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.

The potent immunosuppressive drugs FK506 and rapamycin interfere with signal transduction pathways required for T cell activation and growth. The distinct inhibitory effects of these drugs on the T cell activation program are mediated through the formation of pharmacologically active complexes with members of a family of intracellular receptors termed the FK506 binding proteins (FKBPs). The FKBP12.FK506 complex specifically binds to and inhibits calcineurin, a signaling protein required for transcriptional activation of the interleukin (IL)-2 gene in response to T cell antigen receptor engagement. The FKBP12. rapamycin complex interacts with a recently defined target protein termed the mammalian target of rapamycin (mTOR). Accumulating data suggest that mTOR functions in a previously unrecognized signal transduction pathway required for the progression of IL-2-stimulated T cells from G1 into the S phase of the cell cycle. Here we review the immunopharmacology of rapamycin, with particular emphasis on the characterization of mTOR.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

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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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