Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis.
To investigate the role of intermediate filament (IF) protein phosphorylation by cdc2 kinase during mitosis, we developed a monoclonal antibody 4A4 recognizing Ser55-phosphorylated vimentin. Western blotting indicated that this antibody reacted with vimentin phosphorylated by cdc2 kinase but not with non-phosphorylated vimentin or with vimentin phosphorylated by other kinases such as cAMP-dependent protein kinase, protein kinase C, or Ca(2+)-calmodulin-dependent protein kinase II. Immunofluorescence and immunoelectron microscopy showed that vimentin Ser55 residues distributed in the entire cytoplasmic vimentin filament system are phosphorylated when the cells enter mitosis and dephosphorylated in cytokinesis. All cell lines examined showed a similar appearance of immunoreactivity with antibody 4A4. Fractionation of mitotic cell extracts on Mono-Q Sepharose revealed a single peak of vimentin Ser55 kinase activity, and the anti-p34cdc2 antibody reacted with the 34 kDa band in the kinase containing fractions. Vimentin Ser55 kinase activities were nil in the interphase cell extract. Immunofluorescent evidence using antibody 4A4 and biochemical analysis using vimentin Ser55 peptide showed that the degree of disassembly of vimentin filament of various cell types at early mitotic phase correlated well with the amount of mitotically activated cdc2 kinase.
Tsujimura
K
K
Department of Neurophysiology, Tokyo Metropolitan Institute of Gerontology, Japan.
Ogawara
M
M
Takeuchi
Y
Y
Imajoh-Ohmi
S
S
Ha
M H
MH
Inagaki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008677:Metaphase; D051379:Mice; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014746:Vimentin; D012694:Serine; D016203:CDC2 Protein Kinase