Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1995 Apr;7(4):431–445. doi: 10.1105/tpc.7.4.431

The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

S M Wolniak 1, P M Larsen 1
PMCID: PMC160794  PMID: 7539650

Abstract

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Axton J. M., Dombrádi V., Cohen P. T., Glover D. M. One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell. 1990 Oct 5;63(1):33–46. doi: 10.1016/0092-8674(90)90286-n. [DOI] [PubMed] [Google Scholar]
  2. Colasanti J., Tyers M., Sundaresan V. Isolation and characterization of cDNA clones encoding a functional p34cdc2 homologue from Zea mays. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3377–3381. doi: 10.1073/pnas.88.8.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Davis P. D., Hill C. H., Keech E., Lawton G., Nixon J. S., Sedgwick A. D., Wadsworth J., Westmacott D., Wilkinson S. E. Potent selective inhibitors of protein kinase C. FEBS Lett. 1989 Dec 18;259(1):61–63. doi: 10.1016/0014-5793(89)81494-2. [DOI] [PubMed] [Google Scholar]
  4. Feiler H. S., Jacobs T. W. Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5397–5401. doi: 10.1073/pnas.87.14.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ferreira P. C., Hemerly A. S., Villarroel R., Van Montagu M., Inzé D. The Arabidopsis functional homolog of the p34cdc2 protein kinase. Plant Cell. 1991 May;3(5):531–540. doi: 10.1105/tpc.3.5.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gerace L., Blobel G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell. 1980 Jan;19(1):277–287. doi: 10.1016/0092-8674(80)90409-2. [DOI] [PubMed] [Google Scholar]
  7. Gerace L., Burke B. Functional organization of the nuclear envelope. Annu Rev Cell Biol. 1988;4:335–374. doi: 10.1146/annurev.cb.04.110188.002003. [DOI] [PubMed] [Google Scholar]
  8. Grosskopf D. G., Felix G., Boller T. K-252a inhibits the response of tomato cells to fungal elicitors in vivo and their microsomal protein kinase in vitro. FEBS Lett. 1990 Nov 26;275(1-2):177–180. doi: 10.1016/0014-5793(90)81466-2. [DOI] [PubMed] [Google Scholar]
  9. Harmon A. C., Putnam-Evans C., Cormier M. J. A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 1987 Apr;83(4):830–837. doi: 10.1104/pp.83.4.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harper J. F., Sussman M. R., Schaller G. E., Putnam-Evans C., Charbonneau H., Harmon A. C. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science. 1991 May 17;252(5008):951–954. doi: 10.1126/science.1852075. [DOI] [PubMed] [Google Scholar]
  11. Hashimoto J., Hirabayashi T., Hayano Y., Hata S., Ohashi Y., Suzuka I., Utsugi T., Toh-e A., Kikuchi Y. Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants. Mol Gen Genet. 1992 May;233(1-2):10–16. doi: 10.1007/BF00587555. [DOI] [PubMed] [Google Scholar]
  12. Hepler P. K. Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells. J Cell Biol. 1985 May;100(5):1363–1368. doi: 10.1083/jcb.100.5.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hepler P. K. Calcium transients during mitosis: observations in flux. J Cell Biol. 1989 Dec;109(6 Pt 1):2567–2573. doi: 10.1083/jcb.109.6.2567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herbert J. M., Seban E., Maffrand J. P. Characterization of specific binding sites for [3H]-staurosporine on various protein kinases. Biochem Biophys Res Commun. 1990 Aug 31;171(1):189–195. doi: 10.1016/0006-291x(90)91375-3. [DOI] [PubMed] [Google Scholar]
  15. Holloway S. L., Glotzer M., King R. W., Murray A. W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. doi: 10.1016/0092-8674(93)90364-v. [DOI] [PubMed] [Google Scholar]
  16. Kemp B. E., Pearson R. B. Design and use of peptide substrates for protein kinases. Methods Enzymol. 1991;200:121–134. doi: 10.1016/0076-6879(91)00134-i. [DOI] [PubMed] [Google Scholar]
  17. Kitagawa M., Okabe T., Ogino H., Matsumoto H., Suzuki-Takahashi I., Kokubo T., Higashi H., Saitoh S., Taya Y., Yasuda H. Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase. Oncogene. 1993 Sep;8(9):2425–2432. [PubMed] [Google Scholar]
  18. Larsen P. M., Chen T. L., Wolniak S. M. Quin2-induced metaphase arrest in stamen hair cells can be reversed by 1,2-dioctanoylglycerol but not by 1,3-dioctanoylglycerol. Eur J Cell Biol. 1989 Apr;48(2):212–219. [PubMed] [Google Scholar]
