Use of p112-deficient myoblasts to determine the temporal order of the in vitro expression of myogenic components.

The present investigation examines the function and site(s) of involvement of an ecto-protein kinase and its substrate protein (a cell surface 112 kDa protein) in the in vitro myogenic pathway. The phosphorylated 112 kDa protein (p112) has recently been shown to be involved in myogenesis. Not much information is currently available on the role of the ecto-protein kinase and the 112 kDa protein in modulating the expression of the myogenic factors and various muscle-specific proteins. Five different p112-deficient rat myoblasts were used to examine the temporal order of the in vitro expression of the myogenic components; namely, L6 myoblasts treated with BrdUrd or phloretin, a conditional p112-defective mutant (clone D1), an ecto-protein kinase-deficient mutant (clone F72), and a mutant defective in the 112 kDa protein (clone D1/S4). All these p112-deficient myoblasts were also impaired in myogenesis. The absence of p112, ecto-protein kinase, and/or the 112 kDa protein was found to have no effect on the Myf-5 mRNA level. However, the expected increase in NCAM and Myf-4 mRNAs was not observed in any of the p112-deficient myoblasts examined. This suggests that the p112 site of action is probably located upstream of the Myf-4 and NCAM sites in the myogenic pathway. While 7-28 fold increases in the MLC, MHC, and TnT transcripts were observed during myogenesis, such increases were not observed in the p112-deficient myoblasts. However, when mutant D1/S4 was transfected with the myf-4 cDNA, expression of Myf-4 in the transfectant resulted in increased level of the MLC, MHC, and TnT mRNAs, and in myotube formation, even though the Myf-5 and NCAM mRNA levels and p112 were not altered. This suggests that p112 may function by activating transcription of Myf-4, which will subsequently promote the expression of muscle-specific proteins and myotube formation. In the absence of p112, Myf-5 cannot activate the expression of Myf-4, NCAM, MLC, MHC, TnT, and myotube formation. If all these components are involved in the same myogenic pathway, then p112 may be acting downstream from Myf-5, and upstream from NCAM and Myf-4.