Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.
The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D003893:Desmin; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism*; D014746:Vimentin
D003893:Desmin; D007381:Intermediate Filament Proteins; D014335:Tropomyosin; D014746:Vimentin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D007545:Isoproterenol