Involvement of a cell surface protein and an ecto-protein kinase in myogenesis

Myogenic differentiation is composed of a sequential cascade of multiple steps leading to the formation of multinucleated myotubes. The interference with any one step would abolish myogenesis. The present investigation examined the cell surface components which might be involved in myogenesis. Studies with subconfluent day 2 cultures of rat L6 myoblasts revealed that a cell surface 112 kDa protein was phosphorylated by a Ca(2+)-, F(-)- and Mg(2+)-dependent ecto-protein kinase [Chen & Lo (1991) Biochem. J. 279, 467-474]. We have shown in the present investigation that adequate ATP was present on the cell surface for efficient functioning of this ecto-protein kinase. The phosphorylation of the 112 kDa protein by this ecto-protein kinase was decrease dramatically in confluent cells and in multinucleated myotubes. The following evidence suggests that both the 112 kDa protein and the ecto-protein kinase may play important roles in myogenesis. (i) The highest phosphorylation activity was observed in subconfluent cultures, i.e. before the onset of morphological differentiation. (ii) Treatment of cells with chemical reagents resulted in a corresponding decrease in the ecto-protein kinase, the 112 kDa protein, the phosphorylated 112 kDa protein (p112) and the ability to form myotubes. (iii) The level of p112 in a conditional myogenesis-defective mutant corresponded with the cells' eventual ability to differentiate. (iv) A mutant defective in the ecto-protein kinase was impaired in the phosphorylation of the 112 kDa protein and in myogenesis. (v) A mutant containing only residual levels of the 112 kDa protein was deficient in both p112 and myogenesis. (vi) Since the level of p112 was normal in another myogenesis-defective mutant, the phosphorylation of this protein was not likely to be a consequence of myogenic differentiation. The above findings suggest that the ecto-protein kinase and the 112 kDa protein may directly or indirectly be associated with the myogenic pathway. Since the levels of the ecto-protein kinase, the 112 kDa protein and p112 decreased dramatically upon the formation of myotubes, these proteins were probably not required once morphological differentiation had been initiated.