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J. Biol. Chem., Vol. 275, Issue 44, 34512-34520, November 3, 2000
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From the § Department of Physiology and Biomedical
Imaging Group, University of Massachusetts Medical School,
Worcester, Massachusetts 01655 and
Received for publication, April 10, 2000, and in revised form, August 14, 2000
Myosin plays an important role in mitosis,
especially during cytokinesis. Although it has been assumed that
phosphorylation of regulatory light chain of myosin (RLC) controls
motility of mammalian non-muscle cells, the functional significance of
RLC phosphorylation remains uninvestigated. To address this problem, we
have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of
T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of
proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal
cleavage furrows, resulting in incomplete cytokinesis, which suggests
that myosin phosphorylation is important for the normal recruitment of
myosin molecules into the contractile ring structure. The other
is that separation of chromosomes from the metaphase plate is disrupted
in T18A/S19A RLC expressing cells, thus preventing proper
transition from metaphase to anaphase. These results suggest that, in
addition to cytokinesis, myosin and myosin phosphorylation play a role
in the karyokinetic process.
Myosin is a motor protein that binds actin filaments and produces
force using the chemical energy of ATP. It is known that actin and
myosin undergo reorganizations in cultured mammalian cells in response
to cellular signals in various cellular motile processes such as
chemotaxis, phagocytosis, capping of surface receptors, and cytokinesis
(1-16). For example, actin and myosin form stress fibers in interphase
cells, but this structure disappears at the onset of mitosis. During
cytokinesis, actomyosin is redistributed into the equatorial region
to produce the cleavage furrow (see Refs. 17 and 18 for reviews).
On the other hand, a signal initiated by the binding of a lectin to its
receptor triggers reorganization of actomyosin thus gathering the
lectin receptors to produce a "cap" structure (5-11).
Myosin is essential for the motility activity in the actomyosin system,
so it has been thought that myosin plays a key role in dictating
cellular motile processes. For both smooth muscle and non-muscle cells
(in contrast to highly differentiated striated muscle cells),
actomyosin motor activity is primarily regulated by phosphorylation of
the regulatory light chain of myosin (19-21). Myosin regulatory light
chain (RLC)1 is
phosphorylated at various sites. Serine 19 and threonine 18 are the
sites responsible for activation of myosin motor activity, and the
former site is thought to be physiologically more important (see Refs.
22-24 for reviews). The protein kinase responsible for phosphorylation
at these sites is Ca2+/calmodulin-dependent
myosin light chain kinase (MLCK), and in smooth muscle cells, this
kinase primarily dictates the level of myosin phosphorylation (see
Refs. 22 and 24 for reviews). In non-muscle cells, although MLCK is
thought to be important for myosin phosphorylation, other protein
kinases also play a role in regulating the phosphorylation of myosin
(25-27). On the other hand, phosphorylation at serine 1/serine 2 and
threonine 9 of the regulatory light chain, which is catalyzed by
protein kinase C and Cdc2 kinase, is inhibitory, and phosphorylation at these sites decreases the affinity for actin (28-30).
The bulk of available evidence supports the conclusion that myosin is a
key component in cytokinesis. Immunocytochemistry has revealed that
myosin is present at the equatorial region during cell division (12,
13, 16, 31). Inhibition of cytokinesis by microinjection of anti-myosin
antibodies further suggests the importance of myosin function in cell
division (32). Conclusive evidence was obtained by genetic
manipulation, and it was found that the deletion of the gene for myosin
II in Dictyostelium cells abolishes cytokinesis (33).
A remaining question is whether or not phosphorylation of myosin is
required for cytokinesis. Fishkind et al. (34) reported that
microinjection of constitutively active MLCK into mitotic cells did not
affect the formation of cleavage furrows and cytokinesis, although the
transit time from nuclear envelope breakdown to anaphase onset was
delayed. However, myosin in mitotic cells is phosphorylated (at least
to a certain extent), and injection of active MLCK may not
significantly change the phosphorylation level of myosin at the
cleavage furrow, as the myosin phosphorylation level increases in
non-injected cells at the onset of telophase. Whereas the bulk of
evidence supports the idea that myosin II function is critical for cell
division, the role of serine 19 phosphorylation during cell division in
mammalian cells remains obscure. In the present study, we overexpressed
a myosin regulatory light chain mutant, T18A/S19A RLC, which eliminated
the phosphorylation sites in order to hamper myosin phosphorylation.
