A direct linkage between the phosphoinositide 3-Kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells

The microbially derived antiproliferative agent rapamycin inhibits cell gro wth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR), In this study, we demonstrate that interleukin-3 stimulat ion induces a wortmannin-sensitive increase in mTOR kinase activity in a my eloid progenitor cell line. The involvement of phosphoinositide 3'-kinase ( PI3K) in the regulation of mTOR activity was further suggested by findings that mTOR was phosphorylated in vitro and in vivo by the PI3K-regulated pro tein kinase, AKT/PKB, Although AKT phosphorylated mTOR at two COOH-terminal sites (Thr(2446) and Ser(2448)) in vitro, Ser(2448) was the major phosphor ylation site in insulin-stimulated or -activated AKT-expressing human embry onic kidney cells. Transient transfection assays with mTOR mutants bearing Ala substitutions at Ser(2448) and/or Thr(2446) indicated that AKT-dependen t mTOR phosphorylation was not essential for either PHAS-I phosphorylation or p70(S6K) activation in HEK cells. However, a deletion of amino acids 243 0-2450 in mTOR, which includes the potential AKT phosphorylation sites, sig nificantly increased both the basal protein kinase activity and in vivo sig naling functions of mTOR, These results demonstrate that mTOR is a direct t arget of the PI3K-AKT signaling pathway in mitogen-stimulated cells, and th at the identified AKT phosphorylation sites are nested within a "repressor domain" that negatively regulates the catalytic activity of mTOR, Furthermo re, the activation status of the PI3K-AKT pathway in cancer cells may be an important determinant of cellular sensitivity to the cytostatic effect of rapamycin.