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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/24344203
Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway - PubMed Skip to main page content
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. 2014 Mar;34(5):794-806.
doi: 10.1128/MCB.01473-13. Epub 2013 Dec 16.

Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway

Dana Laor  1 , Adiel Cohen, Metsada Pasmanik-Chor, Varda Oron-Karni, Martin Kupiec, Ronit Weisman
Affiliations

Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway

Dana Laor et al. Mol Cell Biol. 2014 Mar.

Erratum in

  • Mol Cell Biol. 2014 Apr;34(8):1535

Abstract

TOR proteins reside in two distinct complexes, TOR complexes 1 and 2 (TORC1 and TORC2), that are central for the regulation of cellular growth, proliferation, and survival. TOR is also the target for the immunosuppressive and anticancer drug rapamycin. In Schizosaccharomyces pombe, disruption of the TSC complex, mutations in which can lead to the tuberous sclerosis syndrome in humans, results in a rapamycin-sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7(+), a member of the family of 2-oxoglutarate-Fe(II)-dependent oxygenase genes. The transcript level of isp7(+) is negatively regulated by TORC1 but positively regulated by TORC2. Yet we find extensive similarity between the transcriptome of cells disrupted for isp7(+) and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression in a fashion similar to that of TORC1 and opposite that of TORC2. Overexpression of isp7(+) induces TORC1-dependent phosphorylation of ribosomal protein Rps6 while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7(+)-dependent regulatory loops that affect both TORC1 and TORC2.

