Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells

W. U. Yue, F. Erdődi, Andrea Murányi, Kevin D. Nullmeyer, Ronald M. Lynch, David J. Hartshorne

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 (PPlcδ). 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 PPlcδ was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PPlc, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PPlcδ, 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 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4- 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.

Original languageEnglish
Pages (from-to)499-511
Number of pages13
JournalJournal of Muscle Research and Cell Motility
Volume24
Issue number8
DOIs
Publication statusPublished - 2003

Fingerprint

Myosin-Light-Chain Phosphatase
Phosphorylation
Skeletal Muscle Fibers
Myosins
Smooth Muscle Myosins
Cells
Phosphoric Monoester Hydrolases
Muscle
Protein Isoforms
Skeletal Muscle Myosins
Protein Phosphatase 1
Okadaic Acid
Sarcomeres
Muscle Proteins
Muscle Cells
Fusion reactions
Skeletal Muscle
Light
Substrates
Enzymes

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells. / Yue, W. U.; Erdődi, F.; Murányi, Andrea; Nullmeyer, Kevin D.; Lynch, Ronald M.; Hartshorne, David J.

In: Journal of Muscle Research and Cell Motility, Vol. 24, No. 8, 2003, p. 499-511.

Research output: Contribution to journalArticle

Yue, W. U. ; Erdődi, F. ; Murányi, Andrea ; Nullmeyer, Kevin D. ; Lynch, Ronald M. ; Hartshorne, David J. / Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells. In: Journal of Muscle Research and Cell Motility. 2003 ; Vol. 24, No. 8. pp. 499-511.
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