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. 1992 Dec;11(13):4715–4722. doi: 10.1002/j.1460-2075.1992.tb05576.x

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

T Rowe 1, J Kendrick-Jones 1
PMCID: PMC556946  PMID: 1464306

Abstract

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.

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Selected References

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

  1. Ankrett R. J., Rowe A. J., Cross R. A., Kendrick-Jones J., Bagshaw C. R. A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity. J Mol Biol. 1991 Jan 20;217(2):323–335. doi: 10.1016/0022-2836(91)90546-i. [DOI] [PubMed] [Google Scholar]
  2. Baba M. L., Goodman M., Berger-Cohn J., Demaille J. G., Matsuda G. The early adaptive evolution of calmodulin. Mol Biol Evol. 1984 Nov;1(6):442–455. doi: 10.1093/oxfordjournals.molbev.a040330. [DOI] [PubMed] [Google Scholar]
  3. Chantler P. D., Szent-Györgyi A. G. Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects. J Mol Biol. 1980 Apr 15;138(3):473–492. doi: 10.1016/s0022-2836(80)80013-1. [DOI] [PubMed] [Google Scholar]
  4. Craig R., Smith R., Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. 1983 Mar 31-Apr 6Nature. 302(5907):436–439. doi: 10.1038/302436a0. [DOI] [PubMed] [Google Scholar]
  5. Cross R. A., Cross K. E., Sobieszek A. ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi. EMBO J. 1986 Oct;5(10):2637–2641. doi: 10.1002/j.1460-2075.1986.tb04545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cross R. A., Jackson A. P., Citi S., Kendrick-Jones J., Bagshaw C. R. Active site trapping of nucleotide by smooth and non-muscle myosins. J Mol Biol. 1988 Sep 5;203(1):173–181. doi: 10.1016/0022-2836(88)90100-3. [DOI] [PubMed] [Google Scholar]
  7. Goodwin E. B., Leinwand L. A., Szent-Györgyi A. G. Regulation of scallop myosin by mutant regulatory light chains. J Mol Biol. 1990 Nov 5;216(1):85–93. doi: 10.1016/S0022-2836(05)80062-2. [DOI] [PubMed] [Google Scholar]
  8. Henry G. D., Trayer I. P., Brewer S., Levine B. A. The widespread distribution of alpha-N-trimethylalanine as the N-terminal amino acid of light chains from vertebrate striated muscle myosins. Eur J Biochem. 1985 Apr 1;148(1):75–82. doi: 10.1111/j.1432-1033.1985.tb08809.x. [DOI] [PubMed] [Google Scholar]
  9. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  10. Jakes R., Northrop F., Kendrick-Jones J. Calcium binding regions of myosin 'regulatory' light chains. FEBS Lett. 1976 Nov;70(1):229–234. doi: 10.1016/0014-5793(76)80763-6. [DOI] [PubMed] [Google Scholar]
  11. Karess R. E., Chang X. J., Edwards K. A., Kulkarni S., Aguilera I., Kiehart D. P. The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. Cell. 1991 Jun 28;65(7):1177–1189. doi: 10.1016/0092-8674(91)90013-o. [DOI] [PubMed] [Google Scholar]
  12. Katoh T., Lowey S. Mapping myosin light chains by immunoelectron microscopy. Use of anti-fluorescyl antibodies as structural probes. J Cell Biol. 1989 Oct;109(4 Pt 1):1549–1560. doi: 10.1083/jcb.109.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kendrick-Jones J., Cande W. Z., Tooth P. J., Smith R. C., Scholey J. M. Studies on the effect of phosphorylation of the 20,000 Mr light chain of vertebrate smooth muscle myosin. J Mol Biol. 1983 Mar 25;165(1):139–162. doi: 10.1016/s0022-2836(83)80247-2. [DOI] [PubMed] [Google Scholar]
  14. Kendrick-Jones J., Rasera da Silva A. C., Reinach F. C., Messer N., Rowe T., McLaughlin P. Recombinant DNA approaches to study the role of the regulatory light chains (RLC) using scallop myosin as a test system. J Cell Sci Suppl. 1991;14:55–58. doi: 10.1242/jcs.1991.supplement_14.11. [DOI] [PubMed] [Google Scholar]
