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 phosphorylation of the 100-kDa protein dramatically increases during the early period 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 protein phosphorylation. This effect is dose-dependent and can be reversed upon removal of the nucleotide from the culture media. However, cAMP shows little or no effect on accumulation of the 100-kDa protein. Furthermore, phosphorylation of the 100-kDa protein by the partially purified Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP-treated cells occurs to a much lower extent than that from untreated cells. Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase III, because preincubation of cAMP with the myoblast extracts lacking the endogenous 100-kDa protein does not show any effect on activity of CaM kinase III. Similar to its effect on 100-kDa protein phosphorylation, 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 phosphorylation and myoblast fusion. On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as creatine kinase and tropomyosin. These results suggest that cAMP is involved in down-regulation of both 100-kDa protein phosphorylation and membrane fusion of cultured myoblasts. These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be associated with its inhibitory effect on myoblast fusion.
Baek
H J
HJ
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
Jeon
Y J
YJ
Kim
H S
HS
Kang
M S
MS
Chung
C H
CH
Ha
D B
DB
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D000242:Cyclic AMP / Q000502:physiology*; D054736:Elongation Factor 2 Kinase; D008561:Membrane Fusion; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits
D009124:Muscle Proteins; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase