Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Oct 1;109(4):1529–1535. doi: 10.1083/jcb.109.4.1529

The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II

PMCID: PMC2115825  PMID: 2793932

Abstract

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin- II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Adelstein R. S., Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. doi: 10.1146/annurev.bi.49.070180.004421. [DOI] [PubMed] [Google Scholar]
  2. Atkinson M. A., Korn E. D. A model for the polymerization of Acanthamoeba myosin II and the regulation of its actin-activated Mg2+-ATPase activity. J Biol Chem. 1987 Nov 15;262(32):15809–15811. [PubMed] [Google Scholar]
  3. Collins J. H., Côté G. P., Korn E. D. Localization of the three phosphorylation sites on each heavy chain of Acanthamoeba myosin II to a segment at the end of the tail. J Biol Chem. 1982 Apr 25;257(8):4529–4534. [PubMed] [Google Scholar]
  4. Collins J. H., Korn E. D. Actin activation of Ca2+-sensitive Mg2+-ATPase activity of Acanthamoeba myosin II is enhanced by dephosphorylation of its heavy chains. J Biol Chem. 1980 Sep 10;255(17):8011–8014. [PubMed] [Google Scholar]
  5. Collins J. H., Korn E. D. Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba. J Biol Chem. 1981 Mar 10;256(5):2586–2595. [PubMed] [Google Scholar]
  6. Collins J. H., Kuznicki J., Bowers B., Korn E. D. Comparison of the actin binding and filament formation properties of phosphorylated and dephosphorylated Acanthamoeba myosin II. Biochemistry. 1982 Dec 21;21(26):6910–6915. doi: 10.1021/bi00269a045. [DOI] [PubMed] [Google Scholar]
  7. Côté G. P., Collins J. H., Korn E. D. Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II. J Biol Chem. 1981 Dec 25;256(24):12811–12816. [PubMed] [Google Scholar]
  8. Côté G. P., Robinson E. A., Appella E., Korn E. D. Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites. J Biol Chem. 1984 Oct 25;259(20):12781–12787. [PubMed] [Google Scholar]
  9. Deslauriers R., Byrd R. A., Jarrell H. C., Smith I. C. 31P NMR studies of vegetative and encysted cells of Acanthamoeba castellanii. Observation of phosphonic acids in live cells. Eur J Biochem. 1980 Oct;111(2):369–375. doi: 10.1111/j.1432-1033.1980.tb04950.x. [DOI] [PubMed] [Google Scholar]
  10. Hammer J. A., 3rd, Bowers B., Paterson B. M., Korn E. D. Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail. J Cell Biol. 1987 Aug;105(2):913–925. doi: 10.1083/jcb.105.2.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Josephs R., Harrington W. F. Studies on the formation and physical chemical properties of synthetic myosin filaments. Biochemistry. 1966 Nov;5(11):3474–3487. doi: 10.1021/bi00875a013. [DOI] [PubMed] [Google Scholar]
  12. Kendrick-Jones J., Smith R. C., Craig R., Citi S. Polymerization of vertebrate non-muscle and smooth muscle myosins. J Mol Biol. 1987 Nov 20;198(2):241–252. doi: 10.1016/0022-2836(87)90310-x. [DOI] [PubMed] [Google Scholar]
  13. Korn E. D., Hammer J. A., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. doi: 10.1146/annurev.bb.17.060188.000323. [DOI] [PubMed] [Google Scholar]
  14. Kuczmarski E. R., Spudich J. A. Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7292–7296. doi: 10.1073/pnas.77.12.7292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuczmarski E. R., Tafuri S. R., Parysek L. M. Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments. J Cell Biol. 1987 Dec;105(6 Pt 2):2989–2997. doi: 10.1083/jcb.105.6.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuznicki J., Albanesi J. P., Côté G. P., Korn E. D. Supramolecular regulation of the actin-activated ATPase activity of filaments of Acanthamoeba Myosin II. J Biol Chem. 1983 May 25;258(10):6011–6014. [PubMed] [Google Scholar]
  17. Kuznicki J., Korn E. D. Interdependence of factors affecting the actin-activated ATPase activity of myosin II from Acanthamoeba castellanii. J Biol Chem. 1984 Jul 25;259(14):9302–9307. [PubMed] [Google Scholar]
  18. Onishi H., Suzuki H., Nakamura K., Takahashi K., Watanabe S. Adenosine triphosphatase activity and "thick filament" formation of chicken gizzard myosin in low salt media. J Biochem. 1978 Mar;83(3):835–847. doi: 10.1093/oxfordjournals.jbchem.a131980. [DOI] [PubMed] [Google Scholar]
  19. Pagh K., Maruta H., Claviez M., Gerisch G. Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin. EMBO J. 1984 Dec 20;3(13):3271–3278. doi: 10.1002/j.1460-2075.1984.tb02289.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pollard T. D., Korn E. D. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed] [Google Scholar]
  21. Pollard T. D., Stafford W. F., Porter M. E. Characterization of a second myosin from Acanthamoeba castellanii. J Biol Chem. 1978 Jul 10;253(13):4798–4808. [PubMed] [Google Scholar]
  22. Pollard T. D. Structure and polymerization of Acanthamoeba myosin-II filaments. J Cell Biol. 1982 Dec;95(3):816–825. doi: 10.1083/jcb.95.3.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reisler E., Cheung P., Oriol-Audit C., Lake J. A. Growth of synthetic myosin filaments from myosin minifilaments. Biochemistry. 1982 Feb 16;21(4):701–707. doi: 10.1021/bi00533a018. [DOI] [PubMed] [Google Scholar]
  24. Reisler E., Smith C., Seegan G. Myosin minifilaments. J Mol Biol. 1980 Oct 15;143(1):129–145. doi: 10.1016/0022-2836(80)90127-8. [DOI] [PubMed] [Google Scholar]
  25. Sinard J. H., Pollard T. D. Microinjection into Acanthamoeba castellanii of monoclonal antibodies to myosin-II slows but does not stop cell locomotion. Cell Motil Cytoskeleton. 1989;12(1):42–52. doi: 10.1002/cm.970120106. [DOI] [PubMed] [Google Scholar]
  26. Sinard J. H., Stafford W. F., Pollard T. D. The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps. J Cell Biol. 1989 Oct;109(4 Pt 1):1537–1547. doi: 10.1083/jcb.109.4.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stull J. T., Buss J. E. Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977 Feb 10;252(3):851–857. [PubMed] [Google Scholar]
  28. Suzuki H., Onishi H., Takahashi K., Watanabe S. Structure and function of chicken gizzard myosin. J Biochem. 1978 Dec;84(6):1529–1542. doi: 10.1093/oxfordjournals.jbchem.a132278. [DOI] [PubMed] [Google Scholar]
  29. Trybus K. M., Lowey S. Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding. J Cell Biol. 1987 Dec;105(6 Pt 2):3007–3019. doi: 10.1083/jcb.105.6.3007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trybus K. M., Lowey S. Mechanism of smooth muscle myosin phosphorylation. J Biol Chem. 1985 Dec 15;260(29):15988–15995. [PubMed] [Google Scholar]
  31. Yumura S., Fukui Y. Reversible cyclic AMP-dependent change in distribution of myosin thick filaments in Dictyostelium. Nature. 1985 Mar 14;314(6007):194–196. doi: 10.1038/314194a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES