Phosphorylation and disruption of intermediate filament proteins in oligodendrocyte precursor cultures treated with calyculin A.
Treatment of primary cultures of oligodendrocyte precursors with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, caused the phosphorylation of two intermediate filament components, nestin and vimentin. Phosphoamino acid analysis demonstrated that phosphorylation took place mainly on serine and to a lesser extent on threonine residues. In addition, calyculin A treatment caused a shift in the distribution of the two proteins from the Triton-X-100 insoluble fraction to the detergent soluble fraction as demonstrated by immunoblotting. This redistribution, which was evident within 15 min after treatment and was nearly completed by 90 min, was accompanied by a disruption of the intermediate filament network. Thus, both nestin and vimentin retracted from the cytoplasmic processes to form a large perikaryal ring as shown by immunocytochemical analysis. Both morphological and biochemical changes were reversed 2-5 hr after removal of calyculin A from the culture medium.
Almazan
G
G
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
Afar
D E
DE
Bell
J C
JC
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn / Q000502:physiology; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D003720:Densitometry; D005455:Fluorescent Antibody Technique; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; D064231:Nestin; D009836:Oligodendroglia / Q000187:drug effects / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D051381:Rats; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D000596:Amino Acids; D007381:Intermediate Filament Proteins; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D010080:Oxazoles; D014746:Vimentin; C059041:calyculin A; D010749:Phosphoprotein Phosphatases