Skip to main content
Springer Nature Link
Log in

Arabidopsis cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm

  • Cell Biology and Morphogenesis
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

CDK:

Cyclin-dependent kinase

ICK/KRP:

Inhibitor/interactor of cyclin-dependent kinase/KIP-related protein

GFP:

Green fluorescent protein

NLS:

Nuclear localization signal

CaMV:

Cauliflower mosaic virus

DAPI:

4′,6-Diamidino-2-phenylinodole

References

  • Ali GS, Golovkin M, Reddy ASN (2003) Nuclear localization and in vivo dynamics of a plant-specific serine/arginine-rich protein. Plant J 36:883–893

    Article  PubMed  CAS  Google Scholar 

  • Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell 14:1293–1309

    Article  PubMed  CAS  Google Scholar 

  • Cleary AL, Fowke LC, Wang H, John PCL (2002) The effect of ICK1, a plant cyclin-dependent kinase inhibitor, on mitosis in living plant cells. Plant Cell Rep 20:814–820

    Article  CAS  Google Scholar 

  • Coelho CM, Dante RA, Sabelli PA, Sun YJ, Dilkes BP, Gordon-Kamm WJ, Larkins BA (2005) Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication. Plant Physiol 138:2323–2336

    Article  PubMed  CAS  Google Scholar 

  • Cokol M, Nair R, Rost B (2000) Finding nuclear localization signals. EMBO Rep 1:411–415

    Article  PubMed  CAS  Google Scholar 

  • Colasanti J, Cho SO, Wick S, Sundaresan V (1993) Localization of the functional P34(Cdc2) homolog of maize in root-tip and stomatal complex cells—association with predicted division sites. Plant Cell 5:1101–1111

    Article  PubMed  CAS  Google Scholar 

  • Davis SJ, Vierstra RD (1998) Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 36:521–528

    Article  PubMed  CAS  Google Scholar 

  • De Veylder L, Beeckman T, Beemster GT, Krols L, Terras F, Landrieu I, van der Schueren E, Maes S, Naudts M, Inze D (2001) Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13:1653–1668

    Article  PubMed  Google Scholar 

  • Dewitte W, Murray JAH (2003) The plant cell cycle. Annu Rev Plant Biol 54:235–264

    Article  PubMed  CAS  Google Scholar 

  • Docquier S, Tillemans V, Deltour R, Motte P (2004) Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants. Chromosoma 112:255–266

    Article  PubMed  CAS  Google Scholar 

  • Fransz P, de Jong JH, Lysak M, Castiglione MR, Schubert I (2002) Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate. PNAS 99:14584–14589

    Article  PubMed  CAS  Google Scholar 

  • Heese-Peck A, Raikhel NV (1998) The nuclear pore complex. Plant Mol Biol 38:145–162

    Article  PubMed  CAS  Google Scholar 

  • Hemsley R, McCutcheon S, Doonan J, Lloyd C (2001) P34(cdc2) kinase is associated with cortical microtubules from higher plant protoplasts. FEBS Lett 508:157–161

    Article  PubMed  CAS  Google Scholar 

  • Hubner S, Smith HMS, Hu W, Chan CK, Rihs HP, Paschal BM, Raikhel NV, Jans DA (1999) Plant importin alpha binds nuclear localization sequences with high affinity and can mediate nuclear import independent of importin beta. J Biol Chem 274:22610–22617

    Article  PubMed  CAS  Google Scholar 

  • Jakoby MJ, Weinl C, Pusch S, Kuijt SJH, Merkle T, Dissmeyer N, Schnittger A (2006) Analysis of the subcellular localization, function, and proteolytic control of the Arabidopsis cyclin-dependent kinase inhibitor ICK1/KRP1. Plant Physiol 141:1293–1305

    Article  PubMed  CAS  Google Scholar 

  • Jasinski S, Perennes C, Bergounioux C, Glab N (2002) Comparative molecular and functional analyses of the tobacco cyclin-dependent kinase inhibitor NtKIS1a and its spliced variant NtKIS1b. Plant Physiol 130:1871–1882

    Article  PubMed  CAS  Google Scholar 

  • Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005) High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP–ORF fusions. Plant J 41:162–174

    Article  PubMed  CAS  Google Scholar 

  • LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E (1997) New functional activities for the p21 family of CDK inhibitors. Genes Dev 11:847–862

    Article  PubMed  CAS  Google Scholar 

  • Lui H, Wang H, Delong C, Fowke LC, Crosby WL, Fobert PR (2000) The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro. Plant J 21:379–385

    Article  PubMed  CAS  Google Scholar 

  • Macara IG (2001) Transport into and out of the nucleus. Microbiol Mol Biol Rev 65:570–594

    Article  PubMed  CAS  Google Scholar 

  • Mattaj IW, Englmeier L (1998) Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 67:265–306

    Article  PubMed  CAS  Google Scholar 

  • Mews M, Sek FJ, Moore R, Volkmann D, Gunning BES, John PCL (1997) Mitotic cyclin distribution during maize cell division: implications for the sequence diversity and function of cyclins in plants. Protoplasma 200:128–145

    Article  CAS  Google Scholar 

  • Moriguchi K, Suzuki T, Ito Y, Yamazaki Y, Niwa Y, Kurata N (2005) Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations. Plant Cell 17:389–403

