mTOR (
mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (
protein kinase A/
protein kinase G/
protein kinase C) family
kinases such as Akt (
protein kinase B), S6K (p70 ribosomal
S6 kinase) and SGK (serum and
glucocorticoid protein kinase).
mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas
mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule
Ku-0063794, which inhibits both
mTORC1 and
mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other
protein kinases or seven
lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations.
Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal
S6 kinase), an AGC
kinase not regulated by mTOR.
Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast,
Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential
mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that
Ku-0063794 induced a much greater dephosphorylation of the
mTORC1 substrate 4E-BP1 (
eukaryotic initiation factor 4E-
binding protein 1) than
rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from
mTORC1, or
mTORC2 phosphorylates 4E-BP1.
Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that
Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of
cancers in which this pathway is inappropriately activated.