Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase.
The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.
Sahlgren
C M
CM
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521 Turku, Finland.
Mikhailov
A
A
Hellman
J
J
Chou
Y H
YH
Lendahl
U
U
Goldman
R D
RD
Eriksson
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002490:Central Nervous System; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000378:metabolism*; D007399:Interphase; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins; D064231:Nestin; D015739:Nocodazole / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D013912:Threonine / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D010748:Phosphopeptides; D014746:Vimentin; D013912:Threonine; D016203:CDC2 Protein Kinase; D015739:Nocodazole