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.
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.
Green
Charlotte J
CJ
Division of Molecular Physiology, University of Dundee, Dundee DD1 5EH, United Kingdom.
Göransson
Olga
O
Department of Experimental Medical Science, Lund University, Biomedical Center, C11, 22184 Lund, Sweden.
Kular
Gursant S
GS
MRC Protein Phosphorylation Unit, James Black Centre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
Leslie
Nick R
NR
Division of Molecular Physiology, University of Dundee, Dundee DD1 5EH, United Kingdom.
Gray
Alexander
A
Division of Molecular Physiology, University of Dundee, Dundee DD1 5EH, United Kingdom.
Alessi
Dario R
DR
MRC Protein Phosphorylation Unit, James Black Centre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
Sakamoto
Kei
K
MRC Protein Phosphorylation Unit, James Black Centre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
Hundal
Harinder S
HS
Division of Molecular Physiology, University of Dundee, Dundee DD1 5EH, United Kingdom. Electronic address: h.s.hundal@dundee.ac.uk.
D041721:3T3-L1 Cells; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D026924:Amino Acid Transport System A / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D006003:Glycogen / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D020125:Mutation, Missense; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D026924:Amino Acid Transport System A; D000596:Amino Acids; D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D007527:Isoenzymes; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; C505647:Tbc1d4 protein, mouse; D006003:Glycogen; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D005947:Glucose