  15. Kendrick-Jones J., Szentkiralyi E. M., Szent-Györgyi A. G. Regulatory light chains in myosins. J Mol Biol. 1976 Jul 15;104(4):747–775. doi: 10.1016/0022-2836(76)90180-7. [DOI] [PubMed] [Google Scholar]
  16. Kretsinger R. H. Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980;8(2):119–174. doi: 10.3109/10409238009105467. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Kwon H., Goodwin E. B., Nyitray L., Berliner E., O'Neall-Hennessey E., Melandri F. D., Szent-Györgyi A. G. Isolation of the regulatory domain of scallop myosin: role of the essential light chain in calcium binding. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4771–4775. doi: 10.1073/pnas.87.12.4771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kwon H., Melandri F. D., Szent-Györgyi A. G. Role of gizzard myosin light chains in calcium binding. J Muscle Res Cell Motil. 1992 Jun;13(3):315–320. doi: 10.1007/BF01766459. [DOI] [PubMed] [Google Scholar]
  20. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  21. Messer N. G., Kendrick-Jones J. Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain. FEBS Lett. 1988 Jul 4;234(1):49–52. doi: 10.1016/0014-5793(88)81300-0. [DOI] [PubMed] [Google Scholar]
  22. Messer N., Kendrick-Jones J. Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand. J Mol Biol. 1991 Apr 20;218(4):825–835. doi: 10.1016/0022-2836(91)90270-g. [DOI] [PubMed] [Google Scholar]
  23. Morita J., Takashi R., Ikebe M. Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin. Biochemistry. 1991 Oct 1;30(39):9539–9545. doi: 10.1021/bi00103a022. [DOI] [PubMed] [Google Scholar]
  24. Onishi H., Wakabayashi T. Electron microscopic studies of myosin molecules from chicken gizzard muscle I: the formation of the intramolecular loop in the myosin tail. J Biochem. 1982 Sep;92(3):871–879. doi: 10.1093/oxfordjournals.jbchem.a134001. [DOI] [PubMed] [Google Scholar]
  25. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Persechini A., Stull J. T., Cooke R. The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers. J Biol Chem. 1985 Jul 5;260(13):7951–7954. [PubMed] [Google Scholar]
  27. Reinach F. C., Fischman D. A. Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2. J Mol Biol. 1985 Feb 5;181(3):411–422. doi: 10.1016/0022-2836(85)90229-3. [DOI] [PubMed] [Google Scholar]
  28. Reinach F. C., Nagai K., Kendrick-Jones J. Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins. Nature. 1986 Jul 3;322(6074):80–83. doi: 10.1038/322080a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Strynadka N. C., James M. N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem. 1989;58:951–998. doi: 10.1146/annurev.bi.58.070189.004511. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  32. Szent-Györgyi A. G., Szentkiralyi E. M., Kendrick-Jonas J. The light chains of scallop myosin as regulatory subunits. J Mol Biol. 1973 Feb 25;74(2):179–203. doi: 10.1016/0022-2836(73)90106-x. [DOI] [PubMed] [Google Scholar]
  33. Trybus K. M., Huiatt T. W., Lowey S. A bent monomeric conformation of myosin from smooth muscle. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6151–6155. doi: 10.1073/pnas.79.20.6151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Way M., Pope B., Gooch J., Hawkins M., Weeds A. G. Identification of a region in segment 1 of gelsolin critical for actin binding. EMBO J. 1990 Dec;9(12):4103–4109. doi: 10.1002/j.1460-2075.1990.tb07632.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Winkelmann D. A., Lowey S. Probing myosin head structure with monoclonal antibodies. J Mol Biol. 1986 Apr 20;188(4):595–612. doi: 10.1016/s0022-2836(86)80009-2. [DOI] [PubMed] [Google Scholar]

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