Abstract
The antifungal, immunosuppressive compound rapamycin arrests the cell cycle in G1 in both yeast cells and T-lymphocytes. Previous genetic studies in yeast identified mutations in three genes, FPR1 (FKBP12), TOR1, and TOR2, which confer rapamycin resistance, and genetic findings implicated the TOR proteins as direct targets of FKBP12-rapamycin. Consistent with this model, we find that modulating TOR1 and TOR2 expression alters rapamycin sensitivity. We describe several TOR2 mutations that confer rapamycin resistance. These mutations prevent FKBP12-rapamycin binding to TOR2, as assayed with the two-hybrid system. We find that TOR1 and the mammalian TOR homologue (mTOR) also bind FKBP12-rapamycin, and mutations corresponding to those in TOR2 similarly block FKBP12-rapamycin binding. We demonstrate that FKBP12 prolyl isomerase activity is not required for FKBP12-rapamycin binding to TOR and that a composite protein-drug surface contacts the TOR proteins. These studies confirm that the TOR proteins are direct targets of FKBP12-rapamycin, reveal that drug-resistant mutations prevent this association, and define structural features of these complexes.
Highlights
Rapamycin is a natural product with both antifungal and immunosuppressive activities
TOR Gene Expression Levels Alter Rapamycin Sensitivity—As a genetic test of the model that TOR is the target of FKBP12-rapamycin, we examined the effects of altering TOR gene expression on rapamycin toxicity
Overexpression of the essential TOR2 gene from low-copy or multi-copy plasmids increased rapamycin resistance by 2.5–20-fold (Table I), while increasing FKBP12 had no effect. These findings suggest the TOR proteins are limiting for FKBP12-rapamycin action and that TOR1 may compete with TOR2 for FKBP12-rapamycin
Summary
Rapamycin is a natural product with both antifungal and immunosuppressive activities (reviewed in Refs. 1–3). In mammalian T-lymphocytes, rapamycin blocks an unknown step in the signal transduction pathway initiated by interleukin-2 (IL2), leading to G1 cell cycle arrest via inhibition of cyclin D- and cyclin E-dependent p33cdk and p34cdc kinase activities (4 –7). This arrest may result from a block in IL-2-stimulated Cdkinhibitor p27kip degradation [7]. The intracellular receptor for FK506 is the cytoplasmic 12-kDa cis-trans peptidyl-prolyl isomerase FKBP12 Of Genetics, Box 3546, 322 CARL Bldg., Research Dr, Duke University Medical Center, Durham, NC 27710. Inhibition of calcineurin by these complexes prevents Tcell activation by blocking the nuclear import of the cytoplasmic subunit of NFAT, a transcription factor that regulates transcription of genes involved in T-cell activation [21,22,23]
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