Mechanism and Function of the Supercomplex KARATE in Insulin Signaling

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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.