  19. Lee J. C., Kwon Y. G., Lawrence D. S., Edelman A. M. A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6413–6417. doi: 10.1073/pnas.91.14.6413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lohka M. J. Mitotic control by metaphase-promoting factor and cdc proteins. J Cell Sci. 1989 Feb;92(Pt 2):131–135. doi: 10.1242/jcs.92.2.131. [DOI] [PubMed] [Google Scholar]
  21. MacKintosh C., MacKintosh R. W. Inhibitors of protein kinases and phosphatases. Trends Biochem Sci. 1994 Nov;19(11):444–448. doi: 10.1016/0968-0004(94)90127-9. [DOI] [PubMed] [Google Scholar]
  22. Martiny-Baron G., Scherer G. F. Phospholipid-stimulated protein kinase in plants. J Biol Chem. 1989 Oct 25;264(30):18052–18059. [PubMed] [Google Scholar]
  23. Morello L., Gianì S., Coraggio I., Breviario D. Rice membranes contain a calcium-dependent protein kinase activity with biochemical features of animal protein kinase C. Biochem Biophys Res Commun. 1993 Nov 30;197(1):55–61. doi: 10.1006/bbrc.1993.2440. [DOI] [PubMed] [Google Scholar]
  24. Moreno S., Hayles J., Nurse P. Regulation of p34cdc2 protein kinase during mitosis. Cell. 1989 Jul 28;58(2):361–372. doi: 10.1016/0092-8674(89)90850-7. [DOI] [PubMed] [Google Scholar]
  25. Norbury C. J., Nurse P. Control of the higher eukaryote cell cycle by p34cdc2 homologues. Biochim Biophys Acta. 1989 Jul 28;989(1):85–95. doi: 10.1016/0304-419x(89)90036-x. [DOI] [PubMed] [Google Scholar]
  26. Norbury C., Nurse P. Animal cell cycles and their control. Annu Rev Biochem. 1992;61:441–470. doi: 10.1146/annurev.bi.61.070192.002301. [DOI] [PubMed] [Google Scholar]
  27. Ohmi K., Yamashita S., Nonomura Y. Effect of K252a, a protein kinase inhibitor, on the proliferation of vascular smooth muscle cells. Biochem Biophys Res Commun. 1990 Dec 31;173(3):976–981. doi: 10.1016/s0006-291x(05)80881-2. [DOI] [PubMed] [Google Scholar]
  28. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase. Cell. 1990 Mar 9;60(5):791–801. doi: 10.1016/0092-8674(90)90093-t. [DOI] [PubMed] [Google Scholar]
  29. Schaller G. E., Harmon A. C., Sussman M. R. Characterization of a calcium- and lipid-dependent protein kinase associated with the plasma membrane of oat. Biochemistry. 1992 Feb 18;31(6):1721–1727. doi: 10.1021/bi00121a020. [DOI] [PubMed] [Google Scholar]
  30. Strausfeld U., Fernandez A., Capony J. P., Girard F., Lautredou N., Derancourt J., Labbe J. C., Lamb N. J. Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis. J Biol Chem. 1994 Feb 25;269(8):5989–6000. [PubMed] [Google Scholar]
  31. Suprynowicz F. A. Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system. J Cell Sci. 1993 Mar;104(Pt 3):873–881. doi: 10.1242/jcs.104.3.873. [DOI] [PubMed] [Google Scholar]
  32. Svetlov S., Nigam S. Calphostin C, a specific protein kinase C inhibitor, activates human neutrophils: effect on phospholipase A2 and aggregation. Biochim Biophys Acta. 1993 May 8;1177(1):75–78. doi: 10.1016/0167-4889(93)90160-q. [DOI] [PubMed] [Google Scholar]
  33. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  34. Vandré D. D., Borisy G. G. Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly. J Cell Sci. 1989 Oct;94(Pt 2):245–258. doi: 10.1242/jcs.94.2.245. [DOI] [PubMed] [Google Scholar]
  35. Verhey S. D., Gaiser J. C., Lomax T. L. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases). Plant Physiol. 1993 Oct;103(2):413–419. doi: 10.1104/pp.103.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Watillon B., Kettmann R., Boxus P., Burny A. A calcium/calmodulin-binding serine/threonine protein kinase homologous to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells. Plant Physiol. 1993 Apr;101(4):1381–1384. doi: 10.1104/pp.101.4.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wolniak S. M., Bart K. M. The buffering of calcium with quin2 reversibly forestalls anaphase onset in stamen hair cells of Tradescantia. Eur J Cell Biol. 1985 Nov;39(1):33–40. [PubMed] [Google Scholar]
  38. Wolniak S. M., Larsen P. M. Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors. J Cell Sci. 1992 Aug;102(Pt 4):691–715. doi: 10.1242/jcs.102.4.691. [DOI] [PubMed] [Google Scholar]
  39. Wolniak S. M. Lithium alters mitotic progression in stamen hair cells of Tradescantia in a time-dependent and reversible fashion. Eur J Cell Biol. 1987 Oct;44(2):286–293. [PubMed] [Google Scholar]
  40. Wolniak S. M. The regulation of mitotic spindle function. Biochem Cell Biol. 1988 Jun;66(6):490–514. doi: 10.1139/o88-061. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES