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  • KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.

KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.

Molecular cell (2021-09-23)
Hiroshi Senoo, Daisuke Murata, May Wai, Kenta Arai, Wakiko Iwata, Hiromi Sesaki, Miho Iijima
ABSTRACT

AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
H-89, Dihydrochloride, InSolution 10 mM, ≥99%, reversible ATP-competitive inhibitor of protein kinase A
Sigma-Aldrich
3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
Sigma-Aldrich
U0126 ethanolate, ≥98% (HPLC), powder
Millipore
ANTI-FLAG® M2 Affinity Gel, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
Akt1/PKBα Protein, inactive, 50 g, Unactive, N-terminal His6-tagged recombinant full-length human Akt1, for use in Kinase Assays.
Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial