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.
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.
Berndt
N
N
Drug Discovery Department, Moffitt Cancer Center, Tampa, FL 33612, USA.
Yang
H
H
Trinczek
B
B
Betzi
S
S
Zhang
Z
Z
Wu
B
B
Lawrence
N J
NJ
Pellecchia
M
M
Schönbrunn
E
E
Cheng
J Q
JQ
Sebti
S M
SM
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000073:Acenaphthenes / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism* / Q000494:pharmacology*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
D000073:Acenaphthenes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D012265:Ribonucleotides; C014205:triciribine phosphate; D004815:Epidermal Growth Factor; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases