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。对胰岛素的反应，AKT变成 激活和磷酸化关键代谢效应因子，包括TBC1D4、GSK3、TSC2和FOXO。这些 蛋白质通过葡萄糖转运体GLUT4转位到血浆来调节葡萄糖摄取 膜，糖原合成，脂肪和蛋白质合成，以及脂肪组织和骨骼中葡萄糖的产生 肌肉和肝脏。AKT激活异常与2型糖尿病患者的胰岛素抵抗有关。 AKT被另外两个蛋白激酶mTORC2和PDK1激活。MTORC2使疏水分子磷酸化 AKT基序，并打开催化结构域。然后，PDK1使AKT磷酸化，激活其酶活性。 PDK1的激活步骤受AKT和PDK1向质膜募集的控制。 然而，对mTORC2如何被调节以磷酸化AKT的理解是有限的。为了填补这一关键 知识缺口，这项拨款申请检验了KRAS4B、RHOA和mTORC2形成 超复合体(称为空手道)，在胰岛素信号转导中引导mTORC2对AKT的酶活性。 为了实现这一目标，我们将确定空手道组装的机制、本地化和监管。我们会 同时测定空手道在血糖动态平衡中的生理功能。我们将使用多个 创新工具，包括：1)我们最近开发的空手道全生化重建系统- 介导的AKT磷酸化；2)能够对有功能的人进行纯化的网柄苔藓生物反应器 蛋白质具有高度的同质性和关键的翻译后修饰；3)我们的新型空手酸多肽抑制剂 用于体外和细胞研究；4)我们的CRISPR产生的RHOA、KRAS和mTORC2基因敲除细胞系 5)组织特异性RHOA基因敲除小鼠和磷酸化缺陷RHOA小鼠。我们预料到 这项工作的成功完成将极大地促进我们对胰岛素信号转导和 为今后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 以空手道治疗代谢综合征为重点的医疗治疗的信号。

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.