Overexpression of nPKC theta is permissive for myogenic differentiation.
Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.
Miles
K
K
Department of Anatomy and Cell Biology, The State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.
Wagner
M
M
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D013755:Tetradecanoylphorbol Acetate