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. 1988 Jun 1;106(6):1955–1971. doi: 10.1083/jcb.106.6.1955

Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase

PMCID: PMC2115126  PMID: 3290222

Abstract

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P- light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.

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Selected References

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  1. Adelstein R. S. Calmodulin and the regulation of the actin-myosin interaction in smooth muscle and nonmuscle cells. Cell. 1982 Sep;30(2):349–350. doi: 10.1016/0092-8674(82)90232-x. [DOI] [PubMed] [Google Scholar]
  2. Adelstein R. S., Klee C. B. Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem. 1981 Jul 25;256(14):7501–7509. [PubMed] [Google Scholar]
  3. Adelstein R. S., Pato M. D., Conti M. A. The role of phosphorylation in regulating contractile proteins. Adv Cyclic Nucleotide Res. 1981;14:361–373. [PubMed] [Google Scholar]
  4. Adelstein R. S., Pato M. D., Sellers J. R., de Lanerolle P., Conti M. A. Regulation of actin-myosin interaction by reversible phosphorylation of myosin and myosin kinase. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):921–928. doi: 10.1101/sqb.1982.046.01.086. [DOI] [PubMed] [Google Scholar]
  5. Albertini D. F., Herman B. Cell shape and membrane receptor dynamics. Modulation by the cytoskeleton. Cell Muscle Motil. 1984;5:235–253. doi: 10.1007/978-1-4684-4592-3_6. [DOI] [PubMed] [Google Scholar]
  6. Bayley S. A., Rees D. A. Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. Eur J Cell Biol. 1986 Oct;42(1):10–16. [PubMed] [Google Scholar]
  7. Beavo J. A., Bechtel P. J., Krebs E. G. Activation of protein kinase by physiological concentrations of cyclic AMP. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3580–3583. doi: 10.1073/pnas.71.9.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bechtel P. J., Beavo J. A., Krebs E. G. Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977 Apr 25;252(8):2691–2697. [PubMed] [Google Scholar]
  9. Bershadsky A. D., Gelfand V. I., Svitkina T. M., Tint I. S. Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism. Exp Cell Res. 1980 Jun;127(2):421–429. doi: 10.1016/0014-4827(80)90446-2. [DOI] [PubMed] [Google Scholar]
  10. Browne C. L., Lockwood A. H., Su J. L., Beavo J. A., Steiner A. L. Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells. J Cell Biol. 1980 Nov;87(2 Pt 1):336–345. doi: 10.1083/jcb.87.2.336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Browning E. T., Sanders M. M. Vimentin: a phosphoprotein under hormonal regulation. J Cell Biol. 1981 Sep;90(3):803–808. doi: 10.1083/jcb.90.3.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Celis J. E., Larsen P. M., Fey S. J., Celis A. Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis. J Cell Biol. 1983 Nov;97(5 Pt 1):1429–1434. doi: 10.1083/jcb.97.5.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chida K., Kato N., Kuroki T. Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes. J Biol Chem. 1986 Oct 5;261(28):13013–13018. [PubMed] [Google Scholar]
  14. Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature. 1982 Apr 15;296(5858):613–620. doi: 10.1038/296613a0. [DOI] [PubMed] [Google Scholar]
  15. Cohen P. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur J Biochem. 1985 Sep 16;151(3):439–448. doi: 10.1111/j.1432-1033.1985.tb09121.x. [DOI] [PubMed] [Google Scholar]
  16. Conti M. A., Adelstein R. S. The relationship between calmodulin binding and phosphorylation of smooth muscle myosin kinase by the catalytic subunit of 3':5' cAMP-dependent protein kinase. J Biol Chem. 1981 Apr 10;256(7):3178–3181. [PubMed] [Google Scholar]
  17. Cooke R. The mechanism of muscle contraction. CRC Crit Rev Biochem. 1986;21(1):53–118. doi: 10.3109/10409238609113609. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Dabrowska R., Hartshorne D. J. A Ca2+-and modulator-dependent myosin light chain kinase from non-muscle cells. Biochem Biophys Res Commun. 1978 Dec 29;85(4):1352–1359. doi: 10.1016/0006-291x(78)91152-x. [DOI] [PubMed] [Google Scholar]
