Phosphatase Targets in TOR Signaling

  • Estela Jacinto
Part of the Methods in Molecular Biology book series (MIMB, volume 365)


Cells undergo growth or increase in mass in the presence of nutrients. A key signaling molecule that responds to the presence of nutrients is the target of rapamycin (TOR). TOR is a highly conserved protein kinase and is the target of the growth inhibitor rapamycin. In response to nutrients, TOR promotes the phosphorylation of its downstream targets, leading to increased protein synthesis and decreased protein turnover. In yeast, a major mechanism for the downstream regulation of TOR effectors is by inhibition of the type 2A-related phosphatase SIT4. TOR negatively regulates SIT4 by promoting the association of SIT4 with TAP42. When TOR is inactivated by rapamycin treatment or nitrogen starvation, downstream effectors of TOR such as the serine/threonine protein kinase NPR1 and the TAP42 interacting protein TIP41 are dephosphorylated in a SIT4-dependent manner. The phosphorylation state of NPR1 and TIP41 provides a convenient readout in yeast to assay for TOR and SIT4 activities under growth-promoting or growth-inhibitory conditions.

Key Words

Growth TOR rapamycin SIT4 NPR1 TIP41 dephosphorylation nitrogen starvation SDS-PAGE isoelectric focusing two-dimensional gel electrophoresis 


  1. 1.
    Jacinto, E. and Hall, M. N. (2003) TOR signalling in bugs, brain and brawn. Nature Rev. Mol. Cell. Biol. 4, 117–126.CrossRefGoogle Scholar
  2. 2.
    Di Como, C. J. and Arndt, K. T. (1996) Nutrients, via the TOR proteins, stimulate the association of TAP42 with type 2A phosphatases. Genes Dev. 10, 1904–1916.PubMedCrossRefGoogle Scholar
  3. 3.
    Jacinto, E., Guo, B., Arndt, K. T., Schmelzle, T., and Hall, M. N. (2001) TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol. Cell 8, 1017–26.PubMedCrossRefGoogle Scholar
  4. 4.
    Beck, T. and Hall, M. N. (1999) The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689–692.PubMedCrossRefGoogle Scholar
  5. 5.
    Brunn, G. J., Hudson, C. C., Sekulic, A., et al. (1997) Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science 277, 99–101.PubMedCrossRefGoogle Scholar
  6. 6.
    Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H. and Sabatini, D. M. (1998) RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA 95, 1432–1437.PubMedCrossRefGoogle Scholar
  7. 7.
    Kim, D. H., Sarbassov dos, D., Ali, S. M., et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175.PubMedCrossRefGoogle Scholar
  8. 8.
    Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992) Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69, 1227–1236.PubMedCrossRefGoogle Scholar
  9. 9.
    Price, D. J., Grove, J. R., Calvo, V., Avruch, J., and Bierer, B. E. (1992) Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinases. Science 257, 973–977.PubMedCrossRefGoogle Scholar
  10. 10.
    Graves, L. M., Bornfeldt, K. E., Argast, G. M., et al. (1995) cAMP-and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc. Natl. Acad. Sci. USA 92, 7222–7226.PubMedCrossRefGoogle Scholar
  11. 11.
    Beretta, L., Gingras, A. C., Svitkin, Y. V., Hall, M. N., and Sonenberg, N. (1996) Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 15, 658–664.PubMedGoogle Scholar
  12. 12.
    Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. (1999) Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin association protein. Proc. Natl. Acad. Sci. USA 96, 4438–4442.PubMedCrossRefGoogle Scholar
  13. 13.
    Schmidt, A., Beck, T., Koller, A., Kunz, J., and Hall, M. N. (1998) The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J. 17, 6924–6931.PubMedCrossRefGoogle Scholar
  14. 14.
    Bertram, P. G., Choi, J. H., Carvalho, J., et al. (2000) Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J Biol. Chem. 275, 35,727–35,733.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Estela Jacinto
    • 1
  1. 1.Department of Physiology and Biophysics UMDNJRobert Wood Johnson Medical SchoolPiscatawayUSA

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