Journal of Muscle Research & Cell Motility

, Volume 24, Issue 8, pp 499–511 | Cite as

Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells

  • Yue Wu
  • Ferenc Erdődi
  • Andrea Murányi
  • Kevin D. Nullmeyer
  • Ronald M. Lynch
  • David J. Hartshorne


C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, δ isoform (PP1cδ). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1cδ was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1cδ, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by l-(5-chloronaphthalene-l-sulphonyl)-1H-hexahydro-l,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3–5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alessi D, MacDougall LK, Sola MM, Ikebe M and Cohen P (1992)The control of protein phosphatase-1 by targeting subunits.The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.Eur J Biochem 210:1023-1035.PubMedCrossRefGoogle Scholar
  2. Aoki H, Sadoshima J and Izumo S (2000)Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in-vitro.Nat Med 6:183-188.PubMedCrossRefGoogle Scholar
  3. Arnold HH and Winter B (1998)Muscle di.erentiation:more complexity to the network of myogenic regulators.Curr Opin Genet Dev 8:539-544.PubMedCrossRefGoogle Scholar
  4. Aromolaran AS, Albert AP and Large WA (2000)Evidence for myosin light chain kinase mediating noradrenaline-evoked cation current in rabbit portal vein myocytes.J Physiol 524:853-863.PubMedCrossRefGoogle Scholar
  5. Bergstrom DA, Penn BH, Strand A, Perry RLS, Rudnicki MA and Tapscott SJ (2002)Promoter-speci c regulation of MyoD binding and signal transduction cooperate to pattern gene expression.Mol Cell 9:587-600.PubMedCrossRefGoogle Scholar
  6. Black BL and Olson EN (1998)Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2)proteins.Ann Rev Cell Dev Biol 14:167-196.CrossRefGoogle Scholar
  7. Damer CK, Partridge J, Pearson WR and Haystead TAJ (1998)Rapid identi cation of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching.Characterization of a novel holoenzymic form of protein phosphatase 1.J Biol Chem 273:24396-24405.CrossRefGoogle Scholar
  8. Dent P, MacDougall LK, MacKintosh C, Campbell DG and Cotton P (1992)A myo brillar protein phosphatase from rabbit skeletal muscle contains the b isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphory-lation of myosin.Eur J Biochem 210:1037-1044.PubMedCrossRefGoogle Scholar
  9. Dunican DF and Doherty P (2001)Designing cell-permeant phospho-peptides to modulate intracellular signaling pathways.Biopolymers 60:45-60.PubMedCrossRefGoogle Scholar
  10. Epstein HF and Fischman DA (1991)Molecular analysis of protein assembly in muscle development.Science 251:1039-1044.PubMedGoogle Scholar
  11. Etter EF, Eto M, Wardle RL, Brautigan DL and Murphy RA (2001)Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.J Biol Chem 276:34681-34685.PubMedCrossRefGoogle Scholar
  12. Fisher SA and Ikebe M (1995)Developmental and tissue distribution of expression of nonmuscle and smooth muscle isoforms of myosin light chain kinase.Biochem Biophys Res Commun 217:696-703.PubMedCrossRefGoogle Scholar
  13. Foster CJ, Johnson SA, Sunday B and Gaeta FCA (1990)Potent peptide inhibitors of smooth muscle myosin light chain kinase: mapping of the pseudosubstrate and calmodulin binding domains. Arch Biochem Biophys 280:397-404.PubMedCrossRefGoogle Scholar
  14. Fujioka M, Takahashi N, Odai H, Araki S, Ichikawa K, Feng J, Nakamura M, Kabuchi K, Hartshorne DJ, Nakano T and Ito M (1998)A new isoform of human myosin phosphatase targeting/ regulatory subunit (MYPT2):cDNA cloning, tissue expression, and chromosomal mapping.Genomics 49:59-68.PubMedCrossRefGoogle Scholar
  15. Gius DR, Ezhevsky SA, Becker-Hapak M, Nagahara H, Wei MC and Dowdy SF (1999)Transduced p16 INK4a peptides inhibit hypo-phosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1. Cancer Res 59:2577-2580.PubMedGoogle Scholar
  16. Hartshorne DJ (1998)Myosin phosphatase:subunits and interactions. Acta Physiol Scand 164:483-493.PubMedCrossRefGoogle Scholar