  20. Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fey S. J., Larsen M., Celis J. E. Modification of vimentin polypeptides during mitosis. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):379–385. doi: 10.1101/sqb.1982.046.01.037. [DOI] [PubMed] [Google Scholar]
  22. Frearson N., Focant B. W., Perry S. V. Phosphorylation of a light chain component of myosin from smooth muscle. FEBS Lett. 1976 Mar 15;63(1):27–32. doi: 10.1016/0014-5793(76)80187-1. [DOI] [PubMed] [Google Scholar]
  23. Gard D. L., Lazarides E. Analysis of desmin and vimentin phosphopeptides in cultured avian myogenic cells and their modulation by 8-bromo-adenosine 3',5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6912–6916. doi: 10.1073/pnas.79.22.6912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gard D. L., Lazarides E. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells. Mol Cell Biol. 1982 Sep;2(9):1104–1114. doi: 10.1128/mcb.2.9.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Geiger B. Intermediate filaments. Looking for a function. Nature. 1987 Oct 1;329(6138):392–393. doi: 10.1038/329392a0. [DOI] [PubMed] [Google Scholar]
  26. Gilmartin M. E., Mitchell J., Vidrich A., Freedberg I. M. Dual regulation of intermediate filament phosphorylation. J Cell Biol. 1984 Mar;98(3):1144–1149. doi: 10.1083/jcb.98.3.1144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Glass D. B., Krebs E. G. Protein phosphorylation catalyzed by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Annu Rev Pharmacol Toxicol. 1980;20:363–388. doi: 10.1146/annurev.pa.20.040180.002051. [DOI] [PubMed] [Google Scholar]
  28. Hathaway D. R., Adelstein R. S. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1653–1657. doi: 10.1073/pnas.76.4.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hathaway D. R., Eaton C. R., Adelstein R. S. Regulation of human platelet myosin light chain kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. Nature. 1981 May 21;291(5812):252–256. doi: 10.1038/291252a0. [DOI] [PubMed] [Google Scholar]
  30. Hemmings B. A. Regulation of cAMP-dependent protein kinase in cultured cells. Curr Top Cell Regul. 1985;27:117–132. doi: 10.1016/b978-0-12-152827-0.50017-7. [DOI] [PubMed] [Google Scholar]
  31. Kamm K. E., Stull J. T. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25:593–620. doi: 10.1146/annurev.pa.25.040185.003113. [DOI] [PubMed] [Google Scholar]
  32. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  33. Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem. 1982;51:219–250. doi: 10.1146/annurev.bi.51.070182.001251. [DOI] [PubMed] [Google Scholar]
  34. Leitman D. C., Fiscus R. R., Murad F. Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells. J Cell Physiol. 1986 May;127(2):237–243. doi: 10.1002/jcp.1041270208. [DOI] [PubMed] [Google Scholar]
  35. Lockwood A. H., Murphy S. K., Borislow S., Lazarus A., Pendergast M. Cellular signal transduction and the reversal of malignancy. J Cell Biochem. 1987 Apr;33(4):237–255. doi: 10.1002/jcb.240330403. [DOI] [PubMed] [Google Scholar]
  36. Lockwood A. H., Trivette D. D., Pendergast M. Molecular events in cAMP-mediated reverse transformation. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):909–919. doi: 10.1101/sqb.1982.046.01.085. [DOI] [PubMed] [Google Scholar]
  37. Nigg E. A., Schäfer G., Hilz H., Eppenberger H. M. Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell. 1985 Jul;41(3):1039–1051. doi: 10.1016/s0092-8674(85)80084-2. [DOI] [PubMed] [Google Scholar]
  38. Nishikawa M., de Lanerolle P., Lincoln T. M., Adelstein R. S. Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase. J Biol Chem. 1984 Jul 10;259(13):8429–8436. [PubMed] [Google Scholar]
  39. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  40. Otten J., Johnson G. S., Pastan I. Regulation of cell growth by cyclic adenosine 3',5'-monophosphate. Effect of cell density and agents which alter cell growth on cyclic adenosine 3',5'-monophosphate levels in fibroblasts. J Biol Chem. 1972 Nov 10;247(21):7082–7087. [PubMed] [Google Scholar]
