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Analysis of cDNAs encoding Drosophila melanogaster myosin light chain kinase

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Abstract

Myosin light chain kinase regulates the activity of myosin by phosphorylating the myosin regulatory light chain. Here we describe the cloning and characterization of cDNAs encoding Drosophila melanogaster myosin light chain kinase. We amplified a fragment of the Drosophila mlck gene using degenerate primers homologous to a highly conserved region in myosin light chain kinase proteins of vertebrate species. We used the gene fragment to isolate corresponding Drosophila mlck cDNAs. The deduced protein sequence of the cDNAs shows high homology to the catalytic and regulatory domains of vertebrate nonmuscle, smooth muscle and skeletal muscle myosin light chain kinase. Protein motifs I and II, which are present in vertebrate nonmuscle and smooth muscle myosin light chain kinase, but not in skeletal muscle myosin light chain kinase, are also present in Drosophila myosin light chain kinase. Transcript and cDNA analysis shows the gene encodes multiple messages and is expressed in non muscle and muscle cells, including the adult indirect flight muscle. Genomic Southern analysis and chromosome hybridization suggest mlck is a single copy gene which maps to chromosome band 52D, and is not haplo-insufficient for flight

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References

  • ASHBURNER, M. (1989) Drosophila, A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • AYME-SOUTHGATE, A., VIGOREAUX, J., BENIAN, G. PARDUE, M. L. (1991) Drosophilahas a twitchin/titin-related gene that appears to encode projectin. Proc. Natl. Acad. Sci. USA 88, 7973–7.

    Article  PubMed  CAS  Google Scholar 

  • BATE, M. (1990) The embryonic development of larval muscles in Drosophila. Development 110, 791–804.

    PubMed  CAS  Google Scholar 

  • BATE, M. (1993) The mesoderm and its derivatives. In The Development of Drosophila melanogaster II (edited by BATE, M. & MARTINEZ ARIAS, A.) pp. 1013–90. New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • BENIAN, G. M., KIFF, J. E., NECKELMANN, N., MOEMAN, D. G. & WATERSTON, R. H. (1989) Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans. Nature 342, 45–50.

    Article  PubMed  CAS  Google Scholar 

  • BENZER, S. (1973) Genetic dissection of behavior. Sci. Am. 229, 24–37.

    Article  PubMed  CAS  Google Scholar 

  • BERNSTEIN, S. I., O'DONNELL, P. T. & CRIPPS, R. M. (1993) Molecular Analysis of Muscle Development, Structure and function in Drosophila. New York: Academic Press.

    Google Scholar 

  • CAMPOS-ORTEGA, J. A. & HARTENSTEIN, V. (1985) The embryonic development of Drosophila melanogaster. Berlin: Springer-Verlag.

    Google Scholar 

  • CAVENER, D. R. (1987) Comparison of consensus flanking translational start sites in Drosophilaand vertebrates. Nucleic Acids Res. 15, 1353–61.

    PubMed  CAS  Google Scholar 

  • EDWARDS, K. A. & KIEHART, D. P. (1996) Drosophilanonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 122, 1499–511.

    PubMed  CAS  Google Scholar 

  • EINHEBER, S. & FISCHMANN, D. A. (1990) Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily. Proc. Natl Acad. Sci. USA 87, 2157–61.

    Article  PubMed  CAS  Google Scholar 

  • FITZSIMONS, D. P., HERRING, B. P., STULL, J. T. & GALLAGHER, P. J. (1992) Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase. J. Biol. Chem. 267, 23903–9.

    PubMed  CAS  Google Scholar 

  • GALLAGHER, P. J., HERRING, B. P., GRIFFIN, S. A. & STULL, J. T. (1991) Molecular characterization of a mammalian smooth muscle myosin light chain kinase. J. Biol. Chem. 266, 23936–44.

    PubMed  CAS  Google Scholar 

  • GUERRIERO, V., RUSSO, M. A., OLSON, N. J., PUTKEY, J. A. MEANS, A. R. (1986) Domain organization of chicken gizzard mysosin light chain kinase deduced from a cloned cDNA. Biochemistry 25, 8372–81.

    Article  PubMed  CAS  Google Scholar 

  • HANKS, S., QUINN, A. M. HUNTER, T. (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52.

    PubMed  CAS  Google Scholar 

  • HERRING, B. P., STULL, J. T. & GALLAGHER, P. J. (1990) Domain characterization of rabbit skeletal muscle myosin light chain kinase. J. Biol. Chem. 265, 1724–30.

    PubMed  CAS  Google Scholar 

  • IKEBE, M., STEPINSKA, M., KEMP, B. E., MEANS, A. R. & HARTSHORNE, D. J. (1987) Proteolysis of smooth muscle myosin light chain kinase. J. Biol. Chem. 262, 13828–34.

    PubMed  CAS  Google Scholar 

  • IKURA, M., CLORE, G. M., GRONENEBOM, A. M., ZHU, G., KLEE, C. B. & BAX, A. (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–8.

    PubMed  CAS  Google Scholar 

  • JOWETT, T. (1986) Preparation of nucleic acids. In Drosophila, a Practical Approach (edited by ROBERTS, D. B.) pp. 275–86, Oxford/Washington DC: IRL Press.

    Google Scholar 

  • KALDERON, D. & RUBIN, G. M. (1988) Isolation and characterization of DrosophilacAMP-dependent protein kinase genes. Genes Dev. 2, 1539–56.

    PubMed  CAS  Google Scholar 

  • KAMM, K. E. & STULL, J. T. (1985) The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Ann. Rev. Pharmacol. Toxicol. 25, 593–620.

    Article  CAS  Google Scholar 

  • KARESS, R., CHANG, X.-J., EDWARDS, K., KULKARNI, S., AGUILERA, I. & KIEHART, D. (1991) The regulatory light chain of non-muscle myosin is encoded by spaghetti squash, a gene required for cytokinesis in Drosophila. Cell 65, 1177–89.

    Article  PubMed  CAS  Google Scholar 

  • KEMP, B. E. & STULL, J. T. (1990) Myosin Light Chain Kinases. Boca Raton: CRC Press.

    Google Scholar 

  • KEMP, B. E., PEARSON, R. B., GUERRIERO, V., BAGCHI, I. C. & MEANS, A. R. (1987) The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. J. Biol. Chem. 262, 2542–8.

    PubMed  CAS  Google Scholar 

  • KOBAYASHI, H., INOUE, A., MIKAWA, T., KUWAYAMA, H., HOTTA, Y., MASAKI, T. & EBASHI, S. (1992) Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity. J. Biochem. 112, 786–91.

    PubMed  CAS  Google Scholar 

  • LABEIT, S., GAUTEL, M., LAKEY, A. & TRINICK, J. (1992) Towards a molecular understanding of titin. EMBO J. 11, 1711–16.

    PubMed  CAS  Google Scholar 

  • LAKEY, A., FERGUSON, C., LABEIT, S., REEDY, M., LARDINS, A., BUTCHER, G., LEONARD, K. & BULLARD, B. (1990) Identification and localization of high molecular weight proteins in insect flight and leg muscle. EMBO J. 9, 3459–67.

    PubMed  CAS  Google Scholar 

  • LANGER-SAFER, P. R., LEVINE, M. & WARD, D. C. (1982) Immunological method for mapping genes on Drosophilapolytene chromosomes. Proc. Natl Acad. Sci. USA 79, 4381–5.

    Article  PubMed  CAS  Google Scholar 

  • LIN, C. R., KAPILOFF, M. S., DURGERIAN, S., TATEMOTO, K., RUSSO, A. F., HANSON, P., SCHULMAN, H. & ROSENFELD, M. G. (1987) Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase. Proc. Natl Acad. Sci. USA 84, 5962–6.

    Article  PubMed  CAS  Google Scholar 

  • MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1989) Molecular Cloning. A Laboratory Manual. New York: Cold Spring Habor Laboratory Press.

    Google Scholar 

  • MANIATIS, T., HARDISON, R. C., LACY, E., LAUER, J., O'CONNELL, C., QUON, D., SIM, D. K. & EFSTRATIADIS, A. (1978) The isolation of structural genes from libraries of eucaryotic DNA. Cell 15, 687–97.

    Article  PubMed  CAS  Google Scholar 

  • MEADOR, W. E., MEANS, A. R. & QUIOCHO, F. A. (1992) Target enzyme recognition by calmodulin: 2.4 Å structure of a calmodulin-peptide complex. Science 257, 1251–4.

    PubMed  CAS  Google Scholar 

  • MOERMAN, D. G., BENIAN, G. M., BARSTEAD, R. J., SCHRIEFER, L. A. WATERSTON, R. H. (1988) Identification and intracellular localization of the unc-22 gene product of Caenorhabitis elegans. Genes Dev. 2, 93–105.

    PubMed  CAS  Google Scholar 

  • MOUNT, S. M., BURKS, C. HERTZ, G., STORMO, G. D., WHITE, O. & FIELDS, C. (1992) Splicing signals in Drosophila: intron size, information content, and consensus sequence. Nucleic Acids Res. 20, 4255–62.

    PubMed  CAS  Google Scholar 

  • OLSON, N. J., PEARSON, R. B., NEEDLEMAN, D. S., HORVITZ, M. Y., KEMP, B. E. & MEANS, A. R. (1990) Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. Natl Acad. Sci. USA 87, 2284–8.

    Article  PubMed  CAS  Google Scholar 

  • O'NEILL, J. W. & BIER, E. (1994) Double-label in situhybridization using biotin and digoxigenin-tagged RNA probes. BioTechniques 17, 870–5.

    PubMed  Google Scholar 

  • PARKER, V. P., FALKENTHAL, S. & DAVIDSON, N. (1985) Characterization of the myosin light chain-2 gene of Drosophila melanogaster. Mol. Cell. Biol. 5, 3058–68.

    PubMed  CAS  Google Scholar 

  • PEARSON, R. B., WETTENHALL, R. E. H., MEANS, A. R., HARTSHORNE, D. J. & KEMP, B. E. (1988) Autoregulation of enzymes by pseudosubstrate prototypes: myosin light chain kinases. Science 241, 970–3.

    PubMed  CAS  Google Scholar 

  • PEMRICK, S. M. (1980) The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase. J. Biol. Chem. 255, 8836–41.

    PubMed  CAS  Google Scholar 

  • POTIER, M.-C., CHELOT, E., PEKARSKY, Y., GARDINER, K., ROSSIER, J. & TURNELL, W. G. (1995) The human myosin light chain kinase (MLCK) from hippocampus: cloning, sequencing, expression, and localization to 3qcen-q21. Genomics 29, 562–70.

    Article  PubMed  CAS  Google Scholar 

  • PRICE, M. G., BROOKS, C. A. & GOWER, R. H. (1990) Skelemins are members of a family of myosin-associated proteins with immunoglobulin superfamily C2 and fibronecting type III domains. J. Cell Biol. 111, 170a.

    Google Scholar 

  • PRINGLE, J. W. S. (1978) Stretch activation of muscle: function and mechanism. Proc. R. Soc. Lond. B201, 107–30.

    Article  Google Scholar 

  • ROUSH, C. L., KENNELY, P. J., GLACCUM, M. B., HELMAN, D. M., SCOTT, J. D. & KREBS, E. G. (1988) Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. J. Biol. Chem. 263, 10510–16.

    PubMed  CAS  Google Scholar 

  • RUGENDORFF, A., YOUNOSSI-HARTENSTEIN, A. & HARTENSTEIN, V. (1994) Embryonic origin and differentiation of the Drosophilaheart. Roux's Arch. Dev. Biol. 203, 266–80.

    Article  Google Scholar 

  • SANGER, F., MIKLEN, S. & COLSON, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–7.

    Article  PubMed  CAS  Google Scholar 

  • SCHAEFFER, E., SMITH, D., MARDON, G., QUINN, W. & ZUKER, C. (1989) Isolation and characterization of two new protein kinase C genes, including one specifically expressed in photoreceptor cell. Cell 57, 403–12.

    Article  PubMed  CAS  Google Scholar 

  • SELLERS, J. R. & ADELSTEIN, R. S. (1987) Regulation of contractile activity. The Enzymes 18, 381–418.

