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Vol. 14, Issue 5, 1745-1756, May 2003
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* Department of Molecular Biology and Biochemistry, Rutgers University,
Piscataway, New Jersey 08855;
Department of Pharmacology, Kitasato University, Tokyo, Japan; and
Department of Pharmacology, Kyoto University, Kyoto, Japan
Submitted July 25, 2002;
Revised December 11, 2002;
Accepted January 30, 2003
Monitoring Editor: Tom Pollard
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Somlyo and Somlyo, 2000;
Ridley, 2001;
Settleman, 2001). Their main
biological effects are mediated through the regulation and reorganization of
the actin cytoskeleton. Rho-kinase/ROK/ROCK is one of the targets of RhoA and
is involved in the assembly of stress fibers and focal adhesions in serum
stimulated 3T3 cells (Chrzanowska-Wodnicka
and Burridge, 1996; Leung
et al., 1996; Amano
et al., 1997;
Ishizaki et al.,
1997; Nakano et al.,
1999; Watanabe et
al., 1999; Totsukawa
et al., 2000).
Citron is another target of activated Rho
(Di Cunto et al.,
1998; Madaule et al.,
1998,
2000). There are two variants
called citron-N and citron kinase, both of which are produced by the same
transcription unit. Citron kinase is a longer variant of citron-N, including
an amino-terminal serine/threonine kinase domain. It shares a high degree of
structural homology with the ROCK (ROK/Rho-kinase), except that citron kinase
has the SH3 binding and PDZ binding domains at its carboxyl-terminus. A
shorter variant of citron-N is specifically expressed in the nervous system,
and localized to the postsynaptic density, where it forms a stable complex
with the membrane-associated guanylate kinase PSD-95
(Furuyashiki et al.,
1999; Zhang et al.,
1999). The functions of citron-N are unknown, although it has been
suggested to link the Rho signaling cascades to NMDA receptor complexes.
Citron kinase has been suggested to play a role in cytokinesis
(Madaule et al.,
1998; Di Cunto et
al., 2000). Narumiya and coworkers have reported that
overexpression of citron kinase mutants inhibits cytokinesis, suggesting that
it may be a target of RhoA in cytokinesis
(Madaule et al.,
1998). The involvement of citron kinase in cytokinesis is also
supported by the phenotype of mice that are deficient of citron kinase
(Di Cunto et al.,
2000). Mice lacking citron kinase showed severe ataxia and
epilepsy and died within the 3 weeks after birth. Abnormal cytokinesis and
massive apoptosis in certain neuronal precursors are suggested to be probable
causes of defective neurogenesis. Although those results suggest that citron
kinase is involved in cytokinesis, the mechanism is not clear. No
physiological substrate of the kinase has been identified, and this is
essential to elucidate the actions of citron kinase in cytokinesis and other
biological processes.
We found that citron kinase phosphorylated regulatory myosin light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. In vivo, citron kinase generated di-phosphorylated MLC when its kinase domain was expressed in cultured cells. Our results suggest citron kinase may be involved in the regulation of contractile activity and/or organization of cleavage furrows by regulating MLC di-phosphorylation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1, 2, and 3 deletion mutants and kinase-deficient
mutants), as well as pEGFPC1-citron kinase and pCAG-myc-ROCK1 3
deletion mutant, were described previously
(Ishizaki et al.,
1997; Madaule et al.,
1998; Eda et al.,
2001), and the domain structure of citron kinase and its mutants
are depicted in Figure 1.
Although the 3 mutant of citron kinase consists of the kinase domain of
citron kinase alone, the 2 mutant contains both the kinase domain and
half of the coiled-coil domain, which is structurally equivalent to the
3 deletion mutant of ROCK. The 1 mutant of citron kinase has
further extension at the C terminus containing the Rho-binding domain (see
Madaule et al., 1998
for detail). A bacterial expression vector of pGEX-2T V14RhoA was kindly
provided by Dr. A. Hall.
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His-tagged, full-length citron kinase was expressed in Baculovirus using
the Bac-to-Bac system (Invitrogen, Carlsbad, CA). Full-length citron kinase
was cloned at SalI (5' end) and XhoI (3' end) of
the pENTR A1 Gateway vector and then transferred to the pDEST10 vector
(N-terminal His fusion vector). Virus production and citron kinase expression
were followed according to the manufacturer's instruction manual. Full-length
citron kinase was purified by two steps of sequential affinity column
chromatography. Cell lysates of infected Sf9 cells (
2 x
108) were first bound to a Nickel column and eluted with a linear
gradient (50–200 mM) of imidazole. Citron kinase eluted around 80 mM was
bound to a GST RhoA column and eluted as a complex of GST-RhoA-citron kinase.
About 1–2 µg of purified kinase was prepared.
Nonmuscle myosin II was purified from bovine lung as described
(Sellers, 1991). Light chains
of myosin II were purified as described
(Perrie and Perry, 1970). MLCK
was purified from chick gizzard as described
(Adelstein and Klee, 1981a).
MBS of myosin phosphatase was purified from chick gizzard according to Alessi
et al. (1992).
Calmodulin was purchased from Sigma (St. Louis, MO). A specific inhibitor of
ROCK, Y-27632, was kindly provided by Yoshitomi Pharmaceutical Industries,
Ltd. (Oosaka, Japan).
An antimyc polyclonal antibody and an antimyc mAb (9E10) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA) and Covance (Denver, PA),
respectively. Chick antimyc polyclonal antibody was purchased from Aves Lab,
Inc. (Tigard, OR). Monoclonal and polyclonal antibodies against Ser
19-phosphorylated MLC were described previously
(Sakurada et al.,
1994; Matsumura et
al., 1998). A polyclonal antibody against di-phosphorylated
(phosphorylated at both Ser 19 and Thr 18) MLC was described previously
(Sakurada et al.,
1998).
Cell Culture, DNA Transfection, and Immunoprecipitation
PtK2 cells were maintained in a 1:1 mixture of DME and F12 medium
containing 10% fetal calf serum. BHK, NRK, CHO, and COS7 cells were maintained
in DME medium containing 10% fetal calf serum. Transfection was performed
using a GeneJuice (Novagene, Madison, WI) or Lipofectamine (Invitrogen,
Carlsbad, CA) transfection reagent.
For immunoprecipitation of exogenously expressed citron kinase or ROCK, COS7 cells were transfected with the myc-tagged constructs of citron kinase or ROCK according to manufacturer's instructions. After a 24-h incubation, transfected cells were lysed in an immunoprecipitation buffer containing 30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 50 mM sodium pyrophosphate, 1 mM EGTA, 1 mM EDTA, 20 mM beta-glycerophosphate, 1 mM sodium vanadate, 10 mM NaF, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and a mixture of leupeptin, pepstastin, and chymostatin (10 µg/ml each). Cells were homogenized with a Dounce homogenizer and clarified by centrifugation at 16,000 x g for 15 min. The supernatant was incubated with a myc mAb (10 µg/100-mm dish) for 1–2 h at 4°C. The immunocomplex was precipitated with protein A-Sepharose (Pharmacia Biotech, Inc., Piscataway, NJ) during a 1-h incubation. The immunocomplex was washed three times with the immunoprecipitation buffer, washed three times with a buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 5 mM MgCl2, and used for kinase assay.
Phosphorylation Assay
The kinase assays were performed in 25 mM Tris-HCl buffer (pH 7.5)
containing 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.1 mM ATP (0.1
mCi/ml), 0.1 mM EGTA, enzyme, and varying amounts of MLC or bovine lung myosin
II. Kinases used include immunoprecipitated citron kinase and ROCK, purified
MLCK, and Baculovirus-expressed full-length citron kinase. The reaction was
performed at 30°C for 3–60 min and terminated by the addition of
2x SDS sample buffer. After SDS-PAGE, MLC bands were cut out and counted
by the Cerenkov method. Urea/glycerol gel electrophoresis revealed that
purified myosin II was not phosphorylated (our unpublished results).
We also used mono-phosphorylated myosin II as a substrate and prepared it as follows: Purified myosin II was incubated with MLCK (5 µg/ml) in 25 mM Tris-HCl buffer (pH 7.5) containing 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM ATP, and calmodulin (5 µg/ml) at room temperature for 30 min. Phosphorylated myosin II was precipitated by overnight dialysis against 20 mM imidazole buffer (pH 6.5) containing 30 mM KCl and 5 mM MgCl2, washed once with the same buffer, and dissolved in 25 mM Tris-HCl (pH 7.5) containing 0.3 M NaCl. Urea/glycerol gel electrophoresis revealed that more than 90% of myosin II was mono-phosphorylated under these conditions. To compare the specificity of a substrate between mono-phosphorylated and unphosphorylated myosin II, unphosphorylated myosin II was prepared in the same way as described above except that ATP was omitted when myosin II was phosphorylated with MLCK.
Immunofluorescence
Immunofluorescence was performed using formaldehyde fixation as described
(Yamashiro et al.,
1998). Exogenously expressed citron kinase or ROCK was detected by
immunofluorescence using antimyc mAb or antimyc polyclonal antibody. Changes
in phosphorylation of MLC was examined by immunofluorescence using the
polyclonal or monoclonal antibodies against mono-phosphorylated MLC or the
polyclonal antibody against di-phosphorylated MLC, as described previously
(Totsukawa et al.,
2000). F-actin was visualized using fluorescently labeled
phalloidin (Sigma). Cells were examined with a Nikon TE 300 inverted
microscope. Phase and fluorescence images were taken with a Photometric
CoolSnap-fx CCD camera (Roper Scientific Inc., Tucson, AZ) and processed with
IPLab image processing software (Scanalytics, Inc., Fairfax, VA).
Other Procedures
SDS-PAGE was performed as described
(Blatter et al., 1972)
using 12.5% polyacrylamide except that the buffer system of Laemmli
(1970) was used. Protein
concentrations were determined using an Advanced Protein Assay reagent
(Cytoskeleton, Denver, CO) with bovine serum albumin as a standard.
Phosphopeptide mapping of phosphorylated MLC was performed as described
(Yamakita et al.,
1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1deletion
mutant (that contains the N-terminus kinase domain as well as the coiled-coil
and the Rho binding domain; Madaule et
al., 1998) of myc-tagged citron kinase was expressed in COS7
cells, immunoprecipitated by an antimyc antibody, and used for kinase assay
using bovine lung myosin II as a substrate. As
Figure 2A shows,
immunoprecipitated citron kinase was able to phosphorylate MLC of intact
myosin II (lanes 2 and 3). It also phosphorylated isolated MLC (lane 5). As a
control, a kinase defective (KD) mutant of citron kinase was
immunoprecipitated in the same way and used for its kinase activity. The KD
mutant failed to phosphorylate MLC (lane 6). In addition, immunoprecipitates
from MOCK-transfected cells did not phosphorylate MLC (lane 4). These results
suggest that citron kinase itself, rather than an unknown kinase associated or
contaminated with immunoprecipitated citron kinase, phosphorylates MLC. We
examined other constructs of citron kinase including mutants with further
deletion of the C terminus (2, the kinase domain plus half of the
coiled-coil domain, and 3, kinase domain alone) as well as full-length
citron kinase, which all phosphorylated MLC. As the 3 mutant consists
of the kinase domain alone, it is unlikely that an unknown kinase associated
with this mutant phosphorylates MLC.
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The sites of MLC phosphorylated by citron kinase were identified by
one-dimensional phosphopeptide mapping
(Yamakita et al.,
1994). As Figure 2B
shows, citron kinase generated two phosphopeptides, one corresponding to a
peptide phosphorylated at Ser19 and the other to a peptide phosphorylated at
both Ser19 and Thr 18 (lane 3). As a control, phosphopeptide patterns of MLC
phosphorylated by MLCK or PKC are shown in lanes 1 and 2, respectively. It
should be noted that citron kinase produced di-phosphorylated MLC to a higher
extent than did MLCK.
It is possible that citron kinase phosphorylates mono-phosphorylated MLC as well as it does unphosphorylated MLC, thus generating more di-phosphorylated MLC than did MLCK. We thus measured activities of citron kinase, ROCK, and MLCK using both unphosphorylated (0P) and mono-phosphorylated (1P) myosin II as a substrate. As Figure 3 shows, both full-length and mutant citron kinases phosphorylated these two substrates equally well. In contrast, MLCK or ROCK phosphorylated un-phosphorylated MLC much better than they did mono-phosphorylated MLC. The ratios of phosphate incorporation between unphosphorylated and mono-phosphorylated substrate are 0.7–0.8 for citron kinase, 0.2–0.3 for MLCK, and 0.3–0.4 for ROCK.
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Citron Kinase Does Not Phosphorylate MBS of Myosin Phosphatase
It is well known that ROCK phosphorylates MBS of myosin phosphatase.
Because the kinase domain of ROCK shows 46% identity to that of citron kinase
(Madaule et al.,
1998), we examined whether citron kinase was able to phosphorylate
MBS. The kinase domain of myc-tagged ROCK or citron kinase was expressed in
COS7 cells, immunoprecipitated, and assayed for their kinase activities using
both MLC and MBS as a substrate (Figure
4). As Figure 4a shows, phosphorylation of MBS by citron kinase (lane 2) was as low as that of
mock transfection (lane 1). On the other hand, ROCK phosphorylated MBS (lane
3). The lack of phosphate incorporation into the MBS by citron kinase is
neither due to inactive kinase activity of immunoprecipitated citron kinase
nor poor expression of citron kinase. Both citron kinase and ROCK
phosphorylated MLC to a similar extent (lanes 2 and 3 of
Figure 4b). In addition,
Western blotting with a myc antibody (c) revealed that the expression level of
citron kinase (lane 2) in COS7 cells was 5 times higher than that of ROCK
(lane 3). These results indicate that the activity of citron kinase toward MBS
is insignificant.
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Kinetic Analysis of Citron Kinase
To estimate the molecular activity and Km of citron
kinase, full-length citron kinase complexed with constitutively active
GST-RhoA was used for phosphorylation with varying concentrations of MLC
(Figure 5). The apparent
Km and molecular activity of full-length citron kinase
were 6.6 ± 1.0 µM and 0.3 ± 0.09
s–1, respectively. The Km
value is comparable to the values (0.9–2.5 µM) reported for
Rho-kinase and those (5–52 µM) of MLCK
(Adelstein and Klee, 1981b;
Gallagher et al.,
1991; Amano et al.,
1996; Feng et al.,
1999). On the other hand, the molecular activity of citron kinase
is lower than the values reported for Rho-kinase (0.67–2
s–1) and MLCK (2–65
s–1; Adelstein
and Klee, 1981b; Gallagher
et al., 1991; Amano
et al., 1996; Feng
et al., 1999). When intact myosin was used as a
substrate, full-length citron kinase was able to phosphorylate MLC up to
0.5–0.7 mol/mol of MLC.
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Increase in MLC Di-phosphorylation by the Expression of the Kinase
Domain of Citron Kinase in Cultured Cells
To further test whether citron kinase phosphorylates MLC, cultured cells
were transfected with myc-tagged citron kinase and stained with the
phospho-MLC specific antibodies. Immunofluorescence with the phospho-MLC
specific antibodies has allowed us to determine changes in MLC phosphorylation
in transfected cells (detected by a myc antibody;
Totsukawa et al.,
2000). We used two antibodies, one specific to mono-phosphorylated
MLC and the other to di-phosphorylated MLC. It should be noted that the
antibody against mono-phosphorylated MLC did not recognize di-phosphorylated
MLC and the antibody against di-phosphorylated MLC did not recognize
mono-phosphorylated MLC. Actin organization was also examined by phalloidin
staining.
We examined the effects of the 2 mutant of citron kinase on MLC
phosphorylation of PTK cells (Figure
6). This mutant, which consists of the kinase domain and a part of
the coiled-coil domain (see Figure
1), distributed diffusely throughout the cytoplasm (asterisk in A,
D, and G; Eda et al.,
2001). Surprisingly, when transfected cells were stained with the
antibody specific to mono-phosphorylated MLC, they showed lower staining
(asterisk in B). Quantitative measurement showed that staining intensity with
the mono-phosphorylated antibody was reduced to 45 ± 25% of the control
(n = 30). However, phalloidin staining revealed that transfected cells
retained well-developed stress fibers (asterisk in C). Because stress fiber
formation depends on actomyosin contractility and MLC phosphorylation
(Chrzanowska-Wodnicka and Burridge,
1996; Totsukawa et
al., 2000) and because citron kinase produced
di-phosphorylated MLC in vitro, we thought myosin may be di-phosphorylated.
