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Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments.
Yasui Y
,
Amano M
,
Nagata K
,
Inagaki N
,
Nakamura H
,
Saya H
,
Kaibuchi K
,
Inagaki M
.
???displayArticle.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.
Figure 2. Effects of dominant-negative form of Rho-kinase on cytokinesis in EL cells. (A) EL cells were transiently transfected with either pEF-BOS-myc vector (panel a), pEF-BOS-myc-COIL (panel b), or pEF-BOS-myc-RB/PH (TT) (panel c). pME18S-lacZ was cotransfected with control vector to identify the transfected cells. 72 h after the transfection, the cells were fixed and stained for β-galactosidase (panel a) and myc-tagged protein (panels b and c). (B) 72 h (open column) or 120 h (hatched column) after the transfection of plasmids encoding indicated cDNAs, the cells were fixed and stained for β-galactosidase activity. The percentage of LacZ-positive cells bearing multinuclei was scored. Data are means ± SEM of at least triplicate determinations. At least 200 cells per each sample were counted and at least three independent experiments were performed. Bar, 40 μm.
Figure 3. (A) Schemes showing GFAP phosphorylation sites and GFAP mutants produced in this study. Phosphorylation sites for PKA, PKC, CaMKII, cdc2 kinase, and Rho-kinase were identified in in vitro studies, whereas those for CF kinase were identified in in vivo sites. The sites are indicated by P within a circle. Note that Ser-38 of human GFAP (Reeves et al., 1989) corresponds to Ser-34 of bovine GFAP (Inagaki et al., 1996). (B) Cell cycle-dependent site-specific phosphorylation of wild-type GFAP expressed in T24 cells. The cells were immunostained by anti-GFAP antibody MO389 (top), YC10 that recognizes GFAP phosphorylation at Ser-8 (middle), or by KT13 that recognizes GFAP phosphorylation at Ser-13 (bottom). Ser-8 of GFAP was phosphorylated by cdc2 kinase at metaphase, whereas Ser-13 was phosphorylated by CF kinase in the cleavage furrow of cytokinetic cells. The antibodies pG1-T and KT34 that recognize GFAP phosphorylation at Thr-7 and Ser-38, respectively, showed that these sites are also phosphorylated by CF kinase at the cleavage furrow (data not shown). The green fluorescence represents the immunoreactions. The chromosomes were stained by PI (red fluorescence). Bar, 10 μm.
Figure 4. Mutations in the CF kinase/Rho-associated kinase phosphorylation sites impair GFAP segregation into postmitotic daughter cells. (A) Localization of wild-type GFAP and m(7,13,38) in interphase T24 cells. (B) Postmitotic T24 cells expressing wild-type GFAP and the mutant m(7,13,38). The green fluorescence represents GFAP immunoreactivity stained with MO389, whereas the chromosomes were stained by PI (red fluorescence). Bars: (A) 10 μm; (B) 40 μm.
Figure 5. Effects by various mutants on the formation of GFAP bridge-like structure. (A) Population of mitotic cells (open column) and cells forming the bridge-like structure (closed column) expressing GFAP of wild-type, m(8), m(7,13,38), m(8,16,35), m(8,17,35), m(8,16,17), or m(16,17,35). The two daughter cells linked by one bridge-like structure was counted as one cell. (B) Western blot analysis of wild-type and mutant GFAPs expressed in T24 cells.
Figure 6. GFAP bridge-like structures in T24 cells expressing m(7,13,38). The green color represents GFAP immunoreactivity stained with MO389, whereas the red color shows chromosomes stained by PI. Bar, 10 μm.
Figure 7. Analysis of the nuclear membrane, actin filaments, and microtubles in T24 cells forming GFAP bridge-like structures. T24 cells expressing m(7,13,38) were double-stained by anti-lamin A/C antibody (a) and polyclonal anti-GFAP antibody (Dako) (b), rhodamine phalloidin (c), and polyclonal anti-GFAP antibody (Dako) (d), or anti-tubulin antibody (e) and polyclonal anti-GFAP antibody (Dako) (f). Bar, 10 μm.
Figure 8. Signaling pathways from Rho for cytokinesis.
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