  41. Persechini A., Kamm K. E., Stull J. T. Different phosphorylated forms of myosin in contracting tracheal smooth muscle. J Biol Chem. 1986 May 15;261(14):6293–6299. [PubMed] [Google Scholar]
  42. 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]
  43. Porter K. R., Puck T. T., Hsie A. W., Kelley D. An electron microscopy study of the effects on dibutyryl cyclic AMP on Chinese hamster ovary cells. Cell. 1974 Jul;2(3):145–162. doi: 10.1016/0092-8674(74)90089-0. [DOI] [PubMed] [Google Scholar]
  44. Rathke P. C., Seib E., Weber K., Osborn M., Franke W. W. Rod-like elements from actin-containing microfilament bundles observed in cultured cells after treatment with cytochalasin A (CA). Exp Cell Res. 1977 Mar 15;105(2):253–262. doi: 10.1016/0014-4827(77)90123-9. [DOI] [PubMed] [Google Scholar]
  45. Schliwa M. Proteins associated with cytoplasmic actin. Cell. 1981 Sep;25(3):587–590. doi: 10.1016/0092-8674(81)90166-5. [DOI] [PubMed] [Google Scholar]
  46. Sellers J. R., Spudich J. A., Sheetz M. P. Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J Cell Biol. 1985 Nov;101(5 Pt 1):1897–1902. doi: 10.1083/jcb.101.5.1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shacter E., Chock P. B., Stadtman E. R. Regulation through phosphorylation/dephosphorylation cascade systems. J Biol Chem. 1984 Oct 10;259(19):12252–12259. [PubMed] [Google Scholar]
  48. Silver P. J., DiSalvo J. Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin. J Biol Chem. 1979 Oct 25;254(20):9951–9954. [PubMed] [Google Scholar]
  49. Singh T. J., Akatsuka A., Huang K. P. Phosphorylation of smooth muscle myosin light chain by five different kinases. FEBS Lett. 1983 Aug 8;159(1-2):217–220. doi: 10.1016/0014-5793(83)80449-9. [DOI] [PubMed] [Google Scholar]
  50. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Steinberg R. A., Coffino P. Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells: protein modifications, inductions and repressions. Cell. 1979 Nov;18(3):719–733. doi: 10.1016/0092-8674(79)90126-0. [DOI] [PubMed] [Google Scholar]
  52. Steinert P. M., Jones J. C., Goldman R. D. Intermediate filaments. J Cell Biol. 1984 Jul;99(1 Pt 2):22s–27s. doi: 10.1083/jcb.99.1.22s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stossel T. P. Contribution of actin to the structure of the cytoplasmic matrix. J Cell Biol. 1984 Jul;99(1 Pt 2):15s–21s. doi: 10.1083/jcb.99.1.15s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stull J. T. Phosphorylation of contractile proteins in relation to muscle function. Adv Cyclic Nucleotide Res. 1980;13:39–93. [PubMed] [Google Scholar]
  55. 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]
  56. Wagner P. D., George J. N. Phosphorylation of thymus myosin increases its apparent affinity for actin but not its maximum adenosinetriphosphatase rate. Biochemistry. 1986 Feb 25;25(4):913–918. doi: 10.1021/bi00352a026. [DOI] [PubMed] [Google Scholar]
  57. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  58. Weber K., Rathke P. C., Osborn M., Franke W. W. Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB). Exp Cell Res. 1976 Oct 15;102(2):285–297. doi: 10.1016/0014-4827(76)90044-6. [DOI] [PubMed] [Google Scholar]
  59. Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]
  60. Willingham M. C., Pastan I. Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum. J Cell Biol. 1975 Oct;67(1):146–159. doi: 10.1083/jcb.67.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zeilig C. E., Goldberg N. D. Cell-cycle-related changes of 3':5'-cyclic GMP levels in Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1052–1056. doi: 10.1073/pnas.74.3.1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. de Lanerolle P., Nishikawa M., Yost D. A., Adelstein R. S. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science. 1984 Mar 30;223(4643):1415–1417. doi: 10.1126/science.6322302. [DOI] [PubMed] [Google Scholar]

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