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Figures

FIG 1
FIG 1
TORC1 or TORC2 is essential for growth on proline in the absence of TSC. (A) tsc mutant cells are sensitive to rapamycin when the nitrogen source is proline. Serial dilutions of exponentially growing wild-type (WT) and Δtsc1 or Δtsc2 mutant cells were spotted on minimal (EMM) or proline medium, in the presence or absence of rapamycin (R). (B) TORC2-Gad8 is required for growth on proline in the absence of Tsc1 or Tsc2. Serial dilutions of the indicated strains were performed as described for panel A. (C) tor2SE but not tor1SE suppresses rapamycin sensitivity of tsc mutant cells. Serial dilutions of the indicated strains were performed as described for panel A.
FIG 2
FIG 2
The nitrogen starvation response gene isp7+ suppresses rapamycin sensitivity of tsc mutant cells. (A) Overexpression of isp7+ rescues rapamycin sensitivity of tsc mutant cells. Cells lacking either tsc1+ or tsc2+ were transformed with pREP3X-isp7+ and streaked on minimal medium (EMM) or proline medium with or without rapamycin (R). Δtsc1 or Δtsc2 mutant cells containing vector only were used as a control. (B) Synthetic lethality between Δtsc1/2 and Δisp7. Serial dilutions of the indicated strains on minimal (EMM) or proline medium. (C) Isp7 is preferentially transcribed upon shift to proline. Wild-type (WT) or Δtsc2 cells were grown to mid-log phase in minimal medium and shifted into proline or minimal medium with or without rapamycin. Samples were taken after 15 and 60 min. Total RNA was extracted and analyzed by Northern blotting using a probe against isp7+. rRNA was used as a loading control. (D) The protein level of Isp7 is transiently induced in response to poor nitrogen source. Western analysis of Isp7-HA in wild-type cells in minimal medium and after a shift to proline. Samples were taken after 15 and 60 min. Cdc2 was used as a loading control. (E) Northern blot analysis of isp7+ in wild-type versus Δtor1 cells. Cells were treated as described for panel C. (F) Expression of isp7-LacZ fusion constructs in TORC2 mutant cells. Wild-type and cells disrupted for specific subunits of TORC2, tor1+, sin1+, or ste20+, containing LacZ driven by the isp7+ promoter were grown to mid-log phase. β-Galactosidase activity was measured from protein extracts.
FIG 3
FIG 3
Isp7 negatively regulates transcription of the nitrogen starvation-induced amino acid permeases per1+, put4+, and isp5+ but positively regulates cat1+. (A to D) Northern blot analysis of the transcription levels of isp7+, per1+, put4+, isp5+, and cat1+. rRNA was used as a loading control in all samples. (A and C) The indicated strains were grown to mid-log phase at 25°C (0 h), shifted to 32°C, and incubated for 4 h (4 h) (restrictive conditions) before extraction of RNA. For the blot presented in panel C, RNA levels were quantified relative to the rRNA using Gelquant software. All the indicated strains were grown on minimal medium.
FIG 4
FIG 4
Isp7 positively regulates arginine uptake. (A) Serial dilutions of the indicated strains on minimal medium (EMM) plates with or without 60 μg/ml canavanine or 200 μg/ml thialysine. (B) Arginine uptake assays. The indicated strains were grown to mid-log phase on minimal medium. (C) Serial dilutions of the indicated strains on EMM or plates containing 1.14 mM arginine as the sole nitrogen source. (D) The mutant allele rhb1 GS does not restore canavanine sensitivity in Δisp7 cells. Strains were spotted on minimal medium with or without 60 μg/ml canavanine. (E and F) Overexpression of isp7+ compensates for the lack of tsc2+. Cells lacking tsc2+ were transformed with the isp7+ plasmid. Overexpression of isp7+ induced sensitivity to canavanine (60 μg/ml) (E) and restored uptake of radioactively labeled arginine (F).
FIG 5
FIG 5
Isp7 negatively regulates proline uptake. (A to D) Proline or arginine uptake in cells grown in the presence of NH4+ (A, C, and D) or in the presence of proline (B). When rapamycin was used (B), cells were shifted to proline with or without rapamycin (R) for 1 h. Uptake of arginine or proline was measured at 25°C and 4 h after a shift to 32°C (restrictive temperature) (C and D).
FIG 6
FIG 6
Extensive overlap between genes upregulated by loss of Isp7, loss of Tor2, or nitrogen starvation. Venn diagrams presenting overlaps between genes that are upregulated at least 1.5-fold in Δisp7 cells and genes upregulated in tor2-ts6 (A) or under nitrogen starvation (−N) (B).
FIG 7
FIG 7
Overexpression of isp7+ induces Rps6 phosphorylation toward TORC1. Wild-type cells (A) or cells lacking tsc2+ (B) transformed with the isp7+ plasmid or vector only were grown to mid-log phase in minimal medium (M) and shifted into medium without nitrogen (−N) or with proline instead of ammonia (P). Samples were taken after 15 or 30 min. (C) Wild-type cells or cells lacking isp7+ were grown to mid-log phase in minimal medium (M) and shifted to proline for 15 min (P). (D) Wild-type cells or cells lacking tsc2+ transformed with the isp7+ plasmid were grown to mid-log phase in minimal medium (M) and shifted to proline for 30 min (P). Cells were collected 30 min following addition of rapamycin (R). (E) Wild-type, tor1SE, or tor2SE cells transformed with isp7+ were treated as above. Rapamycin was used at a final concentration of 200 ng/ml. The double mutant strain Δrps601 Δrps602SSAA (rps601/2) was used as a negative control. Phosphorylation of Rps6 (Rps6-P) was detected by immunoblotting with the anti-PAS antibody. Tubulin and Rps6 were used as loading controls.
FIG 8
FIG 8
Effects of the Gtr1/2 complex or the Isp7 oxygenase domain on Isp7-dependent activities. (A) Genetic interactions between Δisp7 and Δgtr1/2. Serial dilutions of the indicated strains on rich (YE), minimal (EMM), or proline medium with or without 60 μg/ml canavanine. (B) Overexpression of isp7+ induced Rps6 phosphorylation independent of Gtr1/2. Wild-type cells or cells lacking gtr1+ transformed with the isp7+ plasmid or vector only were grown to mid-log phase in minimal medium (M) and shifted into proline medium (P) for 15 min. Tubulin and Rps6 were used as loading controls. (C) The oxygenase domain of isp7+ is required to induce rapamycin resistance. Cells lacking tsc2+ were transformed with multicopy plasmids containing tsc2+, isp7+, isp7H276A, or vector only. Serial dilutions were performed on minimal plates (EMM) with or without 60 μg/ml canavanine or proline plates with rapamycin (R). (D) Overexpression of isp7H276A induces Rps6 phosphorylation. Cells lacking tsc2+ transformed with plasmids containing isp7+, isp7H276A, or vector only were grown to mid-log phase in minimal medium (M) and shifted into proline medium (P) for 15 min. The double mutant Δrps601 Δrps602SSAA (rps601/2) strain was used as a negative control. Tubulin was used as a loading control.
FIG 9
FIG 9
Isp7 inhibits TORC2-Gad8 activity in vitro and in vivo. (A) Gad8 shows a time-dependent, Tor1-dependent, and kinase active site-dependent phosphorylation toward Fkh2. Protein extracts were immunoprecipitated with anti-HA antibody, and the kinase assay was performed using Fkh2-GST as the substrate. The kinase reaction was stopped after 4 or 15 min of incubation. Phosphorylation of Fkh2-GST was detected with anti-PAS antibody. Gad8-KD (Gad8-K259D) is a kinase-dead allele. A nontagged strain was used as a negative control (No tag). (B) Gad8-dependent kinase activity is affected by the levels of isp7+. Gad8 was immunoprecipitated from cells lacking isp7+ (Δisp7) or overexpressing isp7+ (WT+isp7), and kinase assays were performed as described for panel A. (C) TORC2-dependent phosphorylation of Gad8-Ser456 is reduced by overexpression of isp7+. Wild-type, Δisp7, Δtor1, or Δgad8 cells or cells overexpressing isp7+ (WT+isp7+) or an empty vector (WT+Vector) were grown to mid-log phase, and their crude cell lysates were analyzed by immunoblotting with anti-phospho-Ser546 and anti-Gad8 antibodies. Actin was used as a loading control.
FIG 10
FIG 10
Working model. TORC1 and TORC2 regulate amino acid permease transcription and amino acid uptake via isp7+-dependent regulatory loops. TORC1 and TORC2 oppositely regulate the transcription of isp7+. Overexpression of isp7+ induces phosphorylation of Rps6 in a TORC1-dependent manner but inhibits TORC2-dependent phosphorylation and activation of Gad8.
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References

    1. Soulard A, Cohen A, Hall MN. 2009. TOR signaling in invertebrates. Curr. Opin. Cell Biol. 21:825–836. 10.1016/j.ceb.2009.08.007 - DOI - PubMed
    1. Zoncu R, Efeyan A, Sabatini DM. 2011. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12:21–35. 10.1038/nrm3025 - DOI - PMC - PubMed
    1. Heitman J, Movva NR, Hall MN. 1991. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905–909. 10.1126/science.1715094 - DOI - PubMed
    1. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D, Oppliger W, Jenoe P, Hall MN. 2002. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10:457–468. 10.1016/S1097-2765(02)00636-6 - DOI - PubMed
    1. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. 2004. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14:1296–1302. 10.1016/j.cub.2004.06.054 - DOI - PubMed

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