Isolated mTOR as the FKBP12-rapamycin-associated protein controlling G1-S progression.
| Evidence tier | D Mechanistic / in vitro / review |
| Study type | 5 - Mechanistic / In Vitro |
| Model system | Rat/mouse cells; in vitro |
| Journal | The Journal of biological chemistry |
| Year | 1995 |
| Peer reviewed | Yes |
| Source | DOI 10.1074/jbc.270.2.815 · PMID 7822316 |
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.
| Intervention | Biochemical (FKBP12-rapamycin affinity) |
| Target | mTOR (FRAP/RAFT1) / FKBP12 |
| Model | Rat/mouse cells; in vitro |
| Effect | Isolation of the mammalian FKBP12-rapamycin target protein (mTOR) |