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.
The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.
FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12⅐FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum.Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.FK506 (tacrolimus) is a powerful immunosuppressive drug for treating graft rejection and autoimmune disorders. Rapamycin (RAP, 1 sirolimus) is an immunosuppressant structurally-related to FK506 but with a distinct mechanism of action. Both drugs bind to a family of intracellular receptors, the FK506 binding proteins (FKBPs), whose members include FKBPs 12, 12.6, 13, 25, 51, and 52 (for review, see Ref. 1). All FKBPs are peptidyl-prolyl isomerases, catalyzing the cis-trans isomerization of peptidyl-prolyl bonds in peptides and proteins, an activity inhibited by both FK506 and RAP.Peptidyl-prolyl isomerase inhibition is unrelated to immunosuppression. FK506 and RAP gain function upon binding FKBP12. The FKBP12⅐FK506 and FKBP12⅐RAP complexes are the actual immunosuppressive species whose targets are calcineurin (CaN) and the mammalian target of RAP (mTOR), respectively (for review, see Refs. 1 and 2). CaN is a Ca 2ϩ -dependent, serine-threonine phosphatase required during the commitment phase (G 0 3 G 1 ) of T-cell activation (3). Inhibition of CaN blocks the nuclear translocation of transcription factors such as nuclear factor of activated T-cells and NF-B, controlling the expression of cytokine genes whose products are required for immune response coordination (for review, see Ref.2). RAP, unlike FK506, does not block lymphokine production but inhibits the T-cell proliferative response to cytokines by blocking G 1 3 S-phase progression. The function of mTOR, a 288-kDa protein related to phosphatidylinositol kinases, is unknown.CaN is a ubiquitous protein, and its inhibition at unwanted sites is most responsible for the toxicity associated with FK506 therapy (4). That immunosuppression and toxicity are mechanistica...
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