ISOLATION OF A PROTEIN TARGET OF THE FKBP12-RAPAMYCIN COMPLEX IN MAMMALIAN-CELLS

The immunosuppressive drug, rapamycin, interferes with an undefined si gnaling pathway required for the progression of G(1)-phase T-cells int o S phase. Genetic analyses in yeast indicate that binding of rapamyci n to its intracellular receptor, FKBP12, generates a toxic complex tha t inhibits cell growth in G(1) phase. These analyses implicated two re lated proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin comp lex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian t argets of rapamycin (mTOR) from tissue extracts. In the presence of ra pamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cel l 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 significant ly lower amounts of the candidate mTOR from rapamycin-resistant cell l ines. The latter results suggest that mTOR is a relevant target of rap amycin in these cells. Finally, we report the isolation of a full-leng th mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin com plex. The deduced amino acid sequence of mTOR displays 42 and 45% iden tity to those of yeast TOR1 and TOR2, respectively, These results stro ngly suggest that the FKBP12-rapamycin complex interacts with homologo us ligands in yeast and mammalian cells and that the loss of mTOR func tion is directly related to the inhibitory effect of rapamycin on G(1) - to S-phase progression in T-lymphocytes and other sensitive cell typ es.