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␣ and -␤ 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.It is now widely accepted that the serine/threonine kinase PKB 3 (also known as Akt) acts as a major node in the cellular signaling network downstream of phosphatidylinositol 3-kinase (PI3K) regulating diverse pathways that impact upon cellular metabolism, growth, proliferation, and apoptosis (1). Activation of PKB by insulin and growth factors relies upon its recruitment to the plasma membrane. Precisely how this occurs is unclear, but the process is facilitated by the binding of 3-phosphoinositides (such as phosphatidylinositol 3,4,5-triphosphate (PIP 3 )) to the N-terminal pleckstrin homology (PH) domain of the kinase (2). PIP 3 binding induces conformational changes in PKB (3) that expose two regulatory sites, Thr 308 and Ser 473 (in PKB␣), whose phosphorylation is required for full activation of the kinase. Phosphorylation of Thr 308 is mediated by PDK1, whereas that of Ser 473 relies upon the TORC2 complex (4, 5). Activated PKB is then able to target a number of important downstream molecules including, for example, members of the FOXO family of forkhead transcription factors (6), the pro-apoptotic protein BAD as well as TSC2 and PRAS40, which have been implicated in regulation of the mTOR pathway (7). PKB also targets a protein ca...

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