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 PKBalpha 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 (PKB(W80A)) yields an Akti-resistant kinase. Cellular expression of PKB(W80A) 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3-L1 Cells
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Adipocytes / cytology
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Adipocytes / metabolism*
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Amino Acid Transport System A / genetics
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Amino Acid Transport System A / metabolism*
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Amino Acids / genetics
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Amino Acids / metabolism*
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Animals
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Biological Transport / genetics
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Drug Resistance* / genetics
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Enzyme Activation / genetics
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Extracellular Signal-Regulated MAP Kinases / genetics
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Extracellular Signal-Regulated MAP Kinases / metabolism
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism
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Glucose / metabolism*
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Glucose Transporter Type 4 / genetics
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Glucose Transporter Type 4 / metabolism*
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Glycogen / genetics
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Glycogen / metabolism
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Glycogen Synthase Kinase 3 / genetics
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Glycogen Synthase Kinase 3 / metabolism
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Hypoglycemic Agents / pharmacology*
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Insulin / pharmacology*
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Insulin Receptor Substrate Proteins
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Mice
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Muscle, Skeletal / cytology
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Muscle, Skeletal / metabolism*
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Mutation, Missense
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Phosphatidylinositol 3-Kinases / genetics
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Phosphatidylinositol 3-Kinases / metabolism
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Proto-Oncogene Proteins c-akt / antagonists & inhibitors*
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Proto-Oncogene Proteins c-akt / genetics
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Proto-Oncogene Proteins c-akt / metabolism
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Rats
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Ribosomal Protein S6 Kinases / genetics
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Ribosomal Protein S6 Kinases / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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Amino Acid Transport System A
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Amino Acids
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GTPase-Activating Proteins
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Glucose Transporter Type 4
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Hypoglycemic Agents
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Insulin
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Insulin Receptor Substrate Proteins
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Irs1 protein, mouse
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Irs1 protein, rat
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Isoenzymes
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Slc2a4 protein, mouse
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Slc2a4 protein, rat
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Tbc1d4 protein, mouse
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Glycogen
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Phosphatidylinositol 3-Kinases
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Proto-Oncogene Proteins c-akt
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Ribosomal Protein S6 Kinases
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Extracellular Signal-Regulated MAP Kinases
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Glycogen Synthase Kinase 3
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Glucose