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

Abstract

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.

Rho-kinase contributes to diphosphorylation of myosin II regulatory light chain in nonmuscle cells

“…Here, we demonstrated that Rho-kinase diphosphorylates MRLC and colocalizes with the diphosphorylated MRLC at the cleavage furrow in dividing HeLa cells. Other substrates phosphorylated by Rho-kinase have been reported to accumulate at the cleavage furrow in dividing non-muscle cells (Yasui et al, 1998;Kawano et al, 1999). Recently, Kosako et al (2000) reported that Rho-kinase is involved in cytokinesis through the monophosphorylation of MRLC and not ERM at the cleavage furrow.…”

“…These suggest that the regulation of actin cytoskeleton through MRLC diphosphorylation may be essential for tumor cell invasion. Yasui et al (1998) reported that expression of the dominant-negative form of Rho-kinase inhibited cytokinesis and increased in the multi-nuclei cells, suggesting that Rho-kinase plays an important role in cytokinesis. Here, we demonstrated that Rho-kinase diphosphorylates MRLC and colocalizes with the diphosphorylated MRLC at the cleavage furrow in dividing HeLa cells.…”

Autophosphorylation of a Newly Identified Site of Aurora-B Is Indispensable for Cytokinesis

“…Twenty-four or 48 h after transfection, the cells were fixed for immunocytochemical studies. In some experiments, 48 h after transfection, the mitotic cells were prepared as described (17) and incubated for 3 h to allow for cell cycle progression. Intermediate filament (IF) bridge formation and multinuclear cells were analyzed immunocytochemically.…”

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments

“…Most recently, we found that the expression of mutant GFAP, where the Rho-kinase phosphorylation sites were substituted to alanine residues, impaired cytokinetic segregation of GFAP ®laments, resulting in the formation of an unusually long bridge-like structure between the unseparated daughter cells (Yasui et al, 1998). These observations suggest that the phosphorylation-dependent disassembly of intermediate ®laments is required for proper execution and completion of cytokinesis.…”