Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

Abstract

Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.

Fig. 1.

Tor2p L1310P and E2221K show Rhb1-independent growth and delayed nitrogen starvation response. (A) Strains carrying wild-type tor2⁺ (JUp1273) or the activated mutants tor2^L1310P (JUp1274) and tor2^E2221K (JUp1261) were streaked out onto EMM + AL plates or EMM + AL + FOA plates and incubated at 30°C. EMM, Edinburgh minimal medium. (B) JUp1348 (tor2⁺), JUp1350 (tor2^L1310P), and JUp1352 (tor2^E2221K) were grown to mid-log phase, washed twice with water, and adjusted to an A₅₉₅ of 1. Aliquots of 3 μl were spotted onto SSA plates, incubated at 25°C for 2 days, and stained by using I₂ vapor. Note that two sets of clones were used, and both show similar results. (C) JUp1348 (tor2⁺), JUp1350 (tor2^L1310P), and JUp1352 (tor2^E2221K) were grown to mid-log phase, washed twice with water, and resuspended in SSL media. Samples were taken at the indicated times and analyzed with FACS for cell size and cell cycle. Each profile (purple) was overlayed with the profile outline for the wild-type strain (green line) at each time point.

Fig. 2.

Location of activated mutations in Tor2p and conservation of residues. (A) All identified activating mutations are indicated above the linear representation of Tor2p. Clusters I and II are indicated. (B) TOR protein sequences from Schizosaccharomyces pombe (SpTor1 and SpTor2), S. cerevisiae (ScTor1 and ScTor2), Drosophila (dTOR), and human (mTOR) were aligned with MegAlign. Residues identical or similar to SpTor2 are shaded black or gray, respectively. Mutations found in SpTor2 are indicated above the alignment. The repressor domain region of mTOR is indicated by a line under the alignment. Single and double asterisks indicate the AMPK and S6K phosphorylation sites, respectively.

Fig. 3.

rhb1Δ tor2^L1310P and rhb1Δ tor2^E2221K show sensitivity to stresses. (A) JUp1050 (rhb1⁺tor2⁺), JUp1274 (rhb1⁺tor2^L1310P), JUp1275 (rhb1Δtor2^L1310P), JUp1261 (rhb1⁺tor2^E2221K), and JUp1263 (rhb1Δ tor2^E2221K) were streaked on the indicated plates and incubated at the indicated temperatures. (B) JUp1263 (rhb1Δ tor2^E2221K) and JUp1275 (rhb1Δtor2^L1310P) were transformed with either pREP41 (rhb1Δ) or pRPL-SpRhb1 (rhb1⁺), and transformants were streaked onto the indicated plates and incubated at 30°C.

Fig. 4.

mTOR^L1460P and mTOR^E2419K show constitutive activity. (A–C) HEK293 cells were transfected with pCDNA3 (vector), AU1-mTOR (wt, E2419K, or L1460P), or FLAG-Rheb1 (wt or N153T). To detect the phosphorylation of S6K or 4E-BP1, FLAG-S6K or FLAG-4E-BP1 was cotransfected. Cells then were serum-starved overnight and cultured in PBS for 1 h. The protein levels were detected by anti-FLAG (S6K, 4E-BP1, and Rheb), anti-AU-1 (mTOR), or anti-S6 antibody. The phosphorylation levels of S6K, 4E-BP1, and S6 were detected by phospho-specific antibodies. (D) HEK293 cells were transfected with AU1-mTOR (wt, E2419K, or L1460P). Cells were serum-starved overnight and then treated with rapamycin (100 nM). The expression and phosphorylation levels of S6 were detected by specific antibodies. wt, wild type.

Fig. 5.

Kinase activities of mTOR complexes. (A) HEK293 cells transfected with pCDNA3 (vector) or AU1-mTOR (wt, E2419K, or L1460P) were serum-starved overnight and then cultured in PBS for 1h. AU1-mTOR complexes then were immunoprecipitated with anti-AU1 antibody and used for in vitro kinase assays with 4E-BP1 and Akt as substrates. Phosphorylation of substrates was detected by use of the indicated phospho-specific antibodies. Levels of phosphorylation of substrates were quantitated relative to that by mTOR^E2419K and graphed. (B) HEK293 cells were transfected with pCDNA3 (vector) or AU1-mTOR (wt, E2419K, or L1460P) together with Myc-mLST8. Cells were serum-starved overnight and cultured for 1 h in PBS. AU1-mTOR complexes then were immunoprecipitated with anti-AU1 antibody. mTOR were detected by anti-AU1 antibody. mLST8 were detected by anti-Myc antibody. Raptor and Rictor were detected by specific antibodies. (C) HEK293 cells cotransfected with FLAG-mTOR and AU1-mTOR (wt, E2419K, or L1460P) were serum-starved overnight and cultured for 1 h in PBS. mTOR dimers were immunoprecipitated by using anti-FLAG antibody and used for in vitro kinase assays with 4E-BP1 as substrate. FLAG-mTOR and AU1-mTOR were detected by anti-FLAG and anti-AU1 antibodies, respectively. Phosphorylation of substrate was detected by use of anti-phospho-4E-BP1 (Thr 37/46).