Current Genetics

, Volume 56, Issue 6, pp 479–493

TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae

  • Lieu T. Tran
  • Ruth W. Wang’ondu
  • Jessica B. Weng
  • Grace W. Wanjiku
  • Chi M. Fong
  • Andrew C. Kile
  • Deanna M. Koepp
  • Jennifer K. Hood-DeGrenier
Research Article

Abstract

The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild-type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression.

Keywords

Rapamycin Kip3 Clb5 Cell cycle S phase Microtubules 

Supplementary material

294_2010_316_MOESM1_ESM.doc (882 kb)
Supplementary material 1 (DOC 882 kb)

References

  1. Archambault V, Buchler NE, Wilmes GM, Jacobson MD, Cross FR (2005) Two-faced cyclins with eyes on the targets. Cell Cycle 4:125–130PubMedGoogle Scholar
  2. Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, Hall MN (1996) TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell 7:25–42PubMedGoogle Scholar
  3. Bazan JF (1996) Helical fold prediction for the cyclin box. Proteins 24:1–17CrossRefPubMedGoogle Scholar
  4. Bjornsti MA, Houghton PJ (2004) The TOR pathway: a target for cancer therapy. Nat Rev Cancer 4:335–348CrossRefPubMedGoogle Scholar
  5. Boeke JD, Truehart J, Natsoulis G, Fink G (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Meth Enzymol 154:164–175CrossRefPubMedGoogle Scholar
  6. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132CrossRefPubMedGoogle Scholar
  7. Brown NR, Noble ME, Endicott JA, Johnson LN (1999) The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat Cell Biol 1:438–443CrossRefPubMedGoogle Scholar
  8. Chan TF, Carvalho J, Riles L, Zheng XF (2000) A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR). Proc Natl Acad Sci USA 97:13227–13232CrossRefPubMedGoogle Scholar
  9. Choi JH, Adames NR, Chan TF, Zeng C, Cooper JA, Zheng XF (2000) TOR signaling regulates microtubule structure and function. Curr Biol 10:861–864CrossRefPubMedGoogle Scholar
  10. Cottingham FR, Gheber L, Miller DL, Hoyt MA (1999) Novel roles for Saccharomyces cerevisiae mitotic spindle motors. J Cell Biol 147:335–350CrossRefPubMedGoogle Scholar
  11. Crespo JL, Hall MN (2002) Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol Mol Biol Rev 66:579–591CrossRefPubMedGoogle Scholar
  12. Cross FR, Jacobson MD (2000) Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin. Mol Cell Biol 20:4782–4790CrossRefPubMedGoogle Scholar
  13. Cross FR, Yuste-Rojas M, Gray S, Jacobson MD (1999) Specialization and targeting of B-type cyclins. Mol Cell 4:11–19CrossRefPubMedGoogle Scholar
  14. Donaldson AD, Raghuraman MK, Friedman KL, Cross FR, Brewer BJ, Fangman WL (1998) CLB5-dependent activation of late replication origins in S. cerevisiae. Mol Cell 2:173–182CrossRefPubMedGoogle Scholar
  15. Epstein CB, Cross FR (1992) CLB5: a novel B cyclin from budding yeast with a role in S phase. Genes Dev 6:1695–1706CrossRefPubMedGoogle Scholar
  16. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, Dow S, Lucau-Danila A, Anderson K, Andre B, Arkin AP, Astromoff A, El-Bakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Guldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kotter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang CY, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391CrossRefPubMedGoogle Scholar
  17. Gibson DG, Aparicio JG, Hu F, Aparicio OM (2004) Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae. Mol Cell Biol 24:10208–10222CrossRefPubMedGoogle Scholar
  18. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96CrossRefPubMedGoogle Scholar
  19. Grandin N, Reed SI (1993) Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis. Mol Cell Biol 13:2113–2125PubMedGoogle Scholar