The regulatory light chain was tagged with green fluorescent protein
(GFP) to identify the cells transfected with the RLC-GFP construct.
Overexpression of unphosphorylatable RLC in mammalian cells abolished
surface receptor cap formation. Furthermore, normal cytokinesis was
significantly disrupted by the overexpression of unphosphorylatable RLC
in mammalian cells. These results indicate that the phosphorylation of
myosin regulatory light chain is critical for cell division of
mammalian non-muscle cells.
Materials--
Restriction enzymes and modifying enzymes were
purchased from New England Biolabs (Beverly, MA). Smooth muscle myosin
and MLCK were prepared from frozen turkey gizzards (30, 35). Actin was
prepared from rabbit skeletal muscle according to Spudich and Watt
(36). Xenopus oocyte calmodulin (37) and smooth muscle myosin RLC were expressed in Escherichia coli and purified
as described (38, 39).
Production of Monoclonal Antibody against Phospho-RLC--
A
monoclonal antibody against phosphoserine 19 of RLC was prepared,
basically according to Yano et al. (40). A phosphopeptide KKRPQRATpSNVFAMC (where pS indicates phosphoserine) (p-MIPP 1) coupled to keyhole limpet hemocyanin was used as an antigen.
Production of GFP-Wild Type and GFP-Mutant Regulatory Light Chain
Fusion Constructs--
The cDNA fragment of RLC subcloned into a
pT7-7 vector (38, 41) was excised by XbaI digestion and
inserted into a pTracer-CMV mammalian expression vector downstream of
its CMV promoter (Invitrogen Co.) Green fluorescent protein (GFP)
cDNA in which Ser-65 is mutated to Thr was introduced at the
3'-side of RLC cDNA in a pT7-7 vector. The entire cDNA insert
was then excised and subcloned into a pcDNAI/Amp mammalian
expression vector (Invitrogen Co.). Mutant RLC in which MLCK
phosphorylation sites (Thr-18 and Ser-19) were mutated to Ala was made
by site-directed mutagenesis (42) using the RLC expression vectors as a template.
Expression of GFP-Chimeric RLC and Biochemical
Procedures--
RLC-GFP chimeric proteins were expressed in the BL21
(DE3) E. coli strain that has T7 RNA polymerase and was
purified as described (38). In vitro phosphorylation was
carried out using 20 µg/ml partially purified RLC-GFPs in the
presence of 20 mM NaCl, 2 mM CaCl2,
1 mM MgCl2, 1 µM microcystin-LR,
2 mM ATP, and 20 mM Tris-HCl, pH 7.5. The assay
contained 10 µg/ml calmodulin with or without 20 µg/ml MLCK. The
reaction solutions were incubated for 15 min at 30 °C, and then
phosphorylation of RLC-GFP was detected by Western blotting analysis
using anti-phospho-RLC antibody. SDS-polyacrylamide gel electrophoresis
was carried out on a 7.5-20% polyacrylamide gradient slab gel using
the discontinuous buffer system of Laemmli (43). Western blotting was
carried out as described previously (42, 44), using phospho-RLC
monoclonal antibody and the polyclonal antibody against GFP (MBL Co.,
Ltd., Ina, Japan.) or RLC (Sigma), respectively. Urea/glycerol gel
electrophoresis was performed as described (45). Purified RLC was
phosphorylated by MLCK and PKC (30, 35). After transferring to a
polyvinylidene difluoride membrane (Millipore Co.), Western blotting
was carried out as described above. The bound antibodies were detected
by the enhanced chemiluminescence method (Amersham Pharmacia Biotech).
To produce myosin containing RLC-GFP, RLC-GFP was further purified (38) and was incorporated into myosin as described previously (39). The
actin-activated Mg2+-ATPase activity of myosin was
determined by measuring the liberated 32P as described
previously (35). The effect of free RLC-GFP on myosin phosphorylation
was examined as follows. Myosin II was phosphorylated by MLCK in the
presence of various concentrations of isolated RLC-GFP. The assay
solution contained 0.1 mg/ml myosin, 2 µg/ml calmodulin, and 2 µg/ml MLCK in the presence of 20 mM NaCl, 0.2 mM CaCl2, 1 mM MgCl2, 1 µM microcystin-LR, 0.18 mM
[ Cell Cultures and Synchronization--
Epithelial type NRK cells
(NRK52E; supplied by Dr. Yu-Li Wang, University of Massachusetts
Medical Center) and COS-7 cells were used in the present study. NRK and
COS-7 cells were maintained in Kaighn's modified F-12 medium (Sigma)
containing 10% fetal bovine serum (Life Technologies, Inc.), 2 mM L-glutamine and in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, respectively.
