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MICROSCOPY RESEARCH AND TECHNIQUE 49:173–182 (2000)
Regulation of Intermediate Filament Organization During
Cytokinesis: Possible Roles of Rho-associated Kinase
HIDEMASA GOTO, HIDETAKA KOSAKO,
AND
MASAKI INAGAKI*
Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan
KEY WORDS:
intermediate filament (IF); cleavage furrow; cytokinesis; Rho; Rho-associated
kinase (Rho-kinase)
ABSTRACT
Intermediate filaments (IFs), which form the structural framework of cytoskeleton,
have been found to be dramatically reorganized during mitosis. Some protein kinases activated in
mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of
IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase
Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This
cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and
efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new
models about the molecular mechanism of rearrangements of cytoskeletons including IFs during
cytokinesis. Microsc. Res. Tech. 49:173–182, 2000. © 2000 Wiley-Liss, Inc.
INTRODUCTION
Intermediate filaments (IFs), together with microtubules and actin filaments, form the cytoskeletal framework in the cytoplasm of various eukaryotic cells, and
are also present in nuclei as the major component of
nuclear lamina. Unlike microtubules and actin filaments, the protein components of IFs vary in cell-,
tissue-, and differentiation-dependent manner and are
divided into six groups (type I through type VI). For
example, glial fibrillary acidic protein (GFAP) and
desmin, type III IF proteins, are expressed specifically
in astroglial and muscular cells, respectively. On the
other hand, vimentin, other type III IF protein, is expressed in mesenchymal cells, in most types of cultured
and tumor cells, and transiently in many cells during
development (Eriksson et al., 1992; Fuchs and Weber,
1994; Steinert and Roop, 1988). IFs were thought to be
relatively stable in comparison with microtubules and
actin filaments in the past. However, IFs are far more
dynamic than has been previously considered. There is
increasing evidence that site-specific phosphorylation
of IF proteins alters their filament structures (Inagaki
et al., 1987, 1996). Recent detailed analyses on sitespecific IF phosphorylation in defined subcellular locations at various stages of mitosis have brought new
insights into the molecular mechanisms involved in the
mitotic IF reorganization (Foisner, 1997; Inagaki et al.,
1994, 1997; Ku et al., 1998; Nishizawa et al., 1991).
The small GTPase Rho is implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation, and smooth
muscle contraction (Hall, 1998; Takai et al., 1995). Rho
is also known to play important roles in cytokinesis
(Drechsel et al., 1996; Kishi et al., 1993; Mabuchi et al.,
1993; O’Connell et al., 1999). Upon stimulation with
certain signals, GDP-bound inactive form of Rho is
converted to GTP-bound active form, which presumably binds to specific targets and thereby exerts its
biological functions (Ren et al., 1999). Several groups
have succeeded in identifying putative targets of Rho;
©
2000 WILEY-LISS, INC.
protein kinase N (PKN), rhophilin, rhotekin, citron
kinase/citron-N, the myosin binding subunit (MBS) of
myosin phosphatase, mDia (mammalian homolog of
Drosophila diaphanous), phospholipase D, and Rhoassociated kinase (Rho-kinase)/ROK/ROCK (Lim et al.,
1996; Narumiya et al., 1997; Van Aelst and D’souzaSchorey, 1997). Among such effectors, Rho-kinase
(Matsui et al., 1996) /ROK (Leung et al., 1995, 1996)
/ROCK (Ishizaki et al., 1996; Nakagawa et al., 1996)
was identified as a serine/threonine kinase with a molecular mass of about 160 kDa. GTP-bound active Rho
interacts with Rho-kinase and thereby elevates its kinase activity (Matsui et al., 1996; Ishizaki et al., 1996).
Rho-kinase is shown to mediate some biological effects
of Rho: stress fiber and focal adhesion formation,
smooth muscle contraction, neurite retraction, microvilli formation, and cell migration (Kaibuchi et al.,
1999).
In this review, we will give an overview of spatiotemporal distribution of site-specific IF phosphorylation
during mitosis (especially cytokinesis), and discuss the
relationship between the cleavage furrow-specific IF
phosphorylation and Rho-kinase. We will also speculate on the molecular mechanism of cytoskeletal rearrangements through Rho and its specific targets (especially Rho-kinase) during cytokinesis.
CLEAVAGE FURROW-SPECIFIC
PHOSPHORYLATION OF INTERMEDIATE
FILAMENT (IF) PROTEINS DURING
CYTOKINESIS
In eukaryotes, mitosis is characterized by the formation of two distinct cytoskeletal structures. A bipolar
Contract grant sponsor: Ministry of Education, Science, Sports, and Culture of
Japan; Contract grant sponsor: Japan Society of the Promotion of Science Research for the Future; Contract grant sponsor: Bristol-Myers-Squibb.
*Correspondence to: Masaki Inagaki, Laboratory of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 4648681, Japan. E-mail: [email protected]
Received 1 September 1999; accepted in revised form 2 December 1999
174
H. GOTO ET AL.
Fig. 1. The site-specific phosphorylation of GFAP (A) and vimentin (B) during
mitosis in U251 human astroglial cells. A:
Metaphase or anaphase cells are stained
with the antibody MO389 (anti-GFAP),
YC10 (anti-phosphoSer8 on GFAP),
TMG7 (anti-phosphoThr7 on GFAP),
KT13 (anti-phosphoSer13 on GFAP), or
KT34 (anti-phosphoSer38 on GFAP;
green). DNAs are stained with propidium
iodide (red). Modified with the permission
from Kosako H, Amano M, Yanagida M,
Tanabe K, Nishi Y, Kaibuchi K, Inagaki
M. 1997. Phosphorylation of glial fibrillary acidic protein at the same sites by
cleavage furrow kinase and Rho-associated
kinase.
J
Biol
Chem
272:10333–10336, and Matsuzawa K, Kosako H, Azuma I, Inagaki N, Inagaki M.
1998. Possible regulation of intermediate
filament proteins by Rho-binding kinases.
In: Hermann H, Hariis JR, editors. Intermediate filament subcellular biochemistry, vol. 31. New York: Plenum Press, p
423– 435. B: Anaphase cells are doubly
stained with anti-vimentin (red) and
TM38 (anti-phosphoSer38 on vimentin)
or TM71 (anti-phosphoSer71 on vimentin;
green). DNAs are stained with DAPI
(blue). Bars ⫽ 10 mm. Modified with the
permission from Kosako H, Goto H,
Yanagida M, Matsuzawa K, Fujita M, Tomono Y, Okigaki T, Odai H, Kaibuchi K,
Inagaki M. 1999. Specific accumulation of
Rho-associated kinase at the cleavage furrow: cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 18:2783–2788.
mitotic spindle is composed of microtubules and their
associated proteins, and play a central role in nuclear
division. A contractile ring, composed of actin filaments
and myosin II just beneath the plasma membrane,
appears after nuclear division and partitions the cell
surface and cellular components by pulling the membrane inward (Mabuchi, 1986; Rappaport, 1986). IFs
are also found to be reorganized dramatically during
mitosis but the degree of reorganization differs depending on cell types. During early mitosis (prophase/metaphase), the extent of remodeling varies between a disassembly into soluble oligomers in Xenopus oocytes
(Klymkowsky et al., 1991), a reorganization to nonfilamentous insoluble aggregates (Franke et al., 1982;
175
RHO-ASSOCIATED KINASE DURING CYTOKINESIS
Figure 1.
(Continued)
Rosevear et al., 1990), and a collapse of IF systems into
a cage-like structure around the mitotic spindle without any indications of physical breakage (Blose, 1979;
Jones et al., 1985; Lane et al., 1982). During cytokinesis (late mitosis), in many types of cells, IFs appear to
be interrupted as intact bundles on the plane of the
cleavage furrow (Blose, 1979; Franke et al., 1983; Jones
et al., 1985; Lane et al., 1982).
The mitotic IF reorganization is known to be accompanied with increase in IF phosphorylation (Ce-
lis et al., 1983; Evans and Flik, 1982). The first direct
evidence supporting IF regulation by phosphorylation was obtained for vimentin by in vitro study:
treatment of in vitro polymerized vimentin with purified protein kinase A caused their filament disassembly (Inagaki et al., 1987). Accumulating in vitro
data suggest that the phosphorylation of IF proteins
by various types of protein kinases induces disassembly of the filament structure (Inagaki et al., 1996).