  20. Gupta ML Jr, Carvalho P, Roof DM, Pellman D (2006) Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle. Nat Cell Biol 8:913–923CrossRefPubMedGoogle Scholar
  21. Hildebrandt ER, Hoyt MA (2000) Mitotic motors in Saccharomyces cerevisiae. Biochim Biophys Acta 1496:99–116CrossRefPubMedGoogle Scholar
  22. Hood JK, Silver PA (1998) Cse1p is required for export of Srp1p/importin-α from the nucleus in Saccharomyces cerevisiae. J Biol Chem 273:35142–35146CrossRefPubMedGoogle Scholar
  23. Hu F, Aparicio OM (2005) Swe1 regulation and transcriptional control restrict the activity of mitotic cyclins toward replication proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 102:8910–8915CrossRefPubMedGoogle Scholar
  24. Huang M, Elledge SJ (1997) Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae. Mol Cell Biol 17:6105–6113PubMedGoogle Scholar
  25. Inoki K, Guan KL (2006) Complexity of the TOR signaling network. Trends Cell Biol 16:206–212CrossRefPubMedGoogle Scholar
  26. Jackson LP, Reed SI, Haase SB (2006) Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6. Mol Cell Biol 26:2456–2466CrossRefPubMedGoogle Scholar
  27. Kuhne C, Linder P (1993) A new pair of B-type cyclins from Saccharomyces cerevisiae that function early in the cell cycle. EMBO J 12:3437–3447PubMedGoogle Scholar
  28. Lew DJ, Weinert T, Pringle JR (1997) Chapter 7: cell cycle control in Saccharomyces cerevisiae. In: Pringle JR, Broach JR, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces cerevisiae. Cold Spring Harbor Laboratory Press, Plainview, pp 607–695Google Scholar
  29. Li JJ, Herskowitz I (1993) Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262:1870–1874CrossRefPubMedGoogle Scholar
  30. Loog M, Morgan DO (2005) Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. Nature 434:104–108CrossRefPubMedGoogle Scholar
  31. Lorenz MC, Heitman J (1995) TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin. J Biol Chem 270:27531–27537CrossRefPubMedGoogle Scholar
  32. Meluh PB, Rose MD (1990) KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60:1029–1041CrossRefPubMedGoogle Scholar
  33. Mendenhall MD, Hodge AE (1998) Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1191–1243PubMedGoogle Scholar
  34. Nakashima A, Maruki Y, Imamura Y, Kondo C, Kawamata T, Kawanishi I, Takata H, Matsuura A, Lee KS, Kikkawa U, Ohsumi Y, Yonezawa K, Kamada Y (2008) The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase. PLoS ONE 3:e2223CrossRefPubMedGoogle Scholar
  35. Nigg EA (1993) Targets of cyclin-dependent protein kinases. Curr Opin Cell Biol 5:187–193CrossRefPubMedGoogle Scholar
  36. Pellman D, Bagget M, Tu YH, Fink GR, Tu H (1995) Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae. J Cell Biol 130:1373–1385CrossRefPubMedGoogle Scholar
  37. Putnam CD, Jaehnig EJ, Kolodner RD (2009) Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae. DNA Repair 8:974–982CrossRefPubMedGoogle Scholar
  38. Reinke A, Chen JC, Aronova S, Powers T (2006) Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p. J Biol Chem 281:31616–31626CrossRefPubMedGoogle Scholar
  39. Reneke JE, Blumer KJ, Courchesne WE, Thorner J (1988) The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell 55:221–234CrossRefPubMedGoogle Scholar
  40. Sanchez Y, Desany BA, Jones WJ, Liu Q, Wang B, Elledge SJ (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271:357–360CrossRefPubMedGoogle Scholar
  41. Schmelzle T, Hall MN (2000) TOR, a central controller of cell growth. Cell 103:253–262CrossRefPubMedGoogle Scholar
  42. Schreiber SL (1991) Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251:283–287CrossRefPubMedGoogle Scholar