Transfection of plasmids was carried out by electroporation using a
Gene Pulser II (Bio-Rad). Transfected cells were seeded onto glass
coverslips and cultured for 72-96 h. Mitotic cells were obtained
following 4 h of treatment with nocodazole (NRK52E; 0.75 µg/ml,
COS-7; 0.05 µg/ml, respectively).
Immunofluorescence Staining--
Transfected or untransfected
cells were washed twice with PBS, placed in fixation solution I (4%
formaldehyde, 2 mM MgCl2, and 1 mM
EGTA in PBS), and after extensive washing, permeabilized with 0.1%
Triton X-100 in PBS for 10 min. Myosin was visualized with a rabbit
polyclonal antibody against heavy chain of myosin IIB (supplied by Dr.
R. Adelstein, National Institutes of Health, Bethesda) followed by
Texas Red-conjugated anti-rabbit antibody (Jackson ImmunoResearch). For
visualizing the F-actin structure and nucleus, fixed cells were stained
with Texas Red-conjugated phalloidin (Molecular Probes) and
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular
Probes), respectively. Myosin with GFP-tagged RLC was visualized with
the GFP fluorescence signal or with polyclonal anti-GFP antibody
staining followed by FITC-conjugated anti-rabbit antibody (Jackson
ImmunoResearch). For visualizing the capping structures and
phosphorylated myosin, the cells were treated with ConA as described
below and fixed in solution I. The fixed cells were incubated with
anti-phospho-RLC (Ser19) monoclonal antibody followed by
indodicarbocyanine (Cy5)-conjugated anti-mouse antibody (Jackson
ImmunoResearch). Images of labeled cells were acquired with a Nikon
Diaphot 200 inverted microscope equipped with a 100-watt Hg arc
lamp for epifluorescence microscopy. Cells were viewed with 40 or 100×
Nikon (NA 1.3) Planapo objectives with 2.5 or 5× camera eyepiece, and
images were projected onto the face of a Photometrics
thermoelectrically cooled CCD camera (RCA 501 chip). For data
restorations, three-dimensional digital images were flat
field-corrected, background-subtracted, and normalized for temporal
fluctuations in excitation intensity to constant integrated optical
density. Prepared images were then processed with an iterative
deconvolution algorithm with non-negativity constraints, using
determined point spread function for the microscope to at least
partially reverse the blurring introduced by the optics (46).
Induction of ConA Capping and Determination of Multinuclear
Cells--
Transfected cells were incubated with PBS containing Texas
Red or TRITC-conjugated ConA (0.5 mg/ml; Molecular Probes) at room temperature for 5 min. Unbound ConA was subsequently removed by rinsing
with PBS, and cells were cultured with the medium in an atmosphere of
5% CO2 and 95% air at 37 °C for 40 min. After
induction of cap formation, cells were fixed in solution II (2%
formaldehyde, 2 mM MgCl2, and 1 mM
EGTA in PBS), washed twice with PBS, and then treated with 0.1% Triton
X-100. To determine the number of nuclei in cells, the living cells
were stained with Hoechst 33342 (Molecular Probes) at 72 h after
the transfection. In order to estimate the difference in the myosin
phosphorylation of mutant T18A/S19A RLC expressing cells and wild type
RLC expressing cells, the transfected cells were treated with
non-tagged ConA as described above and then the cells were fixed as
described above. The cells were co-stained with anti-phospho-RLC
antibodies (mouse monoclonal) and anti-non-muscle myosin IIB antibodies
(rabbit polyclonal), using Cy5-labeled anti-mouse and TRITC-labeled
anti-rabbit second antibodies, respectively. The fluorescence intensity
was determined with digital fluorescent microscopy using a cooled CCD camera.
Video Microscopy--
Transfected cells in mitotic phase were
identified by GFP signal and the appearance of a metaphase plate with a
Nikon (Tokyo, Japan) DIAPHOT 300 inverted fluorescence microscope with
a 40× Nikon objective. At 30 min after the release from nocodazole
arrest, phase contrast images of the transfected cells were monitored for 3-4 h in culture medium containing 25 mM Hepes, pH
7.2, or L15 medium (Sigma) by time-lapse video microscopy.
