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© The Rockefeller University Press, 0021-9525/1998//1249 $5.00
The Journal of Cell Biology, Volume 143, Number 5, , 1998 1249-1258

Article

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments



Yoshihiro Yasui*, Mutsuki Amano{ddagger}, Koh-ichi Nagata*, Naoyuki Inagaki*, Hideo Nakamura§, Hideyuki Saya§, Kozo Kaibuchi{ddagger}, and Masaki Inagaki*

* Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-0021, Japan; {ddagger} Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan; § Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, Japan

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

Key Words: intermediate filament • glial fibrillary acidic protein (GFAP) • Rho • Rho-associated kinase • cytokinesis

Abbreviations used in this paper: aa, amino acid(s); CF, cleavage furrow; EL cells, L cells that express E-cadherin; ERM, ezrin, radixin, and moesin; IF, intermediate filament; GFAP, glial fibrillary acidic protein; MBS, myosin-binding subunit of myosin phosphatase; MLC, myosin light chain; PH, pleckstrin homology; PI, propidium iodide; PKN, protein kinase N.

This research was supported in part by Grants-in-Aid for Scientific Research and Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan, Japan Society of the Promotion of Science Research for the Future, special coordination funds from the Science and Technology Agency of the Government of Japan, and a grant from Bristol-Myers-Squibb.


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