Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jan 1;124(1):129–137. doi: 10.1083/jcb.124.1.129

In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells

PMCID: PMC2119899  PMID: 8294496

Abstract

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Adelstein R. S., Klee C. B. Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem. 1981 Jul 25;256(14):7501–7509. [PubMed] [Google Scholar]
  2. Bengur A. R., Robinson E. A., Appella E., Sellers J. R. Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain. J Biol Chem. 1987 Jun 5;262(16):7613–7617. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brizuela L., Draetta G., Beach D. p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 1987 Nov;6(11):3507–3514. doi: 10.1002/j.1460-2075.1987.tb02676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cassidy P., Hoar P. E., Kerrick W. G. Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strips by [35S]ATP gamma S. J Biol Chem. 1979 Nov 10;254(21):11148–11153. [PubMed] [Google Scholar]
  6. Chou Y. H., Rebhun L. I. Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity. J Biol Chem. 1986 Apr 25;261(12):5389–5395. [PubMed] [Google Scholar]
  7. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  8. Erdödi F., Rokolya A., Bárány M., Bárány K. Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle. Biochim Biophys Acta. 1989 Mar 28;1011(1):67–74. doi: 10.1016/0167-4889(89)90080-3. [DOI] [PubMed] [Google Scholar]
  9. Fishkind D. J., Cao L. G., Wang Y. L. Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J Cell Biol. 1991 Sep;114(5):967–975. doi: 10.1083/jcb.114.5.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoar P. E., Kerrick W. G., Cassidy P. S. Chicken gizzard: relation between calcium-activated phosphorylation and contraction. Science. 1979 May 4;204(4392):503–506. doi: 10.1126/science.432654. [DOI] [PubMed] [Google Scholar]
  12. Hosoya N., Hosoya H., Yamashiro S., Mohri H., Matsumura F. Localization of caldesmon and its dephosphorylation during cell division. J Cell Biol. 1993 Jun;121(5):1075–1082. doi: 10.1083/jcb.121.5.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ikebe M., Reardon S. Phosphorylation of bovine platelet myosin by protein kinase C. Biochemistry. 1990 Mar 20;29(11):2713–2720. doi: 10.1021/bi00463a014. [DOI] [PubMed] [Google Scholar]
  15. Ikebe M., Reardon S., Scott-Woo G. C., Zhou Z., Koda Y. Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase. Biochemistry. 1990 Dec 25;29(51):11242–11248. doi: 10.1021/bi00503a013. [DOI] [PubMed] [Google Scholar]
  16. Ishikawa R., Yamashiro S., Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989 May 5;264(13):7490–7497. [PubMed] [Google Scholar]
  17. Kawamoto S., Adelstein R. S. Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study. J Biol Chem. 1987 May 25;262(15):7282–7288. [PubMed] [Google Scholar]
  18. Kawamoto S., Bengur A. R., Sellers J. R., Adelstein R. S. In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C. J Biol Chem. 1989 Feb 5;264(4):2258–2265. [PubMed] [Google Scholar]
  19. Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
  20. Kusubata M., Tokui T., Matsuoka Y., Okumura E., Tachibana K., Hisanaga S., Kishimoto T., Yasuda H., Kamijo M., Ohba Y. p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro. J Biol Chem. 1992 Oct 15;267(29):20937–20942. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Ludowyke R. I., Peleg I., Beaven M. A., Adelstein R. S. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J Biol Chem. 1989 Jul 25;264(21):12492–12501. [PubMed] [Google Scholar]
  23. Mabuchi I. Biochemical aspects of cytokinesis. Int Rev Cytol. 1986;101:175–213. doi: 10.1016/s0074-7696(08)60249-1. [DOI] [PubMed] [Google Scholar]
  24. Mabuchi I., Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. doi: 10.1083/jcb.74.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakabayashi H., Sellers J. R., Huang K. P. Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain. FEBS Lett. 1991 Dec 2;294(1-2):144–148. doi: 10.1016/0014-5793(91)81362-c. [DOI] [PubMed] [Google Scholar]
  26. Nishikawa M., Sellers J. R., Adelstein R. S., Hidaka H. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J Biol Chem. 1984 Jul 25;259(14):8808–8814. [PubMed] [Google Scholar]
  27. Sanger J. M., Mittal B., Dome J. S., Sanger J. W. Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells. Cell Motil Cytoskeleton. 1989;14(2):201–219. doi: 10.1002/cm.970140207. [DOI] [PubMed] [Google Scholar]
  28. Satterwhite L. L., Lohka M. J., Wilson K. L., Scherson T. Y., Cisek L. J., Corden J. L., Pollard T. D. Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis. J Cell Biol. 1992 Aug;118(3):595–605. doi: 10.1083/jcb.118.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sellers J. R., Pato M. D., Adelstein R. S. Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J Biol Chem. 1981 Dec 25;256(24):13137–13142. [PubMed] [Google Scholar]
  30. Sellers J. R. Regulation of cytoplasmic and smooth muscle myosin. Curr Opin Cell Biol. 1991 Feb;3(1):98–104. doi: 10.1016/0955-0674(91)90171-t. [DOI] [PubMed] [Google Scholar]
  31. Shoemaker M. O., Lau W., Shattuck R. L., Kwiatkowski A. P., Matrisian P. E., Guerra-Santos L., Wilson E., Lukas T. J., Van Eldik L. J., Watterson D. M. Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity. J Cell Biol. 1990 Sep;111(3):1107–1125. doi: 10.1083/jcb.111.3.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tan J. L., Ravid S., Spudich J. A. Control of nonmuscle myosins by phosphorylation. Annu Rev Biochem. 1992;61:721–759. doi: 10.1146/annurev.bi.61.070192.003445. [DOI] [PubMed] [Google Scholar]
  33. Tuazon P. T., Traugh J. A. Activation of actin-activated ATPase in smooth muscle by phosphorylation of myosin light chain with protease-activated kinase I. J Biol Chem. 1984 Jan 10;259(1):541–546. [PubMed] [Google Scholar]
  34. Yamashiro S., Matsumura F. Mitosis-specific phosphorylation of caldesmon: possible molecular mechanism of cell rounding during mitosis. Bioessays. 1991 Nov;13(11):563–568. doi: 10.1002/bies.950131103. [DOI] [PubMed] [Google Scholar]
  35. Yamashiro S., Yamakita Y., Hosoya H., Matsumura F. Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis. Nature. 1991 Jan 10;349(6305):169–172. doi: 10.1038/349169a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES