Overexpression of nPKC θ 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 θ, 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 C2C12 as a tissue culture model system to explore the role of nPKC θ in the formation of multinucleated myotubes. We examined endogenous levels of nPKC θ in C2C12 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 θ in C2C12 myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC θ ‐ green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC θ‐GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC θ‐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 θ ‐ GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC θ does not interfere with fusion of myoblasts into myotubes suggesting that nPKC θ activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC θ‐GFP for investigating the function of nPKC θ in living myotubes. J. Cell. Biochem. 79:71–79, 2000. © 2000 Wiley‐Liss, Inc.