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...

Previous studies have shown that the noncatalytic carboxy-terminal tail of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibitory pseudosubstrate domain that is phosphorylated in situ during activation and in vitro by mitogen-activated protein kinases. The present study shows that a recombinant p70 deleted of the carboxy-terminal tail (p70⌬CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin very similar to those of the parent, full-length p70 kinase. Carboxy-terminal deletion reduces the extent of maximal inhibition produced by rapamycin, from >95% in the full-length p70 to 60 to 80% in p70⌬CT104, without altering the sensitivity to rapamycin inhibition (50% inhibitory concentration of 2 nM). Serum activation of p70⌬CT104, as with the parent, full-length p70, is accompanied by an increase in 32 P content (about twofold) in situ and a slowing in electrophoretic mobility; both modifications are inhibited by pretreatment with wortmannin or rapamycin.32 P-peptide maps of p70⌬CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferential dephosphorylation of the same subset of sites. Thus, it is likely that activation of the kinase requires phosphorylation of p70 at sites in addition to those previously identified in the carboxy-terminal tail. Evidence that the carboxy-terminal tail actually functions as a potent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region. Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continued phosphorylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70⌬29-46/⌬CT104) increases activity 10-fold, to a level approaching that of p70⌬CT104. Deletion of residues 29 to 46 also abolishes completely the sensitivity of p70 to inhibition by rapamycin but does not alter the susceptibility to activation by serum or inhibition by wortmannin. Although the mechanisms underlying the effects of the ⌬29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phosphorylation site at Ser-40. Thus, activation of the p70 S6 kinase involves multiple, independent inputs directed at different domains of the p70 polypeptide. Disinhibition from the carboxy-terminal tail requires, in addition to its multisite phosphorylation, an activating input dependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct from that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase. In addition, as exemplified by the rapamycinresistant but mitogen-and wortmannin-sensitive p70⌬29-46/⌬CT104 mutant, a further activating input, which probably involves site-specific phosphorylation in the segment between amino acids 46 to 421, is necessary.Addition of insulin, polypeptide growth factors...

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70ACT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70ACT104 but not Rsk Activation of p7OACT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p7OACT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.The advent of selective inhibitors of phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) (1) has begun to clarify the role of this enzyme in cell signaling. Wortmannin (2) and/or the structurally unrelated LY294002 (3) inhibits mitogenesis (2, 4), receptor-activated secretory (2) and transport responses-e.g., glucose transport and GLUT4 translocation (3, 5)-and receptor regulation of intracellular enzymes-e.g., insulin regulation of adipocyte lipolysis (5) and activation of p70 S6 kinase (4, 6). As to the biochemical mechanisms by which PI 3-kinase participates in these responses, the ability of PI 3-kinase to regulate membrane dynamics is best characterized in yeast, where VPS34, the major PI 3-kinase in Saccharomyces cerevisiae, is an essential protein required for the sorting of polypeptides to the regulated or constitutive secretory pathway (7). The immediate downstream target and effector pathway for PI 3-kinase action in this system remains obscure.The p70 S6 kinase is a ubiquitous mitogen-activated Ser/Thr kinase (8) that is necessary for cells to enter S phase after mitogen stimulation (9, 10). p70 and its closest homologs, the Rsk enzymes, are coordinately activated within minutes after mitogen addition through Ser/Thr phosphorylation of the enzyme (9). Abundant evidence indicates that the Rsks are activated in a Ras-dependent way (11), entirely as a result of their phosphorylation by the mitogen-activated protein kinases, erkl and erk2 (12)(13)(14). In contrast, p70 requires multiple independent inputs for activation. One input is directed at a pseudosubstrate autoinhibitory (SKAIPS) domain in the p70 noncatalytic carboxyl-terminal tail (15,16). Concomitant with activation in situ, this segment undergoes Ser/Thr phosphorThe publication costs of this article were defrayed in part by page...