These observations lead to the notion that IF reor-
TABLE 1. In vivo (CF kinase) and in vitro (Cdc2 kinase, A kinase, C kinase, CaM kinase II, and Rho-kinase) phosphorylation sites on
GFAP and vimentin, and antibodies that recognize site-specific phosphorylation*
GFAP
Vimentin
Antibody site
TMG7
Thr7
YC10
Ser8
KT13
Ser13
KT34
Ser38
MO6
Ser6
YT33
Ser33
TM38
Ser38
TM50
Ser50
4A4
Ser55
TM71
Ser71
MO82
Ser82
Cdc2 kinase
A kinase
C kinase
CaM kinase II
Rho-kinase
CF kinase
⫺
⫹⫹
⫺
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫺
⫺
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
⫺
⫺
⫺
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫺
⫺
⫺
⫹⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
⫹⫹⫹
⫺
⫾
⫺
⫹⫹⫹
⫺
⫺
*CF kinase is a protein kinase that phosphorylates type III IF proteins specifically at the cleavage furrow during cytokinesis (Nishizawa et al., 1991; Matsuoka et
al., 1992; Sekimata et al., 1996). Rho-associated kinase (Matui et al., 1996), one of the serine/threonine kinases downstream of Rho. It is also called ROK (Leung et
al., 1995; 1996) or ROCK (Ishizaki et al., 1995; Nakagawa et al., 1996).
176
H. GOTO ET AL.
Fig. 2. Accumulation of Rho-kinase at the
cleavage furrow. A: Distribution of Rho-kinase in
MDBK cells. Interphase, metaphase, and telophase cells are stained with anti-Rho-kinase
(green; Kosako et al., 1999). DNAs are stained
with propidium iodide (red). Bars ⫽ 10 mm. B: Colocalization of Rho-kinase and vimentin phosphorylated at Ser71 during cytokinesis. Telophase
Swiss 3T3 or NIH3T3 cell is stained with anti-Rhokinase (green) and TM71 (red; Kosako et al., 1999).
ganization may be regulated by phosphorylation of
IF proteins during mitosis.
Site- and phosphorylation state-specific antibodies
that can recognize a phosphorylated residue and its
flanking sequence are of great use in order to analyze
spatiotemporal distribution of site-specific IF phosphorylation at each stage of mitosis. We first established a method to produce site- and phosphorylation
state-specific antibodies, using phosphorylated peptides or synthetic phosphopeptides as antigens for immunization. This method has an advantage in that one
can predesign a phosphorylated residue as an epitope.
Now, these site- and phosphorylation state-specific antibodies are widely utilized to analyze the phosphorylation of various proteins in cellular events (Inagaki et
al., 1994, 1997).
Detailed studies by using site- and phosphorylation
state-specific antibodies revealed that mitotic IF phosphorylation is spatio-temporally regulated by distinct
protein kinase(s) (Foisner, 1997; Inagaki et al., 1994,
1996, 1997; Ku et al., 1998). The first evidence was
obtained with the analysis using these antibodies for
four distinct sites on GFAP (Matsuoka et al., 1992;
Nishizawa et al., 1991). Ser8 phosphorylation of GFAP
in glial cells began in the prometaphase, remained
until metaphase, and declined gradually thereafter.
The phosphorylation was observed diffusely throughout cytoplasm (Fig. 1A). On the other hand, phosphorylation of residues Thr7, Ser13, and Ser38 were spatially and temporally distinct, increasing in anaphase,
maintained until telophase, and decreasing at the exit
of mitosis, and this phosphorylation was localized specifically at the cleavage furrow (Fig. 1A). These observations suggested that GFAP is phosphorylated by at
least two different kinases. It is notable that these
phosphorylation patterns correlate well with the observation concerning IF reorganization during mitosis
(see above). Therefore, these phosphorylations may
play some important roles in mitotic IF reorganization.
