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. 1990 Jan 1;110(1):63–70. doi: 10.1083/jcb.110.1.63

Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation

PMCID: PMC2115990  PMID: 2404023

Abstract

The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM- 34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.

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Selected References

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  1. Bennett P. M. The structure of spindle-shaped paracrystals of light meromyosin. J Mol Biol. 1981 Feb 25;146(2):201–221. doi: 10.1016/0022-2836(81)90432-0. [DOI] [PubMed] [Google Scholar]
  2. Berlot C. H., Devreotes P. N., Spudich J. A. Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity. J Biol Chem. 1987 Mar 15;262(8):3918–3926. [PubMed] [Google Scholar]
  3. Berlot C. H., Spudich J. A., Devreotes P. N. Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium. Cell. 1985 Nov;43(1):307–314. doi: 10.1016/0092-8674(85)90036-4. [DOI] [PubMed] [Google Scholar]
  4. Clarke M., Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum. J Mol Biol. 1974 Jun 25;86(2):209–222. doi: 10.1016/0022-2836(74)90013-8. [DOI] [PubMed] [Google Scholar]
  5. Claviez M., Pagh K., Maruta H., Baltes W., Fisher P., Gerisch G. Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin. EMBO J. 1982;1(8):1017–1022. doi: 10.1002/j.1460-2075.1982.tb01287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Côté G. P., Albanesi J. P., Ueno T., Hammer J. A., 3rd, Korn E. D. Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii. J Biol Chem. 1985 Apr 25;260(8):4543–4546. [PubMed] [Google Scholar]
  7. Côté G. P., McCrea S. M. Selective removal of the carboxyl-terminal tail end of the Dictyostelium myosin II heavy chain by chymotrypsin. J Biol Chem. 1987 Sep 25;262(27):13033–13038. [PubMed] [Google Scholar]
  8. De Lozanne A., Berlot C. H., Leinwand L. A., Spudich J. A. Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment. J Cell Biol. 1987 Dec;105(6 Pt 2):2999–3005. doi: 10.1083/jcb.105.6.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. DeLozanne A., Lewis M., Spudich J. A., Leinwand L. A. Cloning and characterization of a nonmuscle myosin heavy chain cDNA. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6807–6810. doi: 10.1073/pnas.82.20.6807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeRosier D. J., Klug A. Structure of the tubular variants of the head of bacteriophage T4 (polyheads). I. Arrangement of subunits in some classes of polyheads. J Mol Biol. 1972 Apr 14;65(3):469–488. doi: 10.1016/0022-2836(72)90202-1. [DOI] [PubMed] [Google Scholar]
  12. Flicker P. F., Peltz G., Sheetz M. P., Parham P., Spudich J. A. Site-specific inhibition of myosin-mediated motility in vitro by monoclonal antibodies. J Cell Biol. 1985 Apr;100(4):1024–1030. doi: 10.1083/jcb.100.4.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffith L. M., Downs S. M., Spudich J. A. Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. J Cell Biol. 1987 May;104(5):1309–1323. doi: 10.1083/jcb.104.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanson J., O'Brien E. J., Bennett P. M. Structure of the myosin-containing filament assembly (A-segment) separated from frog skeletal muscle. J Mol Biol. 1971 Jun 28;58(3):865–871. doi: 10.1016/0022-2836(71)90045-3. [DOI] [PubMed] [Google Scholar]
  15. Huxley H. E., Faruqi A. R. Time-resolved X-ray diffraction studies on vertebrate striated muscle. Annu Rev Biophys Bioeng. 1983;12:381–417. doi: 10.1146/annurev.bb.12.060183.002121. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Manstein D. J., Titus M. A., De Lozanne A., Spudich J. A. Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 1989 Mar;8(3):923–932. doi: 10.1002/j.1460-2075.1989.tb03453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McLachlan A. D. Structural implications of the myosin amino acid sequence. Annu Rev Biophys Bioeng. 1984;13:167–189. doi: 10.1146/annurev.bb.13.060184.001123. [DOI] [PubMed] [Google Scholar]
  21. Nachmias V. T., Fukui Y., Spudich J. A. Chemoattractant-elicited translocation of myosin in motile Dictyostelium. Cell Motil Cytoskeleton. 1989;13(3):158–169. doi: 10.1002/cm.970130304. [DOI] [PubMed] [Google Scholar]
  22. Pagh K., Gerisch G. Monoclonal antibodies binding to the tail of Dictyostelium discoideum myosin: their effects on antiparallel and parallel assembly and actin-activated ATPase activity. J Cell Biol. 1986 Oct;103(4):1527–1538. doi: 10.1083/jcb.103.4.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Pasternak C., Flicker P. F., Ravid S., Spudich J. A. Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation. J Cell Biol. 1989 Jul;109(1):203–210. doi: 10.1083/jcb.109.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peltz G., Kuczmarski E. R., Spudich J. A. Dictyostelium myosin: characterization of chymotryptic fragments and localization of the heavy-chain phosphorylation site. J Cell Biol. 1981 Apr;89(1):104–108. doi: 10.1083/jcb.89.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peltz G., Spudich J. A., Parham P. Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin. J Cell Biol. 1985 Apr;100(4):1016–1023. doi: 10.1083/jcb.100.4.1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Quinlan R. A., Stewart M. Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry. J Cell Biol. 1987 Jul;105(1):403–415. doi: 10.1083/jcb.105.1.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ravid S., Spudich J. A. Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization. J Biol Chem. 1989 Sep 5;264(25):15144–15150. [PubMed] [Google Scholar]
  29. Stewart P. R., Spudich J. A. Structural states of dictyostelium myosin. J Supramol Struct. 1979;12(1):1–14. doi: 10.1002/jss.400120102. [DOI] [PubMed] [Google Scholar]
  30. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vaillancourt J. P., Lyons C., Côté G. P. Identification of two phosphorylated threonines in the tail region of Dictyostelium myosin II. J Biol Chem. 1988 Jul 25;263(21):10082–10087. [PubMed] [Google Scholar]
  32. Wagle G., Noegel A., Scheel J., Gerisch G. Phosphorylation of threonine residues on cloned fragments of the Dictyostelium myosin heavy chain. FEBS Lett. 1988 Jan 18;227(1):71–75. doi: 10.1016/0014-5793(88)81416-9. [DOI] [PubMed] [Google Scholar]
  33. Warrick H. M., De Lozanne A., Leinwand L. A., Spudich J. A. Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9433–9437. doi: 10.1073/pnas.83.24.9433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]

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