    CAS  Google Scholar 

  • 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. (1990) 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. 111, 1107–25.

    Article  PubMed  CAS  Google Scholar 

  • SOMLYO, A. P. & SOMLYO, A. V. (1994) Signal transduction and regulation in smooth muscle. Nature 372, 231–6.

    Article  PubMed  CAS  Google Scholar 

  • SWEENEY, H. L., BOWMAN, B. F. & STULL, J. T. (1993) Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am. J. Physiol. 264, C1085–95.

    PubMed  CAS  Google Scholar 

  • TAKANO-OHMURO, H., TAKAHASHI, S., HIROSE, G. & MARUYAMA, K. (1990) Phosphorylated and dephosphorylated myosin light chains of Drosophilafly and larva. Comp. Biochem. Physiol. 95B, 171–7.

    CAS  Google Scholar 

  • TAN, J. L. & SPUDICH, J. T. (1991) Characterization and bacterial expression of the Dictyosteliummyosin light chain kinase cDNA. J. Biol. Chem. 266, 16044–9.

    PubMed  CAS  Google Scholar 

  • TAN, J. L., RAVID, S. & SPUDICH, J. A. (1992) Control of nonmuscle myosins by phosphorylation. Ann. Rev. Biochem. 62, 721–59.

    Article  Google Scholar 

  • TANAKA, M., IKEBE, R., MATSUURA, M. & IKEBE, M. (1995) Pseudosubstrate sequence may not be critical for autoinhibition of smooth muscle myosin light chain kinase. EMBO J. 14, 2839–46.

    PubMed  CAS  Google Scholar 

  • TOFFENETTI, J., MISCHKE, D. & PARDUE, M. L. (1987) Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster. J. Cell Biol. 104, 19–28.

    Article  PubMed  CAS  Google Scholar 

  • TOHTONG, R., YAMASHITA, H., GRAHAM, M., HAEBERLE, J., SIMCOX, A. & MAUGHAN, D. (1995) Impairment of muscle function caused by mutations of phosphorylation sites in myosin regulatory light chain. Nature 374, 650–3.

    Article  PubMed  CAS  Google Scholar 

  • TOKUI, T., ANDO, S. & IKEBE, M. (1995) Autophosphorylation of smooth muscle myosin light chain kinase at its regulatory domain. Biochemistry 34, 5173–9.

    Article  PubMed  CAS  Google Scholar 

  • WATTERSON, D. M., COLLINGE, M., LUKAS, T. J., VAN ELDIK, L. J., BIRUKOV, K. G., STEPANOVA, O. V. & SHIRINSKY, V. P. (1995) Multiple gene products are produced from a novel protein kinase transcription region. FEBS Letts 16, 217–20.

    Article  Google Scholar 

  • WHEATLEY, S., KULKARNI, S. & KARESS, R. (1995) Drosophilanomuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos. Development 121, 1937–46.

    PubMed  CAS  Google Scholar 

  • YANO, K., ARAKI, Y., HALES, S. J., TANAKA, M. & IKEBE, M. (1993) Boundary of the autoinhibitory region of smooth muscle myosin light-chain kinase. Biochemistry 32, 12054–61.

    Article  PubMed  CAS  Google Scholar 

  • YOUNG, P. E., PESACRETA, T. C. & KIEHART, D. P. (1991) Dynamic changes in the distribution of cytoplasmic myosin during Drosophilaembryogenesis. Development 111, 1–14.

    PubMed  CAS  Google Scholar 

  • YOUNG, P. E., RICHMAN, A. M., KETCHUM, A. S. & KIEHART, D. P. (1993) Morphogenesis in Drosophilarequires nonmuscle myosin heavy chain function. Genes Dev. 7, 29–41.

    PubMed  CAS  Google Scholar 

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TOHTONG, R., RODRIGUEZ, D., MAUGHAN, D. et al. Analysis of cDNAs encoding Drosophila melanogaster myosin light chain kinase. J Muscle Res Cell Motil 18, 43–56 (1997). https://doi.org/10.1023/A:1018676832164

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