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

    Cite this

    @article{97eb6149c85646139e445356a4fd1606,
    title = "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",
    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.",
    keywords = "SKELETAL-MUSCLE CELLS, PLECKSTRIN-HOMOLOGY-DOMAIN, GROWTH-FACTOR-I, GLYCOGEN-SYNTHASE, PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE, GLUT4 VESICLES, B-ALPHA, TRANSPORT, ACTIVATION, MEMBRANE",
    author = "Green, {Charlotte J.} and Olga Goransson and Kular, {Gursant S.} and Leslie, {Nick R.} and Alexander Gray and Alessi, {Dario R.} and Kei Sakamoto and Hundal, {Harinder S.}",
    year = "2008",
    month = "10",
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    doi = "10.1074/jbc.M802623200",
    language = "English",
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    pages = "27653--27667",
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    TY - JOUR

    T1 - 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

    AU - Green, Charlotte J.

    AU - Goransson, Olga

    AU - Kular, Gursant S.

    AU - Leslie, Nick R.

    AU - Gray, Alexander

    AU - Alessi, Dario R.

    AU - Sakamoto, Kei

    AU - Hundal, Harinder S.

    PY - 2008/10/10

    Y1 - 2008/10/10

    N2 - 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.

    AB - 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.

    KW - SKELETAL-MUSCLE CELLS

    KW - PLECKSTRIN-HOMOLOGY-DOMAIN

    KW - GROWTH-FACTOR-I

    KW - GLYCOGEN-SYNTHASE

    KW - PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE

    KW - GLUT4 VESICLES

    KW - B-ALPHA

    KW - TRANSPORT

    KW - ACTIVATION

    KW - MEMBRANE

    UR - http://www.scopus.com/inward/record.url?scp=55549088220&partnerID=8YFLogxK

    U2 - 10.1074/jbc.M802623200

    DO - 10.1074/jbc.M802623200

    M3 - Article

    VL - 283

    SP - 27653

    EP - 27667

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 41

    ER -