Expression of RLC-GFP Chimeric Protein in Mammalian Cultured
Cells--
The authenticity of the RLC-GFP chimeric protein was
confirmed with Western blot analysis. Partially purified wild type
RLC-GFP and T18A/S19A RLC-GFP were subjected to immunostaining with
anti-GFP polyclonal antibody. As shown in Fig.
1, the 47-kDa peptides were specifically
recognized by the antibody for both wild type and mutant proteins (Fig.
1A, lanes 1 and 2). The expression of the 47-kDa
peptides was not found before
isopropyl-1-thio-
The functional authenticity of GFP-tagged RLC was evaluated in several
respects. The binding of RLC-GFP to myosin heavy chain was examined
using smooth muscle myosin in which the endogenous RLC is removed (39).
The chimeric RLC-GFP stoichiometrically bound to smooth muscle
RLC-deficient myosin (data not shown). The regulatory function of RLC
was examined by measuring the actin-activated ATPase activity of myosin
that is coupled with myosin motor function. The actin-activated ATPase
activity of myosin containing wild type RLC-GFP was activated
significantly by phosphorylation. On the other hand, myosin containing
T18A/S19A RLC-GFP showed little activation of its ATPase activity in
the presence of MLCK and calmodulin (Table
I). The result clearly indicates that
RLC-GFP confers phosphorylation-dependent activation of
myosin motor function.
To examine whether or not GFP-tagged RLC is incorporated into myosin in
transfected cells, RLC-GFP chimera was expressed in COS-7 cells, and
the localization of the GFP signal was observed. COS-7 cells were
transiently transfected with GFP-chimeric RLC constructs as described
under "Experimental Procedures." As shown in Fig.
2, wild type RLC-GFP showed filamentous
localization that coincides with the localization of myosin and F-actin
(Fig. 2, A, B, E, and F). The result
suggests that GFP-tagged RLC is incorporated into myosin molecules in
cells and that myosin containing RLC-GFP localized in stress fibers,
thus retaining authenticity in terms of intracellular localization.
T18A/S19A RLC-GFP also co-localized with myosin and F-actin (Fig. 2,
C, D, G, and H), indicating
that the mutation at the phosphorylation sites of RLC does not change the resting myosin localization. Cells transfected with GFP alone did
not show any filamentous localization, although stress fibers were
observed in these cells when probed by Texas Red-conjugated phalloidin
staining (data not shown).
Effect of Myosin RLC Phosphorylation on Surface Receptor
Capping--
It has been known that lectin binding to surface
receptors induces significant rearrangement of cytoskeletal structures
that in turn results in the aggregation of receptors into clusters, known as "capping" (5-11). Actomyosin function is known to be critical for this process. To investigate the role of myosin RLC phosphorylation during this process, COS-7 cells transfected with either wild type or mutant RLC-GFP were treated with ConA to induce capping of cell surface receptors. The expression level of GFP-RLC was
estimated by digital fluorescence microscopy, and cells expressing similar levels of the wild type or mutant GFP-RLC were compared. For
COS-7 cells expressing wild type RLC-GFP, normal capping of surface
receptors was observed in response to ConA (Fig.
3B). During the cap formation,
the wild type RLC-GFP signal was concentrated under the ConA cap
suggesting that myosin containing GFP-tagged RLC responded to the
ConA-induced signal (Fig. 3A). This suggests that myosin
with GFP-tagged RLC undergoes normal reorganization during the
ConA-induced capping process. In contrast, ConA-induced receptor cap
formation was markedly hampered in T18A/S19A RLC-GFP expressing COS-7
cells (Fig. 3E). In these cells, accumulation of RLC-GFP was
not observed, suggesting that unphosphorylatable myosin having
T18A/S19A RLC-GFP does not proceed with the reorganization observed for
the wild type (Fig. 3D). Immunostaining of untransfected cells as well as wild type RLC-GFP-transfected cells with
anti-phospho-RLC antibody revealed that phosphorylated RLC accumulated
under the ConA cap (Fig. 3C), also suggesting that myosin
phosphorylation is involved in the capping process. At 40 min after
treatment with ConA, 76% of the wild type RLC-GFP-transfected cells
formed capping structures, whereas less than 28% of the cells
transfected with the mutant RLC-GFP were able to form capping
structures (Table II). In order to
determine the difference in phosphorylation of myosin, the GFP-RLC
expressing cells were co-stained with anti-myosin heavy chain antibody
and anti-phospho-RLC antibody, and the ratio of these signals was used