Identifying protein kinases responsible for mitotic IF
phosphorylation is of great importance in order to understand how IF reorganization is regulated during
RHO-ASSOCIATED KINASE DURING CYTOKINESIS
Figure 2.
mitosis. Site- and phosphorylation state-specific antibodies also provide useful information to identify in
vivo IF kinase(s) (Inagaki et al., 1994, 1996, 1997). The
protein kinase activated during early mitosis phosphorylates GFAP only at Ser8 but not at Thr7, Ser13,
and Ser38 (Fig. 1). Cdc2 kinase is known to be activated specifically during early mitosis (Murray and
Hunt, 1993; Norbury and Nurse, 1992) and phosphorylate GFAP only at Ser8 in vitro (Tsujimura et al.,
1994a). Since vimentin-Ser55 was phosphorylated specifically by Cdc2 kinase among known IF kinases in
vitro (Chou et al., 1991; Kusubata et al., 1992), we
further produced a site- and phosphorylation statespecific antibody for this site. Ser55 on vimentin was
also phosphorylated in various types of cells only during early mitotic phase and biochemical analysis of
mitotic cell lysates revealed that Ser55 phosphorylating activity and Cdc2 protein was coeluted as a single
peak (Tsujimura et al., 1994b). Together with data
obtained by tryptic peptide analysis (Chou et al., 1990),
these observations strongly suggest that Cdc2 kinase is
responsible for in vivo IF phosphorylation specifically
during early mitosis.
On the other hand, little was elucidated about the
molecular identity and regulation of a protein kinase,
which phosphorylate Thr7, Ser13, and Ser38 on GFAP
specifically at the cleavage furrow during cytokinesis,
in the past (Fig. 1A). Since this kinase seemed to be
activated specifically at the cleavage furrow during
177
(Continued)
cytokinesis, we tentatively named it cleavage furrow
(CF) kinase (Matsuoka et al., 1992; Nishizawa et al.,
1991). This CF kinase activity was observed not only in
astroglial cells but also in several cultured cells in
which GFAP was ectopically expressed (Sekimata et
al., 1996). These findings indicated that the activation
of CF kinase occurs in a wide range of cell types,
suggesting its important role in cytokinesis. Considering that Rho is implicated in cytokinesis (Drechsel et
al., 1996; Kishi et al., 1993; Mabuchi et al., 1993;
O’Connell et al., 1999), we postulated the possible involvement of Rho in the regulation of CF kinase activity. Recently, Rho-kinase, one of Rho targets (also see
Introduction), was revealed to phosphorylate GFAP at
Thr7, Ser13, and Ser38 but not at Ser8 in vitro (Kosako
et al., 1997). The in vitro Rho-kinase phosphorylation
sites (Ser38 and Ser71) on vimentin were also shown to
be phosphorylated at the cleavage furrow during cytokinesis (Goto et al., 1998; Kosako et al., 1999; Fig. 1B).
Table 1 summarizes known phosphorylation sites of
GFAP and vimentin. Furthermore, we obtained evidence that Rho-kinase phosphorylated desmin, the
other III IF protein, at Thr16, Thr75, and Thr76 in
vitro (Inada et al., 1998), which were phosphorylated
specifically at the cleavage furrow (Inada et al., 1999).
All these observations indicate that Rho-kinase has the
substrate specificity extremely similar to CF kinase,
which phosphorylates at least type III IF proteins during cytokinesis.
178
H. GOTO ET AL.
ACCUMULATION OF RHO AND RHO-KINASE
AT THE CLEAVAGE FURROW
DURING CYTOKINESIS
All accumulating data on the substrate specificity
are consistent with the idea that Rho-kinase is responsible for CF kinase activity (Table 1). These observations raise the question whether Rho-kinase is activated during cytokinesis.
Rho-kinase is thought to be activated by forming a
complex with GTP-bound Rho (Ishizaki et al., 1996;
Matsui et al., 1996). Thus, information on spatiotemporal distribution of Rho and Rho-kinase in the late
mitotic cell is of great importance in order to speculate
where Rho-kinase is activated during cytokinesis. Rho
has been reported to concentrate into the cleavage furrow during cytokinesis (Takaishi et al., 1995). Recently, Rho-kinase was also shown to accumulate into
the cleavage furrow (Fig. 2A). This cleavage furrowspecific accumulation first appears at late anaphase
and is maintained until telophase, then gradually decreases at the exit of mitosis (Kosako et al., 1999). This
subcellular localization of Rho-kinase is very similar to
that of Rho, indicating that Rho-kinase binds to Rho
and thereby is activated at the cleavage furrow.
