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 language | English |
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Pages (from-to) | 27653-27667 |
Number of pages | 15 |
Journal | Journal of Biological Chemistry |
Volume | 283 |
Issue number | 41 |
DOIs | |
Publication status | Published - 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