The targets of the FKBP12-rapamycin complex were first identified in yeast as the TOR1 and TOR2 proteins, which share 67% overall identity, have a C-terminal domain with similarity to both protein and lipid (PI-3 and PI-4) kinases, and directly interact with FKBP12-rapamycin ( Heitman et al., 1991 Cafferkey et al., 1993 Kunz et al., 1993 Helliwell et al., 1994 Cardenas and Heitman, 1995 Zheng et al., 1995). The active intracellular toxin is the resulting FKBP12–rapamycin complex, and yeast mutants lacking FKBP12 are viable and rapamycin resistant ( Heitman et al., 1991 Koltin et al., 1991). Rapamycin binds with high affinity to the prolyl isomerase FKBP12, which is highly conserved from yeast and other microorganisms to man. Rapamycin has attracted much attention because of its potent immunosuppressive, antifungal, and antineoplastic activities, and late-phase III clinical trials are in progress. The immunosuppressants, cyclosporin A, FK506, and rapamycin, form complexes with either cyclophilin A or FKBP12 that inhibit components of signal-transduction pathways conserved from yeast to man ( Heitman et al., 1992 Schreiber and Crabtree, 1992 Cardenas et al., 1994b). In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM. Overexpression of the PLC1gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. Expression of the TOR-toxic domain leads to a G 1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1.
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