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 Göransson, Gursant S Kular, Nick R Leslie, Alexander Gray, Dario R Alessi, Kei Sakamoto, Harinder S Hundal

Research output: Contribution to journalArticle

82 Citations (Scopus)

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

Keywords

  • 3T3-L1 Cells
  • Adaptor Proteins, Signal Transducing
  • Adipocytes
  • Amino Acid Transport System A
  • Amino Acids
  • Animals
  • Biological Transport
  • Drug Resistance
  • Enzyme Activation
  • Extracellular Signal-Regulated MAP Kinases
  • GTPase-Activating Proteins
  • Glucose
  • Glucose Transporter Type 4
  • Glycogen
  • Glycogen Synthase Kinase 3
  • Hypoglycemic Agents
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Isoenzymes
  • Mice
  • Muscle, Skeletal
  • Mutation, Missense
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Rats
  • Ribosomal Protein S6 Kinases

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