Rho is thought to participate in cytokinesis since
inhibition of endogenous Rho by botulium ADP-ribosyltransferase C3 blocked cytokinesis in Xenopus embryos
(Kishi et al., 1993) and sand dollar eggs (Mabuchi et al.,
1993). Rho may be involved in the assembly of actin
filaments and proper constriction of the contractile
ring, which play important roles in defining the spatiotemporal pattern of cleavage (Drechsel et al., 1996;
O’Connell et al., 1999). Recently, Rho-kinase was reported to mediate some biological effects of Rho during
cytokinesis, i.e., expression of the dominant-negative
form of Rho-kinase inhibits cytokinesis in Xenopus embryos and mammalian cells, resulted in multinucleation (Yasui et al., 1998). This suggests that Rho-kinase may be activated and phosphorylate multiple proteins during cytokinesis.
GFAP, vimentin, and desmin serve as excellent substrates for Rho-kinase in vitro (Goto et al., 1998; Inada
et al., 1998; Kosako et al., 1997). The in vitro phosphorylation sites of these type III IF proteins are identical
to the in vivo sites, occurring at the cleavage furrow
(Goto et al., 1998; Inada et al., 1999; Kosako et al.,
1999; Fig. 1 and Table 1). In addition, this cleavage
furrow-specific IF phosphorylation occurs near the area
where Rho-kinase accumulates during cytokinesis (Kosako et al., 1999; Fig. 2B). This evidence led us to
suggest that Rho-kinase phosphorylates at least type
III IF proteins specifically at the cleavage furrow during cytokinesis.
BIOLOGICAL SIGNIFICANCE OF CLEAVAGE
FURROW-SPECIFIC IF PHOSPHORYLATION
During cytokinesis, the extent of IF remodeling varies in cell types, but IFs appear to be interrupted on the
plane of the cleavage furrow (Blose, 1979; Franke et al.,
1983; Jones et al., 1985; Lane et al., 1982). This localized IF reorganization is highly correlated with the
cleavage furrow-specific IF phosphorylation. In vitro
studies revealed that the phosphorylation of type III IF
proteins by Rho-kinase led to disassembly of their fil-
ament structures (Goto et al., 1998; Inada et al., 1998;
Kosako et al., 1997). Recently, we produced a mutant
GFAP in which Rho-kinase/CF kinase phosphorylation
sites (Thr7, Ser13, and Ser38) are changed to Ala residues and introduced this mutant GFAP into type III
IF-negative T24 cells (Yasui et al., 1998). The expression of this mutant GFAP impaired cytokinetic segregation of GFAP filaments, resulting in the formation of
an unusually long bridge-like structure between unseparated daughter cells (Fig. 3). Wild type GFAP and
mutants in which three Ser/Thr different from Rhokinase/CF kinase sites were altered showed no remarkable phenotype (Yasui et al., 1998). These observations
suggest that the phosphorylation of IF proteins by Rhokinase/CF kinase is required for the local IF breakdown and the separation of IFs into daughter cells.
POSSIBLE ROLES OF RHO AND
RHO-KINASE IN CYTOKINESIS
Rho is thought to play an important role in the formation and constriction of the contractile ring (Drechsel et al., 1996; Kishi et al., 1993; Mabuchi et al., 1993;
O’Connell et al., 1999; Takaishi et al., 1995). It is very
likely that Rho binds to its targets specifically at the
cleavage furrow and exerts its biological functions during cytokinesis. We propose a model for the possible
pathways downstream of Rho during cytokinesis in
Figure 4.
