Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast

Abstract

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.

Figure 1

TOR1 has an intrinsic protein kinase activity. (A) TOR1 was immunoprecipitated from protein extracts prepared from spheroplasts of CAY6 yeast cells (MATa ura3–52 trp1 lys2–801 leu2Δ1 his3Δ200 can1 pep4::HIS3 prb1Δ1.6R Δtor1::G418) overexpressing HA-TOR1 from the GAL1 promoter. TOR1 immunoprecipitates were preincubated for 15 min at 4°C with kinase buffer in the absence (−) or presence of FKBP12-FK506 (F/F), FKBP12-rapamycin (F/R), or 250 nM or 500 nM Wortmannin (W250 and W500, respectively). Phosphorylation reactions were started by addition of a radioactive mix containing 10 μM ATP, 10 μCi γ³²P-ATP, 1 μg PHAS-I, and 10 mM MgCl₂ or MnCl₂ as indicated. (B) Immunoprecipitates of the wild-type (WT) TOR1, the S1972I (SI), or the D2275A (DA) TOR1 mutant enzymes were assayed for protein kinase activity with PHAS-I as substrate and MnCl₂ as cofactor. Phosphorylation reactions were performed in the absence (−) or presence of FKBP12-rapamycin (F/R), or 500 nM wortmannin (W) as indicated in panel A. (C) TOR1 Western blot of the protein extracts employed for the immunoprecipitates performed in panel B.

Figure 2

TOR1 kinase domain is essential for TOR1 function and rapamycin resistance, and overexpression of TOR1 kinase-inactive mutant inhibits growth. (A) A tor1 Δsrk1 yeast strain (CAY7) was transformed with 2μ plasmids expressing wild-type TOR1, the S1972I rapamycin-resistant TOR1 mutant, the D2275A kinase-inactive TOR1 mutant, or the S1972I/D2275A double-mutant TOR1 protein. Plasmids expressing TOR1 function complement the tor1 srk1 conditional lethal phenotype and rescue cell viability at 39°C, whereas plasmids lacking TOR1 function do not. Cells were grown for 72 h at 30 and 39°C. (B) Wild-type strain JK9–3da was transformed with 2μ plasmids expressing wild-type TOR1, the kinase-inactive D2275A mutant TOR1, four independent rapamycin-resistant TOR1 mutant isolates (S1972I), and two independent isolates of the S1972I/D2275A TOR1 double mutant. Cells were grown on medium containing 1 μg/ml rapamycin for 72 h at 30°C. (C) Yeast strain CAY6 expressing wild-type TOR1 (TOR1), the TOR1 kinase-inactive mutant (TOR1 D2275A), the TOR1 rapamycin-resistant mutant (TOR1 S1972I), or the TOR1 kinase-inactive rapamycin-resistant double mutant (TOR1 S1972I/D2275A) from the galactose-inducible GAL1 promoter were grown on glucose or galactose medium lacking tryptophan for 96 h at 30°C.

Figure 3

TOR1 structural features required for in vivo function and overexpression toxicity. (A) Diagram of full-length TOR1 with the FRB and kinase domains depicted by open boxes. The S1972I rapamycin- resistant mutation is indicated by a black circle, and the kinase-inactive mutation D2275A by a star. The specific amino acids (AA) mutated or deleted in each mutant, and the ability of these TOR1 derivatives to inhibit growth when overexpressed, or to provide TOR1 function and complement the synthetic lethal growth defect at 39°C in a tor1 Δsrk1 mutant strain (CAY7) are indicated. N and C indicate the amino- and carboxy termini of TOR1, and 1 and 2470 indicate the positions of the first and last amino acids, respectively, of the TOR1 protein. (B) TOR1 mutants and deletion derivates were expressed in the Δtor1 strainCAY6, and the ability to inhibit growth on galactose media was tested. Cells were grown on glucose or galactose medium lacking tryptophan for 96 h at 30°C. DA and SI indicate the D2275A kinase-inactive and the S1972I rapamycin resistance mutations, respectively. The nubers on the plate designation correspond to the construct number indicated to the left in panel A. (C) TOR1 deletion mutant proteins were expressed in the Δtor1 strain CAY6 and detected by Western blot with an α-HA monoclonal antibody. Numbers above the Western blot correspond to the construct numbers listed to the left in panel A. Numbers at left indicate molecular mass in kilodaltons of marker proteins.

