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：
10601093
负责人：
Miho Iijima
金额：
$ 43.72万
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/10601093
关键词：
3-Phosphoinositide Dependent Protein Kinase-1 Adenylate Cyclase Adipocytes Adipose tissue Affect Amoeba genus Applications Grants Binding 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 PIK3CG gene 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是胰岛素信号传导中最重要的蛋白激酶之一。为了响应胰岛素，AKT变成了主动和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转移到等离子体的蛋白质摄取葡萄糖的摄取脂肪组织中的膜，糖原合成，脂质和蛋白质合成以及葡萄糖的产生肌肉和肝脏。 AKT激活的异常已与2型糖尿病中的胰岛素抵抗有关。 AKT被另外两种蛋白激酶MTORC2和PDK1激活。 MTORC2磷酸化疏水性 AKT的主题并打开催化域。然后，PDK1磷酸化AKT以激活其酶活性。 PDK1的激活步骤由Akt和PDK1募集到质膜。但是，了解如何调节MTORC2以磷酸化AKT是有限的。填补这个关键知识差距，此赠款申请检验了KRAS4B，RHOA和MTORC2形成A的假设 SuperComplex（称为空手道）将MTORC2的酶促活性引向胰岛素信号传导中的Akt。为了实现这一目标，我们将确定空手道组装的机制，本地化和调节。我们将还可以确定空手道在葡萄糖稳态中的生理功能。我们将雇用多个创新工具，包括：1）我们最近开发的空手道生化总结系统 - 介导的Akt磷酸化； 2）一个使纯化纯化为人类的生物反应器蛋白质具有高均匀性，并具有关键的翻译后修饰； 3）我们的新型空手道肽抑制剂用于体外和细胞研究； 4）我们为RhoA，KRAS和MTORC2的CRISPR生成的淘汰细胞系亚基； 5）组织特异性的RhoA敲除小鼠和磷酸化的Rhoa小鼠。我们预料到这一点成功完成工作将大大提高我们对胰岛素信号传导和为未来的研究奠定坚实的基础。最终，这将有助于翻译AKT的基本生物学发出信号的医疗治疗，集中于空手道代谢综合征。