Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast.
In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. 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. 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. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. 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. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. 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.
Alarcon
C M
CM
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Heitman
J
J
Cardenas
M E
ME
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D002352:Carrier Proteins; D002455:Cell Division / Q000235:genetics; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D016193:G1 Phase / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D020104:Immunophilins / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020123:Sirolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism; D000077191:Wortmannin; D015003:Yeasts / Q000187:drug effects / Q000502:physiology*
D000730:Androstadienes; D000935:Antifungal Agents; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C083324:TOR1 protein, S cerevisiae; D010738:Type C Phospholipases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D020123:Sirolimus; D000077191:Wortmannin