Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition

Oncogene. 2007 Aug 16;26(38):5655-61. doi: 10.1038/sj.onc.1210343. Epub 2007 Mar 5.

Abstract

Rapamycin, a natural product inhibitor of the Raptor-mammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN- and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2-/- cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.

MeSH terms

  • Cell Line, Tumor
  • Chromones / pharmacology
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Immunoblotting
  • Indazoles / pharmacology*
  • Indoles / pharmacology*
  • Mechanistic Target of Rapamycin Complex 1
  • Morpholines / pharmacology
  • Multiprotein Complexes
  • Mutation
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation / drug effects
  • Proteins
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors*
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA, Small Interfering / genetics
  • Serine / metabolism
  • TOR Serine-Threonine Kinases
  • Time Factors
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transfection
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism

Substances

  • A 443654
  • Chromones
  • Enzyme Inhibitors
  • Indazoles
  • Indoles
  • Morpholines
  • Multiprotein Complexes
  • Phosphoinositide-3 Kinase Inhibitors
  • Proteins
  • RNA, Small Interfering
  • TSC2 protein, human
  • Transcription Factors
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Serine
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1
  • Proto-Oncogene Proteins c-akt