Modulation of vimentin containing intermediate filament distribution and phosphorylation in living fibroblasts by the cAMP-dependent protein kinase.
Microinjection of the purified catalytic subunit of the cAMP-dependent protein kinase (A-kinase) into living rat embryo fibroblasts leads to dramatic changes in vimentin intermediate filament (IF) organization, involving the collapse of the filaments into tight bundles. In some cell types, this rearrangement of the IF proceeds further, leading to an apparent loss of filament integrity, resulting in a punctate staining pattern throughout the cytoplasm. Both these types of IF rearrangement are fully reversible, and similar to structural changes previously described for IF during mitosis. As shown by electron microscopy, in rat embryo fibroblasts these changes in IF structure do not involve the loss of the 10-nM filament structure but instead correspond to the bundling together of 25 or more individual filaments. Metabolic pulse labeling of injected cells reveals that accompanying these changes in IF organization is a dramatic increase in vimentin phosphorylation which appears maximal when the IF are fully rearranged. However, this increase in IF phosphorylation is not accompanied by any significant increase in soluble vimentin. Analysis of the sites of phosphorylation on vimentin from injected cells by either V8 protease cleavage, or two-dimensional tryptic peptide mapping, revealed increased de novo phosphorylation of two vimentin phosphopeptides after microinjection of A-kinase. These data strongly suggest that the site-specific phosphorylation of vimentin by A-kinase is responsible for the dynamic changes in IF organization observed after injection of the kinase into living cells, and may be involved in similar rearrangement of the IF previously described during mitosis or after heat shock.
Lamb
N J
NJ
Cold Spring Harbor Laboratory, New York 11724.
Fernandez
A
A
Feramisco
J R
JR
Welch
W J
WJ
D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure*; D008845:Microinjections; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000008:administration & dosage / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D014746:Vimentin / Q000378:metabolism*
D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D014746:Vimentin; D011494:Protein Kinases