to estimate the difference in the extent of phosphorylation. After ConA
treatment, the cells expressing T18A/S19A RLC-GFP had approximately
50% less RLC phosphorylation than those expressing wild type or
non-transfected cells (not shown). The disruption of the cap formation
in T18A/S19A RLC-GFP expression is thus explained by a decrease in
phosphorylated myosin molecules. Although the decrease is explained by
the substitution of the endogenous RLC by T18A/S19A RLC-GFP in myosin
molecules, it is also possible that free T18A/S19A RLC-GFP inhibits
MLCK, thus decreasing the phosphorylation of RLC actually incorporated in myosin molecules. To address this possibility, myosin II was phosphorylated by MLCK in the presence of excess concentrations of free
RLC-GFP (Fig. 4). The myosin
phosphorylated by MLCK was subjected to SDS-polyacrylamide gel
electrophoresis followed by autoradiography. Since RLC-GFP has
molecular mass of 47 kDa, the phosphorylation of the intrinsic RLC in
myosin molecule is easily distinguished by SDS-polyacrylamide gel
electrophoresis. Neither wild type nor T18A/S19A RLC-GFP significantly
affected the phosphorylation of native myosin bound RLC by MLCK,
suggesting that free RLC-GFP molecules do not inhibit MLCK to any
measurable extent. The results also suggest that a significant number
of the myosin light chains in myosin are substituted by RLC-GFP. The
present results demonstrate that myosin RLC phosphorylation is critical
for gathering surface receptors into cap structures, indicating that
phosphorylation of RLC plays an essential role in non-muscle myosin
function in capping of surface receptors.
Effects of Myosin RLC Phosphorylation in Cell Division--
The
bulk of evidence has indicated that myosin plays a critical role in
cell division. It is less clear, however, whether or not myosin RLC
phosphorylation to activate myosin motor activity is critical for cell
division in mammalian cells. We addressed this question by
overexpressing unphosphorylatable RLC in mitotic mammalian cells. It
has been known that myosin II accumulates at the contractile ring
during cell division (12-16, 31). We first examined whether or not
myosin containing RLC-GFP localizes at the contractile ring during
mitosis. As shown in Fig. 5, A (early telophase) and C (late telophase), the GFP signal of
the wild type RLC-GFP-transfected cells was concentrated at the
contractile ring, suggesting that RLC-GFP containing myosin shows
authentic localization during mitosis. Immunostaining of COS-7 cells
with anti-phospho-RLC antibody during cytokinesis showed staining at the equator of dividing cells, indicating that myosin in the
contractile ring is phosphorylated at its RLC. Therefore, we
anticipated that the introduction of unphosphorylatable RLC into myosin
in the cells would decrease the fraction of phosphorylated myosin and thus hamper proper mitosis. Wild type RLC-GFP or T18A/S19A RLC-GFP was
transfected into COS-7 cells, and the number of nuclei per cell was
observed at 72 h after transfection. A number of cells transfected
by T18A/S19A RLC-GFP became multinuclear cells. Fig. 6 shows such a transfected cell. Cells
transfected by both wild type and mutant RLC-GFP showed filamentous
localization of GFP fluorescence (Fig. 6A, panels
1 and 2), indicating that both chimeric RLCs are
incorporated into myosin molecules and confer proper myosin
localization. However, mutant RLC-GFP-transfected COS-7 cells became
multinuclear (Fig. 6A, panel 4) compared with wild type
RLC-GFP-transfected cells (Fig. 6A, panel 3) and
GFP-transfected control cells (data not shown). Approximately 40% of
the mutant RLC-GFP-transfected cells had multiple nuclei. At 72 h
after transfection, the fraction of multinuclear cells was 2.5-fold
greater for mutant RLC-GFP-transfected cells than for wild type
RLC-GFP-transfected cells (Fig. 6B). Although GFP-tagged RLC
demonstrated normal myosin function using various criteria,
i.e. normal binding to the heavy chain, normal distribution
of myosin in cells, and normal response to ConA signal during the
capping process, it is possible that the GFP tag may induce abnormal
cell division. To address this possibility, we subcloned RLC cDNA
into a pTracer-CMV mammalian expression vector and transfected cells
with this construct. The pTracer-CMV expression vector contains a super
GFP expression cassette under the control of an SV40 promoter, with the
inserted foreign gene under the control of a cytomegalovirus promoter. Therefore, transfected cells expressing non-GFP-tagged RLC can be
selected by the fluorescence of the independently expressed GFP.
