Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells.
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.
Mesh Terms:
1-Phosphatidylinositol 3-Kinase, Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins, Cell Cycle Proteins, DNA Primers, DNA, Complementary, DNA-Binding Proteins, Fungal Proteins, Heat-Shock Proteins, Humans, Lymphoma, T-Cell, Molecular Sequence Data, Phosphotransferases (Alcohol Group Acceptor), Polyenes, Protein Kinases, Rats, Rats, Sprague-Dawley, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Sirolimus, Tacrolimus Binding Proteins, Tumor Cells, Cultured
1-Phosphatidylinositol 3-Kinase, Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins, Cell Cycle Proteins, DNA Primers, DNA, Complementary, DNA-Binding Proteins, Fungal Proteins, Heat-Shock Proteins, Humans, Lymphoma, T-Cell, Molecular Sequence Data, Phosphotransferases (Alcohol Group Acceptor), Polyenes, Protein Kinases, Rats, Rats, Sprague-Dawley, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Sirolimus, Tacrolimus Binding Proteins, Tumor Cells, Cultured
J. Biol. Chem.
Date: Jan. 13, 1995
PubMed ID: 7822316
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