Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells


Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [γ-32P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 μM Ca2+ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 μg/ml) in the presence of calmodulin (10 μM) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca2+ in digitonin-treated cells. In the presence of 1 μM Ca2+, myosin light chain kinase (400 μg/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 μM. Ca2+. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.

This is a preview of subscription content, log in to check access.



ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid


N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid


solution containing potassium glutamate, EGTA, PIPES and MgCl2




piperazine-N,-N′-bis-(2-ethanesulfonic acid)


physiological salt solution


  1. 1.

    Baker, P. F. and Knight, D. E. (1978).Nature 276:620–622.

  2. 2.

    Knight, D. E. and Baker, P. F. (1982).J. Membrane Biol. 68:107–140.

  3. 3.

    Dunn, L. A. and Holz, R. W. (1983).J. Biol. Chem. 258:4989–4993.

  4. 4.

    Wilson, S. P. and Kirshner, N. (1983).J. Biol. Chem. 258:4994–5000.

  5. 5.

    Brooks, J. C. and Treml, S. (1983).J. Neurochem. 40:468–473.

  6. 6.

    Kelner, K., Morita, K., Rossen, J. S. and Pollard, H. B. (1986).Proc. Nat. Acad. Sci. (USA) 83:2998–3002.

  7. 7.

    Douglas, W. W. and Rubin, R. R. (1964).J. Physiol. (Lond.) 175:231–241.

  8. 8.

    Douglas, W. W. (1975). In:Handbook of Physiology Endocrinology, Sec. 7, Vol. 6 (H. Blaschko, G. Sayers and A. D. Smith, Eds.), American Physiological Society, Washington DC, pp. 367–388.

  9. 9.

    Trifaro, J. M. (1978).Neuroscience 3:1–24.

  10. 10.

    Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R. and Hartshorne, D. J. (1978).Biochemistry 17:4411–4418.

  11. 11.

    Sellers, J. R., Pato, M. D. and Adelstein, R. S. (1981).J. Biol. Chem. 256: 13137–13142.

  12. 12.

    Adelstein, R. S. and Eisenberg, E. (1980).Annu. Rev. Biochem. 49:921–956.

  13. 13.

    Hartshorne, D. J. and Siemankowski, R. F. (1981).Annu. Rev. Physiol. 43:519–530.

  14. 14.

    Scholey, J. M., Taylor, K. A. and Keadrick-Jones, J. (1980).Nature,287:233–235.

  15. 15.

    Daniel, J. L., Malish, I. R. and Holmsen, H. (1981).J. Biol. Chem. 256:7510–7514.

  16. 16.

    Trifaró, J. M., Lee, R. W. H., Kenigsberg, R. L. and Coté, A. (1982). In:Advances in the Biosciences (F. Izumiet al. Eds.), Vol. 36, pp. 151–158.

  17. 17.

    Schneider, A. S., Cline, H. T., Rosenheck, K. and Sonenberg, M. J. (1981).J. Neurochem. 37:567–575.

  18. 18.

    Holz, R. W., Senter, R. A. and Frye, R. A., (1982).J. Neurochem. 39:635–646.

  19. 19.

    Waymire, J. C., Bennett, W. F., Boehme, R., Hanteins, L., Gilmer-Waymire, K. and Haycock, J. (1983).J. Neurosci. Methods 7:329–351.

  20. 20.

    Laemmli, U. K. (1970).Nature 227:680–685.

  21. 21.

    O'Farrell, P. H. (1975).J. Biol. Chem. 250:4007–4021.

  22. 22.

    Ueda, T., Maeno, H. and Greengard, P. (1973).J. Biol. Chem. 248:8295–8305.

  23. 23.

    Hathaway, D. R., Konicki, M. V. and Coolican, S. A. (1985).J. Mol. Cell. Cardiol 17: 841–850.

  24. 24.

    Haeberle, J. R., Hott, J. W. and Hathaway, D. R. (1984).Biochem. Biophys. Acta 790: 78–86.

  25. 25.

    Ikebe, M. and Hartshorne, D. J. (1985).J. Biol. Chem. 260:10027–10031.

  26. 26.

    Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y. (1982).J. Biol. Chem. 257:7847–7851.

  27. 27.

    Pocotte, S. L., Frye, R. A., Senter, R. A., TerBush, D. R., Lee, S. A. and Holz, R. W. (1985).Proc. Natl. Acad. Sci. USA 82:930–934.

  28. 28.

    Cheek, T. R. and Burgoyne, R. D. (1986).FEBS Lett. 207:110–114.

  29. 29.

    Perrin, D. and Aunis, D. (1985).Nature,315:589–592.

Download references

Author information

Correspondence to Ronald W. Holz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, S.A., Holz, R.W. & Hathaway, D.R. Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells. Biosci Rep 7, 323–332 (1987).

Download citation

Key Words

  • myosin
  • light chain
  • phosphorylation
  • catecholamine
  • chromaffin cell
  • protein kinase
  • exocytosis