  17. Hartshorne DJ, Ito M and Erdodi F (1998)Myosin light chain phosphatase:subunit composition, interactions and regulation.J Mus Res Cell Motil 19:325-341.CrossRefGoogle Scholar
  18. Hathaway DR and Haeberle JR (1983)Selective puri cation of the 20, 000-Da light chains of smooth muscle myosin.Anal Biochem 135: 37-43.PubMedCrossRefGoogle Scholar
  19. Herring BP, Dixon S and Gallagher PJ (2000)Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle.Am J Physiol 279:C1656-C1664.Google Scholar
  20. Hirano K, Phan BC and Hartshorne DJ (1997)Interactions of the subunits of smooth muscle myosin phosphatase.J Biol Chem 272: 3683-3688.PubMedCrossRefGoogle Scholar
  21. Ichikawa K, Hirano K, Ito M, Tanaka J, Nakano T and Hartshorne DJ (1996)Interactions and properties of smooth muscle myosin phosphatase.Biochemistry 35:6313-6320.PubMedCrossRefGoogle Scholar
  22. Ikebe M and Hartshorne DJ (1985)Effects of Ca 2 + on the conforma-tion and enzymatic activity of smooth muscle myosin.J Biol Chem 260:13146-13153.PubMedGoogle Scholar
  23. Itoh T, Ikebe M, Kargacin GJ, Hartshorne DJ, Kemp BE and Fay FS (1989)E.ects of modulators of myosin light-chain kinase activity in single smooth muscle cells.Nature 338:164-167.PubMedCrossRefGoogle Scholar
  24. Kamm KE and Stull JT (2001)Dedicated myosin light chain kinases with diverse cellular functions.J Biol Chem 276:4527-4530.PubMedCrossRefGoogle Scholar
  25. Lazar V and Garcia JG (1999)A single human myosin light chain kinase gene.Genomics 57:256-267.PubMedCrossRefGoogle Scholar
  26. Leachman SA, Gallagher PJ, Herring BP, McPhaul MJ and Stull JT (1992)Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.J Biol Chem 267:4930-4938.PubMedGoogle Scholar
  27. Mabuchi K, Gong B-J, Langsetmo K, Ito M, Nakano T and Tao T (1999)Isoforms of the small non-catalytic subunit of smooth muscle myosin light chain phosphatase.Biochim Biophys Acta 1434:296-303.PubMedGoogle Scholar
  28. Margossian SS and Lowey S (1982)Preparation of myosin and its sub-fragments from rabbit skeletal muscle.Method Enzymol 85:55-71.Google Scholar
  29. Martinez-Zaguilan R, Tompkins LS, Gillies RJ and Lynch RM (1999)Simultaneous measurements of calcium and pH in cell popula-tions.In:Lambert DG (ed)Calcium Signaling Protocols.(vol.114, pp.287-306)Humana Press, NJ.Google Scholar
  30. McKinsey TA, Zhang CL and Olson EN (2001)Control of muscle development by dueling HATS and HDACs.Curr Opin Genet Dev 11:497-504.PubMedCrossRefGoogle Scholar
  31. McKinsey TA, Zhang CL and Olson EN (2002)MEF2:a calcium-dependent regulator of cell division, di.erentiation and death. Trends Biochem Sci 27:40-47.PubMedCrossRefGoogle Scholar
  32. Molkentin JD and Olson EN (1996)De ning the regulatory networks for muscle development.Curr Opin Genet Dev 6:445-453.PubMedCrossRefGoogle Scholar
  33. Moorhead G, Johnson D, Morrice N and Cohen P (1998)The major myosin phosphatase in skeletal muscle is a complex between the b isoform of protein phosphatase 1 and the MYPT2 gene product. FEBS Lett 438:141-144.PubMedCrossRefGoogle Scholar
  34. Mura´nyi A, Zhang R, Liu F, Hirano K, Ito M, Epstein H and Hartshorne DJ (2001)Myotonic dystrophy protein kinase phos-phorylates the myosin phosphatase targeting subunit and inhibits phosphatase activity.FEBS Lett 493:80-84.CrossRefGoogle Scholar
  35. Nunnally MH, Rybicki SB and Stull JT (1985)Characterization of chicken skeletal muscle myosin light chain kinase.Evidence for muscle-speci c isozymes.J Biol Chem 260:1020-1026.PubMedGoogle Scholar
  36. Paterson B and Strohman RC (1972)Myosin synthesis in cultures of di.erentiating chicken embryo skeletal muscle.Dev Biol 29:113-138.PubMedCrossRefGoogle Scholar
  37. Persechini A, Kamm KE and Stull JT (1986)Different phosphorylated forms of myosin in contacting tracheal smooth muscle.J Biol Chem 261:6293-6299.PubMedGoogle Scholar
  38. Porter GA, Makuek RF and Rivkees SA (2002)Reduction in intracellular calcium levels inhibit myoblast di.erentiation.J Biol Chem 277:28942-28947.PubMedCrossRefGoogle Scholar
  39. Sabourin LA and Rudnicki MA (2000)The molecular regulation of myogenesis.Clin Genet 57:16-25.PubMedCrossRefGoogle Scholar
