Site-specific antibodies block kinase A phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes.
Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP-dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino-terminal headpiece domains; in chicken smooth muscle desmin the most kinase A-reactive residues are ser-29 and ser-35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti-peptide antibodies directed against residues 26-36. The antibodies, which we call anti-D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26-36. Pre-incubation of desmin with affinity purified anti-D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75-80%. When antibody-treated IFs are subjected to phosphorylation, no filament break-down is observed after 3 hours. Thus anti-D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti-D26 at the mononucleated, myoblast stage. By 24 h post-injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation-induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.
Tao
J X
JX
Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Ohio 45267-0521.
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D000242:Cyclic AMP / Q000502:physiology; D003893:Desmin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000378:metabolism; D007382:Intermediate Filaments / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011784:Quail
D000906:Antibodies; D003893:Desmin; D007381:Intermediate Filament Proteins; D010761:Phosphorus Radioisotopes; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP