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. 1999 May 17;18(10):2782–2792. doi: 10.1093/emboj/18.10.2782

Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast.

Y Jiang 1, J R Broach 1
PMCID: PMC1171359  PMID: 10329624

Abstract

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22. Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barbet N. C., Schneider U., Helliwell S. B., Stansfield I., Tuite M. F., Hall M. N. TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell. 1996 Jan;7(1):25–42. doi: 10.1091/mbc.7.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beretta L., Gingras A. C., Svitkin Y. V., Hall M. N., Sonenberg N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 1996 Feb 1;15(3):658–664. [PMC free article] [PubMed] [Google Scholar]
  3. Berset C., Trachsel H., Altmann M. The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4264–4269. doi: 10.1073/pnas.95.8.4264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertram P. G., Zeng C., Thorson J., Shaw A. S., Zheng X. F. The 14-3-3 proteins positively regulate rapamycin-sensitive signaling. Curr Biol. 1998 Nov 19;8(23):1259–1267. doi: 10.1016/s0960-9822(07)00535-0. [DOI] [PubMed] [Google Scholar]
  5. Brown E. J., Beal P. A., Keith C. T., Chen J., Shin T. B., Schreiber S. L. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature. 1995 Oct 5;377(6548):441–446. doi: 10.1038/377441a0. [DOI] [PubMed] [Google Scholar]
  6. Brunn G. J., Fadden P., Haystead T. A., Lawrence J. C., Jr The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus. J Biol Chem. 1997 Dec 19;272(51):32547–32550. doi: 10.1074/jbc.272.51.32547. [DOI] [PubMed] [Google Scholar]
  7. Brunn G. J., Hudson C. C., Sekulić A., Williams J. M., Hosoi H., Houghton P. J., Lawrence J. C., Jr, Abraham R. T. Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science. 1997 Jul 4;277(5322):99–101. doi: 10.1126/science.277.5322.99. [DOI] [PubMed] [Google Scholar]
  8. Burnett P. E., Barrow R. K., Cohen N. A., Snyder S. H., Sabatini D. M. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1432–1437. doi: 10.1073/pnas.95.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cardenas M. E., Heitman J. FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity. EMBO J. 1995 Dec 1;14(23):5892–5907. doi: 10.1002/j.1460-2075.1995.tb00277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen J., Peterson R. T., Schreiber S. L. Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun. 1998 Jun 29;247(3):827–832. doi: 10.1006/bbrc.1998.8792. [DOI] [PubMed] [Google Scholar]
  11. Di Como C. J., Arndt K. T. Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 1996 Aug 1;10(15):1904–1916. doi: 10.1101/gad.10.15.1904. [DOI] [PubMed] [Google Scholar]
  12. Fernandez-Sarabia M. J., Sutton A., Zhong T., Arndt K. T. SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2, and HCS26 RNAs during late G1. Genes Dev. 1992 Dec;6(12A):2417–2428. doi: 10.1101/gad.6.12a.2417. [DOI] [PubMed] [Google Scholar]
  13. Hall M. N. The TOR signalling pathway and growth control in yeast. Biochem Soc Trans. 1996 Feb;24(1):234–239. doi: 10.1042/bst0240234. [DOI] [PubMed] [Google Scholar]
  14. Hara K., Yonezawa K., Weng Q. P., Kozlowski M. T., Belham C., Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem. 1998 Jun 5;273(23):14484–14494. doi: 10.1074/jbc.273.23.14484. [DOI] [PubMed] [Google Scholar]
  15. Healy A. M., Zolnierowicz S., Stapleton A. E., Goebl M., DePaoli-Roach A. A., Pringle J. R. CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol. 1991 Nov;11(11):5767–5780. doi: 10.1128/mcb.11.11.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heitman J., Movva N. R., Hall M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991 Aug 23;253(5022):905–909. doi: 10.1126/science.1715094. [DOI] [PubMed] [Google Scholar]
  17. Heitman J., Movva N. R., Hiestand P. C., Hall M. N. FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1948–1952. doi: 10.1073/pnas.88.5.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helliwell S. B., Schmidt A., Ohya Y., Hall M. N. The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton. Curr Biol. 1998 Nov 5;8(22):1211–1214. doi: 10.1016/s0960-9822(07)00511-8. [DOI] [PubMed] [Google Scholar]
  19. Helliwell S. B., Wagner P., Kunz J., Deuter-Reinhard M., Henriquez R., Hall M. N. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell. 1994 Jan;5(1):105–118. doi: 10.1091/mbc.5.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jefferies H. B., Fumagalli S., Dennis P. B., Reinhard C., Pearson R. B., Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J. 1997 Jun 16;16(12):3693–3704. doi: 10.1093/emboj/16.12.3693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunz J., Henriquez R., Schneider U., Deuter-Reinhard M., Movva N. R., Hall M. N. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. doi: 10.1016/0092-8674(93)90144-f. [DOI] [PubMed] [Google Scholar]
  22. Lorenz M. C., Heitman J. TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin. J Biol Chem. 1995 Nov 17;270(46):27531–27537. doi: 10.1074/jbc.270.46.27531. [DOI] [PubMed] [Google Scholar]
  23. Luke M. M., Della Seta F., Di Como C. J., Sugimoto H., Kobayashi R., Arndt K. T. The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase. Mol Cell Biol. 1996 Jun;16(6):2744–2755. doi: 10.1128/mcb.16.6.2744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murata K., Wu J., Brautigan D. L. B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10624–10629. doi: 10.1073/pnas.94.20.10624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muslin A. J., Tanner J. W., Allen P. M., Shaw A. S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell. 1996 Mar 22;84(6):889–897. doi: 10.1016/s0092-8674(00)81067-3. [DOI] [PubMed] [Google Scholar]
  26. Proud C. G. p70 S6 kinase: an enigma with variations. Trends Biochem Sci. 1996 May;21(5):181–185. [PubMed] [Google Scholar]
  27. Ronne H., Carlberg M., Hu G. Z., Nehlin J. O. Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol Cell Biol. 1991 Oct;11(10):4876–4884. doi: 10.1128/mcb.11.10.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rubin G. M. The nucleotide sequence of Saccharomyces cerevisiae 5.8 S ribosomal ribonucleic acid. J Biol Chem. 1973 Jun 10;248(11):3860–3875. [PubMed] [Google Scholar]
  29. Schmidt A., Beck T., Koller A., Kunz J., Hall M. N. The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J. 1998 Dec 1;17(23):6924–6931. doi: 10.1093/emboj/17.23.6924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schmidt A., Bickle M., Beck T., Hall M. N. The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell. 1997 Feb 21;88(4):531–542. doi: 10.1016/s0092-8674(00)81893-0. [DOI] [PubMed] [Google Scholar]
  31. Schmidt A., Kunz J., Hall M. N. TOR2 is required for organization of the actin cytoskeleton in yeast. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13780–13785. doi: 10.1073/pnas.93.24.13780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scott P. H., Brunn G. J., Kohn A. D., Roth R. A., Lawrence J. C., Jr Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7772–7777. doi: 10.1073/pnas.95.13.7772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shu Y., Yang H., Hallberg E., Hallberg R. Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A. Mol Cell Biol. 1997 Jun;17(6):3242–3253. doi: 10.1128/mcb.17.6.3242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sneddon A. A., Cohen P. T., Stark M. J. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J. 1990 Dec;9(13):4339–4346. doi: 10.1002/j.1460-2075.1990.tb07883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stan R., McLaughlin M. M., Cafferkey R., Johnson R. K., Rosenberg M., Livi G. P. Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue. J Biol Chem. 1994 Dec 23;269(51):32027–32030. [PubMed] [Google Scholar]
  36. Sutton A., Immanuel D., Arndt K. T. The SIT4 protein phosphatase functions in late G1 for progression into S phase. Mol Cell Biol. 1991 Apr;11(4):2133–2148. doi: 10.1128/mcb.11.4.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thomas G., Hall M. N. TOR signalling and control of cell growth. Curr Opin Cell Biol. 1997 Dec;9(6):782–787. doi: 10.1016/s0955-0674(97)80078-6. [DOI] [PubMed] [Google Scholar]
  38. Thorson J. A., Yu L. W., Hsu A. L., Shih N. Y., Graves P. R., Tanner J. W., Allen P. M., Piwnica-Worms H., Shaw A. S. 14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity. Mol Cell Biol. 1998 Sep;18(9):5229–5238. doi: 10.1128/mcb.18.9.5229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vandenbol M., Jauniaux J. C., Grenson M. The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol Gen Genet. 1990 Jul;222(2-3):393–399. doi: 10.1007/BF00633845. [DOI] [PubMed] [Google Scholar]
  40. Zheng X. F., Florentino D., Chen J., Crabtree G. R., Schreiber S. L. TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell. 1995 Jul 14;82(1):121–130. doi: 10.1016/0092-8674(95)90058-6. [DOI] [PubMed] [Google Scholar]
  41. van Zyl W., Huang W., Sneddon A. A., Stark M., Camier S., Werner M., Marck C., Sentenac A., Broach J. R. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Nov;12(11):4946–4959. doi: 10.1128/mcb.12.11.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. von Manteuffel S. R., Gingras A. C., Ming X. F., Sonenberg N., Thomas G. 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4076–4080. doi: 10.1073/pnas.93.9.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]

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