Indeed, transfected cells (asterisks in E) exhibited significantly higher
staining with the antibody specific to di-phosphorylated MLC, indicating that
di-phosphorylation was increased. Again phalloidin staining (F) revealed that
transfected cells had well-developed stress fibers. To confirm the changes in
MLC phosphorylation, transfected cells were triple-labeled with the antibodies
against myc (G), mono- (H), and di-phosphorylated MLC (I). The increase in
di-phosphorylation (I) with simultaneous decrease in mono-phosphorylation (H)
was clearly seen in transfected cells (asterisks in G). A similar result was
obtained with 3 mutant of citron kinase (which contains the kinase
domain alone). These observations indicate that the expression of kinase
domain of citron kinase caused an increase in MLC di-phosphorylation.
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The increase in di-phosphorylation becomes more evident when ROCK activity
is blocked by a specific inhibitor, Y-27632. The inhibition of ROCK is known
to result in the disassembly of stress fibers
(Ishizaki et al.,
2000). As Figure 6,
J–L shows, however, cells expressing 2 citron kinase
(asterisks in J) retained well-developed stress fibers (asterisks in L). At
the same time, these transfected cells clearly exhibited an increase in MLC
di-phosphorylation (asterisks in K), indicating that di-phosphorylation
efficiently restores the assembly of stress fibers. In contrast, surrounding
cells expressing no citron kinase lost staining with the antibody against
di-phosphorylated MLC (K), and stress fibers of these nontransfected cells
were disassembled (L). These results indicate that citron kinase is able to
generate di-phosphorylation of MLC even when myosin phosphatase is activated
by the inhibition of ROCK.
To compare the effects of citron kinase with those of ROCK, we examined how
the expression of the kinase domain of ROCK (3 mutant) alters MLC
phosphorylation (Figure 7). As
revealed by phalloidin staining (C and F), expression of ROCK frequently
induced the formation of characteristic stellar stress fibers (C), which is
consistent with the previous reports
(Leung et al., 1996;
Amano et al., 1997;
Ishizaki et al.,
1997). These stellar stress fibers were highly stained with the
antibodies against either mono- (B) or di-phosphorylated MLC (E). The increase
in both mono- and di-phosphorylation of MLC by ROCK overexpression is probably
due to the two activities of ROCK: ROCK can directly phosphorylate MLC
(Amano et al., 1996;
Totsukawa et al.,
2000) and at the same time inhibit myosin phosphatase
(Kimura et al.,
1996).
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The changes in MLC phosphorylation by citron kinase or by ROCK were
confirmed biochemically using Western blotting
(Figure 8). BHK cells were used
for this purpose because they are more efficient for transfection. Cells were
transfected with either citron kinase 2 mutant or ROCK 3 mutant.
Total cell lysates were then immunoblotted using the antibodies specific to
mono- or di-phosphorylated MLC (middle panels) as well as the myc antibody to
detect levels of expression (top panels). Similar amounts of lysates were
loaded for each lane (bottom panels). Cells transfected with citron kinase
showed lower reactivity to the antibody against mono-phosphorylated MLC (lane
2 of Figure 8a) than did
mock-transfected cells (lane 1). On the other hand, the immunoblot with the
antibody against di-phosphorylated MLC revealed higher reactivity with citron
kinase-transfected cells (lane 2 of Figure
8b) than with mock-transfected cells (lane 1). ROCK-transfection
increased both mono- and di-phosphorylation of MLC to a great extent (lane 3
of Figure 8, a and b,
respectively), which is probably due to the inhibition of myosin phosphatase
by ROCK. These data are consistent with the immunofluorescence observations
(Figures 6 and
7).
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We then examined whether the expression of full-length citron kinase alters
MLC phosphorylation. Previous work has demonstrated that, although full-length
citron kinase is present as protein aggregates in interphase cells, it becomes
dispersed during prophase and localized in cleavage furrows during cytokinesis
(Eda et al., 2001).
Because RhoA is likely to activate citron kinase during cytokinesis, we
examined whether the expression of full-length citron kinase alters MLC
di-phosphorylation during cytokinesis. To this end, we chose CHO cells because
CHO cells are relatively easily synchronized and show good transfection
efficiency. As Figure 9 shows,
we have expressed GFP-tagged full-length citron kinase in synchronized CHO
cells (A) and stained them with the antibody against di-phosphorylated MLC (B)
and DAPI (C). As a control, GFP alone was expressed (D) and stained with the
anti–di-phosphorylated MLC antibody (E) and DAPI (F). GFP-citron kinase
was localized in cleavage furrows (A) where strong staining of MLC
di-phosphorylation was observed (B). Quantitative analyses of
immunofluorescence revealed that staining intensities of di-phosphorylation
more than doubled. On the other hand, control cells expressing GFP alone (D)
showed di-phosphorylation indistinguishable from untransfected cells (E).
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In interphase cells, GFP-tagged citron kinase showed punctate localization,
as reported previously (Madaule et
al., 1998; Eda et
al., 2001). Such cells showed no changes in MLC mono- or
di-phosphorylation (our unpublished results). This lack of effects is likely
due to the fact that the localization of full-length citron kinase is
constrained to protein aggregates (Eda
et al., 2001), which would restrict the access of the
kinase to a substrate.
Localization of Mono- and Di-phosphorylated MLC during Cell
Division
We examined the localization of di-phosphorylated MLC during cell division.
Figure 10 shows double-labeled
immunofluorescence localization of both mono-phosphorylated (A, D, and G) and
di-phosphorylated (B, E, and H) MLC in NRK cells at different stages of
cytokinesis. From metaphase (our unpublished results) to early anaphase
(A–C), both mono- and di-phosphorylated MLC showed diffuse staining
though anti–di-phosphorylated MLC antibody stained centrioles. At
telophase (D–F), both mono- (D) and di-phosphorylated (E) MLC
colocalized at cleavage furrows. It is interesting to note that
di-phosphorylated MLC showed more constrained localization in cleavage furrows
than did mono-phosphorylated MLC. At late telophase (G and H), the constrained
localization of di-phosphorylated MLC became clearer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Kosako et al.,
1999; Kosako et al.,
2000; Fukata et al.,
2001), it is likely to activate myosin during cytokinesis. The
presence of the two RhoA-activated MLC kinases, citron kinase and ROCK, in
cleavage furrows may explain why the ROCK inhibitor, Y-27632, does not
effectively inhibit cytokinesis (Madaule
et al., 2000). The direct phosphorylation of MLC by
citron kinase may also account for abnormal contractility of "push and
pull" movements during cytokinesis when a kinase active mutant of citron
kinase is overexpressed (Madaule et
al., 2000). Although both ROCK and citron kinase phosphorylate MLC, there are two important differences in their activities. First, citron kinase does not phosphorylate MBS of myosin phosphatase (Figure 4) and thus does not inhibit myosin phosphatase activity. In contrast, ROCK phosphorylates MBS, inhibiting the activity of myosin phosphatase. This indicates that ROCK would block the turnover of MLC phosphorylation, making most MLC to be the phosphorylated form of MLC. Both immunofluorescence (Figure 6) and biochemical analysis (Figure 8) support this notion. When constitutively active ROCK is expressed in cells, stellar actin fibers are assembled (Figure 7), which is likely to be a result of contraction of actomyosin due to a very high extent of MLC phosphorylation (Figure 8). In contrast, citron kinase would not block turnover of MLC phosphorylation. Consistent with this notion, cells expressing the kinase domain of citron kinase exhibit parallel stress fibers with apparently normal morphology (Figure 6).
Second, the expression of citron kinase in cells resulted in an increase in di-phosphorylation of MLC but a decrease in MLC mono-phosphorylation (Figure 6). This is in contrast to the expression of ROCK in which both mono- and di-phosphorylation were increased (Figure 7). The increase in di-phosphorylated MLC by the citron kinase domain is apparently explained by the result that citron kinase phosphorylated monophosphorylated MLC as well as it did un-phosphorylated MLC (Figure 3). This ability of citron kinase may also explain the decrease in mono-phosphorylation of MLC because citron kinase would effectively convert mono-phosphorylated myosin to di-phosphorylated myosin.
Other interpretations are also possible. For example, citron kinase may
activate a myosin phosphatase that is specific for mono-phosphorylated myosin,
although no such phosphatase has been known. Another possibility is that
citron kinase phosphorylates and inhibits other major MLC kinases. If citron
kinase, for example, inhibited MLCK, then mono-phosphorylated MLC may be
reduced. We found, however, that citron kinase did not phosphorylate MLCK or
ROCK, which are believed to be two major MLC kinases inside cells. Further
studies are necessary to determine whether citron kinase modulates other MLC
kinases including PAK (Chew et
al., 1998; Zeng et
al., 2000) and ZIP kinase (Murata-Hori et al.,
1999,
2001).