  40. Saitoh M, Ishikawa T, Matsushima S, Naka M and Hidaka H (1987)Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase.J Biol Chem 262:7796-7801.PubMedGoogle Scholar
  41. Schwarze SR and Dowdy SF (2000)In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA.Trends Pharmacol Sci 21:45-48.PubMedCrossRefGoogle Scholar
  42. Schwarze SR, Ho A, Vocero-Akbani A and Dowdy SF (1999)In vivo transduction:delivery of a biologically active protein into the mouse.Science 285:1569-1572.PubMedCrossRefGoogle Scholar
  43. Shainberg A, Yagil G and Yae D (1969)Control of myogenesis in vitro by Ca 2 + concentration in nutritional medium.Exp Cell Res 58:163-167.PubMedCrossRefGoogle Scholar
  44. Shen M-R, Furla P, Chou C-Y and Ellory JC (2002)Myosin light chain kinase modulates hypotonicity-induced Ca 2 + entry and Cl ) channel activity in human cervical cancer cells.Pflu ¨gers Arch 444: 276-285.CrossRefGoogle Scholar
  45. Shima H, Hatano Y, Chun Y-S, Sugimura T, Zhang Z, Lee EYC and Nagao M (1993)Identi cation of PP1 catalytic subunit isotypes PP1 c PP1 d and PP1 a in various rat tissues.Biochem Biophys Res Commun 192:1289-1296.PubMedCrossRefGoogle Scholar
  46. Shimizu H, Ito M, Miyahara M, Ichikawa K, Okubo S, Konishi T, Naka M, Tanaka T, Hirano K, Hartshorne DJ and Nakano T (1994)Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase.J Biol Chem 269:30407-30411.PubMedGoogle Scholar
  47. Shoemaker MD, Lau W, Shattuck RL, Kwiatkowski AP, Matrisian PE, Guerra-Santos L, Wilson E, Lukas TJ, van Eldik LJ and Watterson DM (1990)Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhi-bition, calmodulin recognition, and activity.J Cell Biol 111:1107-1125.PubMedCrossRefGoogle Scholar
  48. Takahashi N, Ito M, Tanaka J, Nakano T, Kaibuchi K, Odai H and Takemura K (1997)Localization of the gene coding for myosin phosphatase target subunit 1 (MYPT1)to human chromosome 12q15-q21.Genomics 44:150-152.PubMedCrossRefGoogle Scholar
  49. Takeda K, Yu Z-X, Qian S, Chin TK, Adelstein RS and Ferrans VJ (2000)Nonmuscle myosin II localizes to the Z-lines and interca-lated discs of cardiac muscle and to the Z-lines of skeletal muscle. Cell Motil Cytoskeleton 46:59-68.PubMedCrossRefGoogle Scholar
  50. Tognarini M and Villa-Moruzzi E (1998)Protein phosphatase 1 isoforms in di.erentiating C2C12 myocytes.Eur J Cell Biol 76: 212-219.PubMedGoogle Scholar
  51. To´th A, Kiss E, Herberg FW, Gergely P, Hartshorne DJ and Erdodi F (2000)Study of the subunit interactions in myosin phosphatase by surface plasmon resonance.Eur J Biochem 267:1687-1697.CrossRefGoogle Scholar
  52. Tran Q-K, Watanabe H, Le H-Y, Pan L, Seto M, Takeuchi K and Ohashi K (2001)Myosin light chain kinase regulates capacitative Ca 2 + entry in human monocytes/macrophages.Arterioscler Thromb Vasc Biol 21:509-515.PubMedGoogle Scholar
  53. Tullio AN, Accili D, Ferrans VJ, Yu Z-X, Takeda K, Grinberg A, Westphal H, Preston YA and Adelstein RS (1997)Nonmuscle myosin II-B is required for normal development of the mouse heart.Proc Natl Acad Sci USA 94:12407-12412.PubMedCrossRefGoogle Scholar
  54. Walsh MP, Hinkins S, Dabrowska R and Hartshorne DJ (1983)Smooth muscle myosin light chain kinase.Method Enzymol 99: 279-288.CrossRefGoogle Scholar
  55. Weber LP, Van Lierop JE and Walsh MP (1999)Ca 2 +-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myo laments.J Physiol 516:805-824.PubMedCrossRefGoogle Scholar
  56. Wu H, Naya FJ, McKinsey TA, Mercer B, Shelton JM, Chin ER, Simard AR, Michel RN, Bassel-Duby R, Olson EN and Williams RS (2000)MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle ber type.EMBO J 19:1963-1973.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Yue Wu
    • 1
  • Ferenc Erdődi
    • 2
  • Andrea Murányi
    • 1
  • Kevin D. Nullmeyer
    • 3
  • Ronald M. Lynch
    • 3
  • David J. Hartshorne
    • 3
  1. 1.Muscle Biology GroupUniversity of ArizonaTucsonUSA
  2. 2.Department of Medical ChemistryUniversity of DebrecenDebrecenUSA
  3. 3.Department of PhysiologyUniversity of ArizonaTucsonUSA

Personalised recommendations