CYCLIC-AMP NEGATIVELY MODULATES BOTH CA2+ CALMODULIN-DEPENDENT PHOSPHORYLATION OF THE 100-KDA PROTEIN AND MEMBRANE-FUSION OF CHICK EMBRYONIC MYOBLASTS/

We have previously shown that Ca2+/calmodulin-dependent phosphorylatio n of the 100-kDa protein dramatically increases during the early perio d of myoblast fusion and treatment of calmodulin antagonists, such as trifluoperazine, blocks the fusion. Here, we show that cAMP treatment of primary cultures of chick embryonic myoblasts blocks 100-kDa protei n phosphorylation. This effect is dose-dependent and can be reversed u pon removal of the nucleotide from the culture media. However, cAMP sh ows little or no effect on accumulation of the 100-kDa protein. Furthe rmore, phosphorylation of the 100-kDa protein by the partially purifie d Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP- treated cells occurs to a much lower extent than that from untreated c ells. Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase II I, because preincubation of cAMP with the myoblast extracts lacking th e endogenous 100-kDa protein does not show any effect on activity of C aM kinase III. Similar to its effect on 100-kDa protein phosphorylatio n, cAMP reversibly inhibits the fusion of cultured myoblasts. Moreover , treatment with forskolin or theophylline, which is known to elevate the intracellular cAMP level, also reversibly blocks both protein phos phorylation and myoblast fusion. On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as crea tine kinase and tropomyosin. These results suggest that cAMP is involv ed in down-regulation of both 100-kDa protein phosphorylation and memb rane fusion of cultured myoblasts. These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be as sociated with its inhibitory effect on myoblast fusion. (C) 1994 Acade mic Press,Inc.