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.
Sabers
C J
CJ
Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905.
Martin
M M
MM
Brunn
G J
GJ
Williams
J M
JM
Dumont
F J
FJ
Wiederrecht
G
G
Abraham
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D016399:Lymphoma, T-Cell / Q000378:metabolism; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011090:Polyenes / Q000378:metabolism*; D011494:Protein Kinases; D051381:Rats; D017207:Rats, Sprague-Dawley; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011090:Polyenes; D011494:Protein Kinases; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; C546842:MTOR protein, human; C546845:mTOR protein, rat; C083324:TOR1 protein, S cerevisiae