The activity of citron kinase is likely to be regulated in a cell
cycle–dependent manner. Citron kinase has been reported to be present as
protein aggregates in interphase cells
(Eda et al., 2001),
and as such, it may not be active. In contrast, ROCK is diffusely localized in
interphase cells. ROCK is, at least in part, active as its activity is known
to be essential for the formation of stress fibers
(Leung et al., 1996;
Amano et al., 1997;
Ishizaki et al.,
1997; Nakano et al.,
1999; Watanabe et
al., 1999; Totsukawa
et al., 2000). During prophase, citron kinase becomes
dispersed and is translocated to cleavage furrows during cytokinesis
(Eda et al., 2001).
Because Rho A has been reported to be greatly activated during cytokinesis
(Kimura et al.,
2000), citron kinase is likely to become activated. This
activation probably accounts, at least in part, for the di-phosphorylation of
MLC at cleavage furrows (Figures
9 and
10) although other kinases
such as ROCK may also be involved in the di-phosphorylation of MLC.
Di-phosphorylated myosin showed more constrained localization at cleavage
furrows than did mono-phosphorylated myosin
(Figure 10). Although
di-phosphorylated myosin showed twofold higher ATPase activity than
mono-phosphorylated myosin (Ikebe and
Hartshorne, 1985), the velocity of actin filaments in an in vitro
motility assay is the same for both types of phosphorylated myosin
(Umemoto et al.,
1989). On the other hand, di-phosphorylation significantly
increases thick filament assembly and actomyosin superprecipitation
(Ikebe and Hartshorne, 1985),
suggesting that di-phosphorylated myosin may play a role in cross-linking of
actin filaments rather than stimulation of motor activity. Further studies are
required to elucidate the exact role of di-phosphorylation of MLC in cleavage
furrows.
It now appears that three different kinases including MLCK, ROCK, and
citron kinase are all localized in cleavage furrows
(Madaule et al.,
1998; Kosako et al.,
1999,
2000;
Poperechnaya et al.,
2000; Fukata et al.,
2001; Chew et al.,
2002) and are functioning as MLC kinases. In addition, MBS of
myosin phosphatase phosphorylated by Rho-kinase (ROCK) is also localized in
cleavage furrows (Kawano et al.,
1999). These results reinforce the importance of the regulation of
MLC phosphorylation during cytokinesis. An important question is why citron
kinase–deficient mice show a cytokinesis defect in some neuronal
precursor cells even though these cells express abundant ROCK
(Di Cunto et al.,
2000). This observation indicates that citron kinase plays a role
distinct from ROCK. As discussed above, citron kinase, unlike ROCK, does not
block turnover of MLC phosphorylation while generating di-phosphorylated MLC.
This ability of citron kinase to allow turnover may be important for certain
cells to complete cytokinesis. For example, ROCK may be activated by Rho to a
much higher extent in the absence of citron kinase than in its presence
because Rho may not be shared between ROCK and citron kinase. Such
overactivation of ROCK may result in the MLC phosphorylation to a very high
extent while blocking dephosphorylation of MLC or turnover of MLC
phosphorylation. Blockage of MLC dephosphorylation may hinder the proper
execution of cytokinesis because cytokinesis is known to be associated with
simultaneous contraction and disassembly of contractile rings, and the
disassembly is likely to require MLC dephosphorylation or turnover. It is also
quite possible that citron kinase controls cytokinesis by phosphorylating
unique substrates that ROCK cannot phosphorylate. Future studies should be
directed at elucidating whether citron kinase indeed regulates MLC
di-phosphorylation during cytokinesis in a way distinct from ROCK or whether
citron kinase has other substrates that are critical in regulating other
aspects of cell division.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: MBS, myosin binding subunit or myosin targeting subunit of myosin phosphatase; MLC, regulatory light chain of myosin II; ROCK, Rho-associated kinase, Rho-kinase or ROK.
Corresponding authors. E-mail addresses:
yamashiro{at}mbcl.rutgers.edu;
matsumura{at}mbcl.rutgers.edu.
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