Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase

The intermediate filament protein nestin is expressed during early stages o f development in the central nervous system and in muscle tissues. Nestin e xpression is associated with morphologically dynamic cells, such as dividin g 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 n euronal progenitor cell line, ST15A. Confocal microscopy of nestin organiza tion and P-32 in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosph orylation-induced alterations in nestin organization during mitosis in ST15 A cells are associated with partial disassembly of nestin filaments. Compar ative in vitro and in vivo phosphorylation studies identified cdc2 as the p rimary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the p hosphorylated form of this site. By using this antibody we observed that ne stin shows constitutive phosphorylation at Thr316, which is increased durin g mitosis. This study shows that nestin is reorganized during mitosis and t hat cdc2-mediated phosphorylation is an important regulator of nestin organ ization and dynamics during mitosis.