Mechanism and Function of the Supercomplex KARATE in Insulin Signaling

超级复合物空手道在胰岛素信号传导中的机制和功能

基本信息

批准号：
10444290
负责人：
Miho Iijima
金额：
$ 45.46万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-05 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10444290
关键词：
1-Phosphatidylinositol 3-Kinase 3-Phosphoinositide Dependent Protein Kinase-1 Adenylate Cyclase Adipocytes Adipose tissue Affect Amoeba genus Applications Grants Biochemical Biology Bioreactors Blood Blood Glucose Catalytic Domain Cell Fractionation Cell Line Cell membrane Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Cryoelectron Microscopy Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Diabetes Mellitus Dictyostelium Dictyostelium discoideum Event FRAP1 gene Foundations Future GLUT 4 protein GTP Binding Glucose Glucose Transporter Glycogen Goals Homologous Gene Human Hydrophobicity In Vitro Insulin Insulin Receptor Insulin Resistance KRAS2 gene Knock-out Knockout Mice Knowledge Link Lipids Liver Mammalian Cell Mediating Medical Metabolic Metabolic syndrome Molecular Monomeric GTP-Binding Proteins Mus Non-Insulin-Dependent Diabetes Mellitus Obesity Organ PH Domain Pancreas Peptides Persons Phosphatidylinositols Phosphorylation Phosphotransferases Physiological Post-Translational Protein Processing Prevalence Protein Biosynthesis Protein Kinase Protein-Serine-Threonine Kinases Proteins Proto-Oncogene Proteins c-akt RHOA gene Race Regulation Reporting Role Serine Signal Transduction Signal Transduction Pathway Skeletal Muscle Solid Structure System TSC2 gene Testing Textbooks Therapeutic Intervention Threonine Tissues Translating Tyrosine Phosphorylation United States Work biological systems blood glucose regulation cell motility glucose production glucose uptake glucose-regulated proteins in vivo inhibitor innovation insight insulin signaling live cell imaging novel protein activation reconstitution recruit response rho social tool

项目摘要

Abstract AKT is one of the most important protein kinases in insulin signaling. In response to insulin, AKT becomes active and phosphorylates critical metabolic effectors, including TBC1D4, GSK3, TSC2, and FOXO. These proteins regulate glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane, glycogen synthesis, lipid and protein synthesis, and glucose production in adipose tissues, skeletal muscles, and livers. Abnormalities in AKT activation have been linked to insulin resistance in type 2 diabetes. AKT is activated by two other protein kinases, mTORC2 and PDK1. mTORC2 phosphorylates the hydrophobic motif of AKT and opens the catalytic domain. PDK1 then phosphorylates AKT to activate its enzymatic activity. The activation step by PDK1 is controlled by the recruitment of AKT and PDK1 to the plasma membrane. However, understanding of how mTORC2 is regulated to phosphorylate AKT is limited. To fill this critical knowledge gap, this grant application tests the hypothesis that KRAS4B, RHOA, and mTORC2 form a supercomplex (termed KARATE) to direct the enzymatic activity of mTORC2 toward AKT in insulin signaling. Toward this goal, we will identify the mechanism, localization, and regulation of the KARATE assembly. We will also determine the physiological function of KARATE in glucose homeostasis. We will employ multiple innovative tools, including: 1) our recently developed total biochemical reconstitution system for KARATE- mediated AKT phosphorylation; 2) a Dictyostelium bioreactor that enables the purification to functional human proteins to high homogeneity with critical post-translational modification; 3) our novel KARATE peptide inhibitor for in vitro and cellular studies; 4) our CRISPR-generated knockout cell lines for RHOA, KRAS and mTORC2 subunits; and 5) tissue-specific RHOA-knockout mice and phospho-defective RHOA mice. We anticipate that the successful completion of the work will significantly advance our understanding of insulin signaling and establish a solid foundation for future studies. Ultimately, this will help translate the fundamental biology of AKT signaling into medical treatments focused on KARATE for metabolic syndrome.

抽象的 AKT 是胰岛素信号转导中最重要的蛋白激酶之一。为了响应胰岛素，AKT 变为激活并磷酸化关键代谢效应子，包括 TBC1D4、GSK3、TSC2 和 FOXO。这些蛋白质通过葡萄糖转运蛋白 GLUT4 易位至血浆来调节葡萄糖摄取脂肪组织、骨骼中的膜、糖原合成、脂质和蛋白质合成以及葡萄糖产生肌肉、肝脏。 AKT 激活异常与 2 型糖尿病的胰岛素抵抗有关。 AKT 由另外两种蛋白激酶 mTORC2 和 PDK1 激活。 mTORC2 磷酸化疏水性 AKT 基序并打开催化结构域。然后 PDK1 磷酸化 AKT 以激活其酶活性。 PDK1 的激活步骤是通过将 AKT 和 PDK1 募集到质膜来控制的。然而，对 mTORC2 如何调节 AKT 磷酸化的了解还有限。为了填补这个关键为了弥补知识差距，该拨款申请测试了 KRAS4B、RHOA 和 mTORC2 形成一个假设的假设超级复合物（称为 KARATE）在胰岛素信号转导中将 mTORC2 的酶活性引导至 AKT。为了实现这一目标，我们将确定空手道集会的机制、本地化和监管。我们将还决定了空手道在葡萄糖稳态中的生理功能。我们将聘请多名创新工具，包括：1）我们最近开发的空手道总生化重建系统- 介导的 AKT 磷酸化； 2) 盘基网柄菌生物反应器，能够纯化功能性人类通过关键的翻译后修饰使蛋白质具有高度同质性； 3) 我们的新型空手道肽抑制剂用于体外和细胞研究； 4) 我们的 CRISPR 生成的 RHOA、KRAS 和 mTORC2 敲除细胞系亚基； 5) 组织特异性 RHOA 敲除小鼠和磷酸化缺陷型 RHOA 小鼠。我们预计这项工作的成功完成将显着增进我们对胰岛素信号传导的理解为今后的学习打下坚实的基础。最终，这将有助于转化 AKT 的基础生物学信号传导到以空手道治疗代谢综合征为重点的医学治疗中。