The Akt activation inhibitor TCN-P inhibits Akt phosphorylation by binding to the PH domain of Akt and blocking its recruitment to the plasma membrane

Cell Death Differ. 2010 Nov;17(11):1795-804. doi: 10.1038/cdd.2010.63. Epub 2010 May 21.

Abstract

Persistently hyperphosphorylated Akt contributes to human oncogenesis and resistance to therapy. Triciribine (TCN) phosphate (TCN-P), the active metabolite of the Akt phosphorylation inhibitor TCN, is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mammalian target of rapamycin or phosphoinositide-dependent kinase 1. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance shows that TCN, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (K(D): 690 nM). Furthermore, nuclear magnetic resonance spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3-binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN and rescue from its inhibition of proliferation and induction of apoptosis. Thus, the results of our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane, and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P antiproliferative and proapoptotic activities, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3-Phosphoinositide-Dependent Protein Kinases
  • Acenaphthenes / metabolism*
  • Acenaphthenes / pharmacology*
  • Animals
  • Apoptosis
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Epidermal Growth Factor / metabolism
  • Epidermal Growth Factor / pharmacology
  • Fluorescent Antibody Technique
  • Gene Amplification
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / metabolism
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism
  • Phosphorylation / drug effects
  • Polymerase Chain Reaction
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / chemistry
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Ribonucleotides / metabolism*
  • Ribonucleotides / pharmacology*
  • Signal Transduction
  • Surface Plasmon Resonance
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • Acenaphthenes
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Phosphoproteins
  • Ribonucleotides
  • triciribine phosphate
  • Epidermal Growth Factor
  • TOR Serine-Threonine Kinases
  • 3-Phosphoinositide-Dependent Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt