使用完整的生化和生物物理策略，包括动力学分析， 质谱和X射线晶体学。同样，ERK1/2的氧化还原调节也是一个主题 在这个项目中开发。共同期望这些实验为 了解ERK1/2对BCAT氧化还原调节的磷酸化的机制，结构 磷酸化对HBCAT的影响。此外，这些研究将建立一个工作流程，以检查各种 HBCAT-激酶相互作用并帮助我们进一步研究磷酸化介导的构象变化 其他激酶的HBCAT将帮助我们更好地了解细胞内的功能后果 HBCAT的磷酸化。该项目将提供对分子的首次独特见解 HBCAT-ERK1/2相互作用的氧化还原调节的基础机制，这将从根本上告知其 在乳腺癌和阿尔茨海默氏病等疾病中的作用。