The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p70⌬2-46/⌬CT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase.

PDK1 is one of the components of the signaling pathway recruited by Pl 3-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pl 3-kinase.

The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/ RAFT-1/FRAP is the target through which the rapamycin⅐FKBP12 complex acts to dephosphorylate/ deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin⅐FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivation in situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner.Rapamycin is an immunosurpressive macrolide whose major cellular receptor is the cytosolic 12-kDa FK506-binding protein (FKBP12) 1 (1, 2). Rapamycin binds to FKBP12 at a single site, identical to the site bound by the structurally related drug FK506, and both agents, acting in situ as a drug-protein complex, are immunosurpressive through inhibition of T-cell proliferation. Despite these similarities, the two drugs operate through distinct mechanisms. The FK506⅐FKBP12 complex blocks T-cell receptor signal transduction by directly inhibiting protein phosphatase 2B/calcineurin. The rapamycin⅐FKBP12 complex does not inhibit T-cell receptor signal transduction or calcineurin activity but rather inhibits interleukin-2-stimulated signal transduction, concomitant with a potent (Ͼ95%) and selective inhibition in situ of the p70 S6 kinase (3, 4), an enzyme critical for the G 1 to S transition, at least in some cells (5, 6). Rapamycin, acting indirectly in situ, causes a partial dephosphorylation and deactivation of p70; the direct target of rapamycin⅐FKBP12 complexes in situ relevant to the rapamycin-inhibition of the p70 S6 kinase is the protein known variously as RAFT-1/FRAP/RAPT-1 or mTOR.The TOR proteins were first identified in Saccharomyces cerevisiae, where rapamycin (but not FK506) is growth-inhibitory. The TOR proteins are the product of one class of mutant genes that confer resistance to rapamycin-induced growth inhibition as a dominant phenotype (7,8). The yeast (7-9) and mammalian TOR proteins (specifically, FRAP (10); RAFT-1 (11); RAPT-1 (12); and mTOR (13)) are Ͼ250-kDa polypeptides that contain at their carboxyl terminus a protein and/or lipid kinase catalytic domain, most closely related to those of the DNA protein kinase and the ATM, MEC1, and Tel1 checkpoint gene products, and somewhat more distantly related to the PI-3 kinases (14). The rapamycin/FKBP12 complex b...