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

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 phosphatidyl-inositol (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 (elF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G(1) 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 mu M), 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 mu M) 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.