Rho-kinase is one of the targets mediating significant biological effects of Rho during cytokinesis, i.e.,
expression of the dominant-negative form of Rhokinase inhibited the cytokinesis, resulting in the production of multinuclei (Yasui et al., 1998). Rho-kinase is shown to regulate the phosphorylation of
regulatory light chain of myosin II (RMLC) at Ser19
by direct phosphorylation of RMLC and by inactivation of myosin phosphatase through MBS phosphorylation (Amano et al., 1996; Chihara et al., 1997;
Kimura et al., 1996). During cytokinesis, RMLC
phosphorylation at Ser19 occurred specifically at the
cleavage furrow (Matsumura et al., 1998) and near
the area where Rho-kinase accumulated (Kosako et
al., 1999). Since RMLC phosphorylation at Ser19 is
believed to promote the contractility of actomyosin in
the cells (Huttenlocher et al., 1995), these observations indicate that Rho-kinase may play important
roles not only in proper IF separation into daughter
cells but also in the formation and constriction of the
contractile ring.
Citron kinase, a Rho target with the structural similarity to Rho-kinase, was recently reported to localize
to the cleavage furrow (Madaule et al., 1998). Overexpression of its mutants results in production of
multinuclei and abnormal contraction during cytokinesis (Madaule et al., 1998). These observations suggest
that citron kinase may play an important role in the
contractile process of cytokinesis. However, the putative substrate(s) have not be identified yet, at least,
type III IF proteins might not be phosphorylated by
citron kinase (Inagaki, unpublished observation).
Thus, little is known about the molecular mechanism
by which citron kinase regulates the contractile process
of cytokinesis.
A mammalian homolog of Drosophila diaphanous
(mDia), one of the putative targets of Rho, was re-
RHO-ASSOCIATED KINASE DURING CYTOKINESIS
179
Fig. 3. GFAP bridge-like structures in T24 cells expressing a mutant GFAP with mutations in Rho-kinase phosphorylation sites (Thr7,
Ser13, and Ser38 on GFAP are changed into Ala). Green and red
colors represent GFAP stained with MO389 and propidium iodide,
respectively. Bar ⫽ 10 mm. Modified with the permission from Yasui
Y, Amano M, Nagata K, Inagaki N, Nakamura H, Saya H, Kaibuchi
K, Inagaki M. 1998. Roles of Rho-associated kinase in cytokinesis;
mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J Cell Biol 143:1249 –1258.
ported to concentrate weakly into the cleavage furrow in some mitotic cells (Watanabe et al., 1997).
This study also showed that Rho regulated actin
polymerization by targeting profilin via mDia beneath the plasma membranes. Since disruption of
diaphanous or profilin gene results in cytokinesisdefective phenotype (Balasubramanian et al., 1994;
Castrillon and Wasserman, 1994; Haugwitz et al.,
1994; Verheyen and Cooley, 1994), mDia is thought
to associate with profilin and regulate the assembly
of actin filaments at the cleavage furrow.
FUTURE PROSPECTS
Accumulating evidence suggests that site-specific
phosphorylation of IF proteins alters their filament
structure. Recent analyses on site-specific IF phosphorylation in defined subcellular locations at various mitotic stages demonstrates that several protein kinases
180
H. GOTO ET AL.
Fig. 4.
A scheme showing possible signaling pathways from Rho during cytokinesis.
phosphorylate IF proteins in a spatio-temporally regulated manner. Mutational analysis has revealed that
cleavage furrow-specific IF phosphorylation by CF kinase/Rho-kinase plays an important role in efficient
separation of IFs to daughter cells. Recent analyses on
Rho signalling pathways have promoted our understanding of how Rho regulates cytoskeletal rearrangements during cytokinesis. Rho-kinase may be essential, not only for separation of IFs into daughter cells,
but also for the formation and constriction of contractile ring.
On the other hand, little is known about the effects of
IF phosphorylation by other mitotic kinases on IF
structure and cellular behavior. We speculate that IF
phosphorylation during early mitosis may be necessary
for mitotic morphological change, together with rearrangements of other cytoskeletal filament systems and
regulation of interaction of IFs and other cytoskeletal
filaments. These possibilities need to be addressed in
future studies.
ACKNOWLEDGMENTS
We thank Dr. K. Nagata for critically reading the
manuscript. This work was supported in part by
Grants-in-Aid for Scientific Research and Cancer Re-
search from the Ministry of Education, Science, Sports,
and Culture of Japan, Japan.
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