    Article  PubMed  CAS  Google Scholar 

  • Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–35

    Article  PubMed  CAS  Google Scholar 

  • Ormenese S, Engler JD, De Groodt R, De Veylder L, Inze D, Jacqmard A (2004) Analysis of the spatial expression pattern of seven kip related proteins (KRPs) in the shoot apex of Arabidopsis thaliana. Ann Bot 93:575–580

    Article  PubMed  CAS  Google Scholar 

  • Pines J (1999) Four-dimensional control of the cell cycle. Nat Cell Biol 1:E73-E79

    Article  PubMed  CAS  Google Scholar 

  • Platani M, Goldberg I, Swedlow JR, Lamond AI (2000) In vivo analysis of Cajal body movement, separation, and joining in live human cells. J Cell Biol 151:1561–1574

    Article  PubMed  CAS  Google Scholar 

  • Robbins J, Dilworth SM, Laskey RA, Dingwall C (1991) Two interdependent basic domains in nucleoplasmin nuclear targeting sequence—identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623

    Article  PubMed  CAS  Google Scholar 

  • Schnittger A, Weinl C, Bouyer D, Schobinger U, Hulskamp M (2003) Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death. Plant Cell 15:303–315

    Article  PubMed  CAS  Google Scholar 

  • Sherr CJ, Roberts JM (1999) CDK inhibitors positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512

    PubMed  CAS  Google Scholar 

  • Stals H, Bauwens S, Traas J, VanMontagu M, Engler G, Inze D (1997) Plant CDC2 is not only targeted to the pre-prophase band, but also co-localizes with the spindle, phragmoplast, and chromosomes. FEBS Lett 418:229–234

    Article  PubMed  CAS  Google Scholar 

  • Stepanova L, Leng XH, Parker SB, Harper JW (1996) Mammalian p50(Cdc37) is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4. Genes Dev 10:1491–1502

    Article  PubMed  CAS  Google Scholar 

  • Vandepoele K, Raes J, De Veylder L, Rouze P, Rombauts S, Inze D (2002) Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell 14:903–916

    Article  PubMed  CAS  Google Scholar 

  • Verkest A, Manes CLD, Vercruysse S, Maes S, Van der Schueren E, Beeckman T, Genschik P, Kuiper M, Inze D, De Veylder L (2005) The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during Arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes. Plant Cell 17:1723–1736

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Fowke LC, Crosby WL (1997) A plant cyclin-dependent kinase inhibitor gene. Nature 386:451–452

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Qi Q, Schorr P, Cutler AJ, Crosby WL, Fowke LC (1998) ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid. Plant J 15:501–510

    Article  PubMed  Google Scholar 

  • Wang H, Zhou Y, Gilmer S, Whitwill S, Fowke LC (2000) Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology. Plant J 24:613–623

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Zhou Y, Fowke LC (2006) The emerging importance of CDK inhibitors in the regulation of the plant cell cycle and related processes. Can J Bot 84:640–650

    Article  CAS  Google Scholar 

  • Weingartner M, Binarova P, Drykova D, Schweighofer A, David JP, Heberle-Bors E, Doonan J, Bogre L (2001) Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis. Plant Cell 13:1929–1943

    Article  PubMed  CAS  Google Scholar 

  • Weinl C, Marquardt S, Kuijt SJH, Nowack MK, Jakoby MJ, Hulskamp M, Schnittger A (2005) Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis. Plant Cell 17:1704–1722

    Article  PubMed  CAS  Google Scholar 

  • Zhou Y, Fowke LC, Wang H (2002a) Plant CDK inhibitors: studies of interactions with cell cycle regulators in the yeast two-hybrid system and functional comparisons in transgenic Arabidopsis plants. Plant Cell Rep 20:967–975

    Article  CAS  Google Scholar 

  • Zhou Y, Wang H, Gilmer S, Whitwill S, Keller W, Fowke LC (2002b) Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants. Planta 215:248–257

    Article  CAS  Google Scholar 

  • Zhou Y, Li G, Brandizzi F, Fowke LC, Wang H (2003) The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition, protein instability and nuclear localization. Plant J 35:476–489

    Article  PubMed  CAS  Google Scholar 

  • Zhou Y, Niu H, Brandizzi F, Fowke L, Wang H (2006) Molecular control of nuclear and subnuclear targeting of the plant CDK inhibitor ICK1 and ICK1-mediated nuclear transport of CDKA. Plant Mol Biol 62:261–278

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

L.C.F. and H.W. gratefully acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada. We thank Dr. F. Brandizzi at the University of Saskatchewan for advice on confocal imaging and transient expression in tobacco. We thank Dr. Xianzong Shi for preparing several initial constructs and Ron Chan for providing the Arabidopsis GFP–ICK7 transformants.

Author information

Author notes

  1. David A. Bird

    Present address: Department of Botany, University of British Columbia, Vancouver, Canada, V6T 1Z4

  2. Yongming Zhou

    Present address: The National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 4300070, China

Authors and Affiliations

  1. Department of Biology, University of Saskatchewan, Saskatoon, Canada, SK S7N 5E2

    David A. Bird, Miruna M. Buruiana, Yongming Zhou & Larry C. Fowke

  2. Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada, S7N 5E5

    Hong Wang

Authors
  1. David A. Bird
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Miruna M. Buruiana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yongming Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Larry C. Fowke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Larry C. Fowke.

Additional information

Communicated by P.P. Kumar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bird, D.A., Buruiana, M.M., Zhou, Y. et al. Arabidopsis cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm. Plant Cell Rep 26, 861–872 (2007). https://doi.org/10.1007/s00299-006-0294-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-006-0294-3

Keywords

Search

Navigation