Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

Abstract

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3‐kinases. This study demonstrates that mTOR is a component of a cytokine‐triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor‐4E (eIF‐4E) binding protein, PHAS‐1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF‐4E‐dependent translation initiation. A mutant YAC‐1 T lymphoma cell line, which was selected for resistance to the growth‐inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS‐1 phosphorylation. In contrast, the PI 3‐kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS‐1 in both rapamycin‐sensitive and ‐resistant T cells. At similar drug concentrations (0.1–1 microM), wortmannin irreversibly inhibited the serine‐specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3‐kinase inhibitor, LY294002, at concentrations (1–30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85‐p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3‐kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

“…In tissue culture cells and in cell free systems, the dependence of mitosis on the completion of S phase (Schlegel and Pardee, 1986) as well as the G2 DNA damage checkpoint (Downes et al, 1994) can be uncoupled by ca eine. We found that levels of ca eine which bypass the replication checkpoint inhibited both basal and DNA-stimulated ATR kinase activity, almost to levels achieved with another inhibitor of PIK family kinases, LY294002 (Brunn et al, 1996). We suggest that ATR may be one target for the ca eine-mediated bypass of S phase and G2 checkpoints.…”

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-γ1-driven activation of mTOR/p70S6-kinase pathway

“…Furthermore, suppression of PI3Ks by wortmannin led to inhibition of PLCg1 phosphorylation (left panel) suggesting involvement of PI3Ks in its stimulation. The downregulation of S6 phosphorylation detected here is most likely result of inactivation of multiple upstream molecules-PI3Ks, PLC-g, Akt, but also consequence of direct suppression of mTOR by wortmannin because its catalytic domain shares high homology to the lipid kinase domain of PI3Ks (Brunn et al, 1996). Downregulation of p70S6-kinase/S6 by inhibition of PLC-g1 signaling Activation of PLC-g results in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to diacylglycerol and inositol 1,4,5-triphosphate (IP 3 ), which in turn leads to activation of different PKC isoforms and increase in Ca-dependent signaling, including Ca/Calmodulin-dependent-kinase (CaMK) (Rhee et al, 2000;Rhee, 2001) (please see the scheme in Figure 4a).…”

Rapamycin and mTOR kinase inhibitors

“…Not surprisingly, considering the similarity between the PI3K and PIKK catalytic domains, wortmannin also irreversibly inhibits PIKK family members. DNA-PK is the most sensitive (IC 50 =16 nM), followed by SMG-1 (IC 50 ∼ 60 nM), ATM (IC 50~1 00-150 nM), mTOR (IC 502 00 nM) and ATR (IC 50 =1.8 μM) [65][66][67][68]. Wortmannin forms a covalent bond with mTOR, presumably at Lys2187 in the ATP-binding site [65].…”