Mitotic Reorganization of the Intermediate Filament Protein Nestin Involves Phosphorylation by cdc2 Kinase

Cecilia M Sahlgren,John E Eriksson,Jukka Hellman,Andrey Mikhailov,Ying-Hao Chou,Urban Lendahl,Robert D Goldman

doi:10.1074/jbc.m009669200

Abstract

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.

Highlights

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues
Because nestin is expressed predominantly in cells that undergo mitosis or rapidly change morphology, we examined the regulation of nestin morphology and organization in comparison with the well characterized class III intermediate filament (IF) protein vimentin in a rat immortalized central nervous system precursor cell line, ST15A, during different stages of the cell cycle
Morphological Changes of Nestin Organization during Interphase-Mitosis Transition—During interphase, ST15A cells contained IF networks that extended from the perinuclear region to the cell surface

Summary

Introduction

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. 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. Coexpression of nestin and the class III IF proteins, vimentin, GFAP, and desmin, has been documented in developing neuronal and embryonic cells In these cells, nestin shows a similar intracellular organization as the class III IF proteins [1, 8]. Intermediate filament proteins form dynamic structures that change their intracellular organization during various conditions, such as mitosis, differentiation, and different pathological situations The role of phosphorylation in regulating IF organization is supported by studies showing that several kinases, including protein kinase C, cAMP-dependent kinase, Ca2ϩ-calmodulin-dependent protein kinase, and cdc kinase, induce disassembly of IFs in vitro (18 –21). Phosphorylation may be involved in regulating the specific cellular distribution of IFs, as demonstrated by the preferential localization of phosphorylated neurofilaments in axons [32, 33], and

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