Figure 4

A central region of TOR1 is required for toxicity of TOR1 deletion derivatives. (A) Diagram of TOR1 internal deletions with the FRB and kinase domains depicted by boxes. The D2275A kinase-inactive mutation is represented by a star. The specific amino acids (AA) deleted in each mutant, the ability to inhibit growth when overexpressed, and the ability to complement and restore growth at 39°C in a synthetic lethal tor1 Δsrk1 mutant strain (CAY7) are indicated. (B) TOR1 deletion mutants were expressed in the Δtor1 strain CAY6, and ability to inhibit growth on galactose medium was assessed. Cells were grown on glucose or galactose medium lacking tryptophan for 96 h at 30°C. DA indicates the D2275A kinase-active site mutation. The numbers on the plate designation correspond to the construct number indicated to the left in panel A. (C) TOR1 deletion mutant proteins were expressed in the Δtor1 strain CAY6 and detected by Western blot with α-HA antibody. Numbers above the Western blot correspond to the construct numbers listed to the left in panel A. Numbers at the left indicate molecular mass in kilodaltons.

Figure 5

Identification of a TOR1-toxic effector domain distinct from the FRB and kinase domains. (A) Diagram of TOR1 fragments with the FRB and kinase domains depicted by open boxes. The rapamycin-resistant mutation S1972I is indicated by a black circle, and the kinase-inactive mutation D2275A is indicated by a star. The specific amino acids expressed, the ability to inhibit growth when overexpressed, and the ability to complement growth at 39°C in a tor1 Δsrk1 mutant strain (CAY7) are indicated. (B) TOR1 fragments were expressed in the Δtor1 strain CAY6, and the ability to inhibit growth on galactose medium was tested. Cells were grown on glucose or galactose medium lacking tryptophan for 96 h at 30°C. DA and SI indicate the D2275A and the S1972I rapamycin resistance mutations, respectively. The numbers on the plate designation correspond to the construct number indicated to the left in panel A. (C) TOR1 fragments were expressed in the Δtor1 strain CAY6, and proteins were detected by Western blot with α-HA antibody. Numbers above the Western blot correspond to the construct numbers listed to the left in panel A. Numbers at left indicate molecular mass in kilodaltons.

Figure 6

Overexpression of TOR1 and TOR2 effector domains inhibits growth and is suppressed by increased expression of wild-type TOR1. Yeast Δtor1 strain CAY6 expressing wild-type TOR1 or the TOR1 or TOR2 central toxic domain from the galactose-inducible GAL1 promoter was cotransformed with a 2μ URA vector alone (−) or vector expressing wild-type TOR1 (+). Cells were grown on glucose or galactose medium lacking tryptophan and uracil for 96 h at 30°C. (A) Wild-type TOR1 and vector; (B) TOR1 fragment 1207–1961 and vector; (C) TOR1 frag-ment 1207–1961 and 2μ TOR1; (D) TOR2 fragment 1216–1782 and vector; (E) TOR2 fragment 1216–1782 and 2μ TOR1; (F) TOR2 fragment 1216–1964 and vector; (G) TOR2 fragment 1216–1964 and 2μ TOR1.

Figure 7

Summary of the TOR deletion derivatives used to define a central toxic effector domain common to TOR1 and TOR2. Diagram of full-length TOR1, TOR1 deletion mutants, and TOR1 and TOR2 fragments. The toxic effector, FRB, and kinase domains are depicted by boxes. The rapamycin-resistant mutation S1972I is indicated by a black circle, and the kinase-inactive D2275A mutation is indicated by a star. The specific amino acids deleted in each mutant, or the amino acids included in each fragment, protein kinase activity employing PHAS-I as substrate, ability to inhibit growth, and ability to complement the conditional synthetic lethal phenotype at 39°C in a tor1 Δsrk1 deletion strain (CAY7) are indicated. N.D. indicates not determined.

Figure 8

Overexpression of phospholipase C suppresses the toxicity of the TOR1 toxic domain. Yeast strain CAY6 was cotransformed with plasmids expressing the TOR1-toxic domains (AA 1207–1961 in panel A and AA 1207–2340 in panel B), and plasmids overexpressing the STT4 (PGALHA-STT4), MSS4 (pGMSS4), and PLC1 (pJF137), proteins, respectively, under the control of the galactose promoter, were grown in minimal medium containing glucose or galactose for 4 d at 30°C.

Figure 9

The TOR toxic domain is conserved and shares identity with regions of the PIK family members, ATR, Rad3, mei-41, and ATM. The TOR1 (amino acids 1376–1614), TOR2 (amino acids 1383–1620), and mTOR/RAFT/FRAP-toxic domains (amino acids 1427–1663) were used for BLAST searches of the NCBI database, revealing sequence similarity with ATR (amino acids 1688–1923) and Rad3 (amino acids 725–950), mei-41 (amino acids 1396–1623), and ATM (amino acids 1985–2148). The alignment shown here was generated using the ClustalW program of MacVector software. Residues that are conserved in four of the seven proteins are boxed, shaded, and in bold. Similar residues are boxed.