Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake

Charlotte J. Green, Olga Goransson, Gursant S. Kular, Nick R. Leslie, Alexander Gray, Dario R. Alessi, Kei Sakamoto, Harinder S. Hundal (Lead / Corresponding author)

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    Abstract

    Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKB alpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKBW80A) yields an Akti-resistant kinase. Cellular expression of PKBW80A antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.

    Original languageEnglish
    Pages (from-to)27653-27667
    Number of pages15
    JournalJournal of Biological Chemistry
    Volume283
    Issue number41
    DOIs
    Publication statusPublished - 10 Oct 2008

    Keywords

    • SKELETAL-MUSCLE CELLS
    • PLECKSTRIN-HOMOLOGY-DOMAIN
    • GROWTH-FACTOR-I
    • GLYCOGEN-SYNTHASE
    • PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE
    • GLUT4 VESICLES
    • B-ALPHA
    • TRANSPORT
    • ACTIVATION
    • MEMBRANE

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