  43. Schulman BA, Lindstrom DL, Harlow E (1998) Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc Natl Acad Sci USA 95:10453–10458CrossRefPubMedGoogle Scholar
  44. Schwob E, Nasmyth K (1993) CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev 7:1160–1175CrossRefPubMedGoogle Scholar
  45. Segal M, Clarke DJ, Reed SI (1998) Clb5-associated kinase activity is required early in the spindle pathway for correct preanaphase nuclear positioning in Saccharomyces cerevisiae. J Cell Biol 143:135–145CrossRefPubMedGoogle Scholar
  46. Segal M, Clarke DJ, Maddox P, Salmon ED, Bloom K, Reed SI (2000) Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast. J Cell Biol 148:441–452CrossRefPubMedGoogle Scholar
  47. Shen C, Lancaster CS, Shi B, Guo H, Thimmaiah P, Bjornsti MA (2007) TOR signaling is a determinant of cell survival in response to DNA damage. Mol Cell Biol 27:7007–7017CrossRefPubMedGoogle Scholar
  48. Sherman F (1991) Getting started with yeast. In: Guthrie C, Fink GR (eds) Guide to yeast genetics and molecular biology (Methods in Enzymology). Academic Press, Inc., Boston, pp 12–15Google Scholar
  49. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  50. Sproul LR, Anderson DJ, Mackey AT, Saunders WS, Gilbert SP (2005) Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends. Curr Biol 15:1420–1427CrossRefPubMedGoogle Scholar
  51. Straight AF, Marshall WF, Sedat JW, Murray AW (1997) Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 277:574–578CrossRefPubMedGoogle Scholar
  52. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294:2364–2368CrossRefPubMedGoogle Scholar
  53. Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow JD, Shah K, Shokat KM, Morgan DO (2003) Targets of the cyclin-dependent kinase Cdk1. Nature 425:859–864CrossRefPubMedGoogle Scholar
  54. Varga V, Helenius J, Tanaka K, Hyman AA, Tanaka TU, Howard J (2006) Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner. Nat Cell Biol 8:957–962CrossRefPubMedGoogle Scholar
  55. Wang PJ, Chabes A, Casagrande R, Tian XC, Thelander L, Huffaker TC (1997) Rnr4p, a novel ribonucleotide reductase small-subunit protein. Mol Cell Biol 17:6114–6121PubMedGoogle Scholar
  56. Wilmes GM, Archambault V, Austin RJ, Jacobson MD, Bell SP, Cross FR (2004) Interaction of the S-phase cyclin Clb5 with an “RXL” docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch. Genes Dev 18:981–991CrossRefPubMedGoogle Scholar
  57. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484CrossRefPubMedGoogle Scholar
  58. Xie MW, Jin F, Hwang H, Hwang S, Anand V, Duncan MC, Huang J (2005) Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. Proc Natl Acad Sci USA 102:7215–7220CrossRefPubMedGoogle Scholar
  59. Zeng X, Kahana JA, Silver PA, Morphew MK, McIntosh JR, Fitch IT, Carbon J, Saunders WS (1999) Slk19p is a centromere protein that functions to stabilize mitotic spindles. J Cell Biol 146:415–425CrossRefPubMedGoogle Scholar
  60. Zinzalla V, Graziola M, Mastriani A, Vanoni M, Alberghina L (2007) Rapamycin-mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae. Mol Microbiol 63:1482–1494CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Lieu T. Tran
    • 1
  • Ruth W. Wang’ondu
    • 1
  • Jessica B. Weng
    • 1
  • Grace W. Wanjiku
    • 1
  • Chi M. Fong
    • 2
  • Andrew C. Kile
    • 2
    • 3
  • Deanna M. Koepp
    • 2
  • Jennifer K. Hood-DeGrenier
    • 1
  1. 1.Department of Biological SciencesWellesley CollegeWellesleyUSA
  2. 2.Department of Genetics, Cell Biology, and DevelopmentUniversity of MinnesotaMinneapolisUSA
  3. 3.Department of Chemical and Systems BiologyStanford University School of MedicineStanfordUSA

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