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 involve ment of an ecto-protein kinase and its substrate protein (a cell surfa ce 112 kDa protein) in the in vitro myogenic pathway. The phosphorylat ed 112 kDa protein (p112) has recently been shown to be involved in my ogenesis. Not much information is currently available on the role of t he ecto-protein kinase and the 112 kDa protein in modulating the expre ssion of the myogenic factors and various muscle-specific proteins. Fi ve different p112-deficient rat myoblasts were used to examine the tem poral order of the in vitro expression of the myogenic components; nam ely, 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/S 4). All these p112-deficient myoblasts were also impaired in myogenesi s. The absence of p112, ecto-protein kinase, and/or the 112 kDa protei n was found to have no effect on the Myf-5 mRNA level. However, the ex pected increase in NCAM and Myf-4 mRNAs was not observed in any of the p112-deficient myoblasts examined. This suggests that the pl 12 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 T nT transcripts were observed during myogenesis, such increases were no t observed in the p112-deficient myoblasts. However, when mutant D1/S4 was transfected with the myf-4 cDNA, expression of Myf-4 in the trans fectant resulted in increased level of the MLC, MHC, and TnT mRNAs, an d in myotube formation, even though the Myf-5 and NCAM mRNA levels and p112 were not altered. This suggests that pl 12 may function by activ ating transcription of Myf-4, which will subsequently promote the expr ession of muscle-specific proteins and myotube formation. In the absen ce 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 pl 12 may be acting downstream from Myf-5, and upstream from NCAM and Myf-4. (C) 1993 Wiley-Liss, Inc.