Mechanism and Function of the Supercomplex KARATE in Insulin Signaling

摘要 AKT是胰岛素信号转导中最重要的蛋白激酶之一。在对胰岛素的反应中，AKT成为 活性和磷酸化关键代谢效应物，包括TBC 1D 4，GSK 3，TSC 2和FOXO。这些 蛋白质通过葡萄糖转运蛋白GLUT 4转运至血浆来调节葡萄糖摄取 膜，糖原合成，脂质和蛋白质合成，以及脂肪组织，骨骼 肌肉和肝脏AKT激活的减少与2型糖尿病的胰岛素抵抗有关。 AKT由另外两种蛋白激酶mTORC 2和PDK 1激活。mTORC 2磷酸化疏水的 AKT基序并打开催化结构域。然后PDK 1磷酸化AKT以激活其酶活性。 PDK 1的激活步骤由AKT和PDK 1向质膜的募集控制。 然而，对mTORC 2如何调节磷酸化AKT的理解是有限的。为了填补这个关键的 知识差距，这项拨款申请测试的假设，KRAS 4 B，RHOA，和mTORC 2形成一个 在胰岛素信号传导中，mTORC 2与超复合物（称为KARATE）结合以将mTORC 2的酶活性导向AKT。 为了实现这一目标，我们将确定的机制，本地化，和调节的KARATE大会。我们将 也决定了KARATE在葡萄糖稳态中的生理功能。我们将雇用多名 创新的工具，包括：1）我们最近开发的KARATE全生化重建系统- 介导的AKT磷酸化; 2）能够纯化功能性人细胞的网骨藻生物反应器， 蛋白质的高度同质性与关键的翻译后修饰; 3）我们的新KARATE肽抑制剂 用于体外和细胞研究; 4）我们的CRISPR产生的RHOA、KRAS和mTORC 2敲除细胞系 亚基;和5）组织特异性RHOA敲除小鼠和磷酸缺陷型RHOA小鼠。我们预计 这项工作的成功完成将大大促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，这将有助于将AKT的基础生物学 将信号传导引入针对代谢综合征KARATE的医学治疗。

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.