Transfection of COS-7 cells with untagged RLCs showed the same effects
in ConA-induced capping formation as those obtained with the
overexpression of GFP-tagged RLCs, i.e. T18A/S19A RLC overexpression inhibited cap formation (data not shown). As shown in
Fig. 7A, transfection of COS-7
cells with non-tagged T18A/S19A RLC significantly increased (5.5-fold)
the fraction of multinuclear cells compared with wild type RLC
expressing cells. In NRK cells, the fraction of multinuclear cells was
8 times higher for cells transfected with mutant RLC versus
wild type RLC (Fig. 7B). These results suggest that
phosphorylation of myosin at Ser-19 and/or Thr-18 of RLC plays an
important role in the normal cell division of mammalian somatic
cells.
Disruption of RLC Phosphorylation of Myosin Causes Abnormal
Cytokinesis and Chromosome Segregation--
A remaining question is
how the disruption of myosin phosphorylation at Ser-19/Thr-18 of RLC,
which is critical for the activation of myosin motor activity, hampers
cell division. To determine the effects of the disruption of myosin
phosphorylation during mitosis in the somatic cell cycle, we monitored
either wild type RLC or T18A/S19A RLC expressing cells during cell
division by video microscopy. For both COS-7 and NRK cells, wild type
RLC-transfected cells had no detectable changes in cell division
progression after release from nocodazole arrest (Fig.
8, A-E and F-J).
The transit times between anaphase and cytokinesis were similar to
those of untransfected cells (data not shown). A majority of both
untransfected and wild type RLC-transfected cells exhibited the onset
of anaphase within 40 min and completed cell division within 49 min
after the release of mitotic arrest (Table
III). In contrast, the T18A/S19A RLC
expressing cells showed significantly delayed progression from
metaphase to telophase (Table III), and many cells stayed at metaphase
at 49 min after release (Fig. 9,
A-C). The cells expressing T18A/S19A RLC showed a
significantly hampered phenotype at cytokinesis, in which a distorted
cleavage furrow was formed and normal cytokinesis failed (Fig. 9,
D-H) (Table IV).
Whereas the equatorial plane of the cell narrowed to certain extent,
the arrangement of the contractile ring was hampered, and the
contraction of the contractile ring was not completed to divide the
cell. On the other hand, cells expressing the wild type RLC showed a normal arrangement of the contractile ring and proceeded through normal
cytokinesis. These observations suggest that the inhibition of myosin
phosphorylation at Thr-18/Ser-19 of RLC hampers both normal progression
of mitosis and cytokinesis.
Actomyosin motor activity is thought to play a critical role in
various aspects of cell motility such as cell locomotion, receptor
capping, and cell division. In vertebrate and non-muscle cells, myosin
motor activity in vitro is regulated by phosphorylation of
RLC at Ser-19 and Thr-18, especially at the former site. Therefore, the
cell motile processes that are governed by actomyosin contractile activity are assumed to be controlled by a dynamic change in RLC phosphorylation during these processes. Yet the relationship between myosin phosphorylation and these cell movements is not well understood. In the present study, we have approached this question by
overexpressing unphosphorylatable RLC in mammalian somatic cells. It is
known that phosphorylation at Ser-19 and Thr-18 of RLC is mandatory for
the motor activity of vertebrate non-muscle and smooth muscle myosins
(see Refs. 22-24 for reviews). Previously, it was shown that
substitution of these residues by Ala abolishes myosin motor activity
(41). Furthermore, it was shown that this substitution also hampers the
thick filament formation of myosin in vitro (41). Therefore,
the effects of overexpression of T18A/S19A RLC in cell motile activity
may be due to either diminished actomyosin contractile activity or
diminished recruitment of myosin into filamentous structures. One way
to obtain RLC overexpressing cells is to produce RLC overexpressing
cell lines, but we avoided this approach in this study because of the
following. 1) The cell phenotype may change during the establishment of
a cell line due to secondary effects of overexpression. 2) The
overexpression of unphosphorylatable RLC may cause the cell not to
proliferate properly because of the disruption of mitosis. In order to
identify transiently transfected cells, GFP fluorescence was utilized
in the present study. Two approaches were taken. First, RLC was tagged
with GFP. The advantage of this approach is that the localization of
myosin during cell motility can be directly monitored. Therefore, we
employed this approach in most of the experiments in this study. The
GFP-tagged RLC showed normal myosin heavy chain binding activity
in vitro. Furthermore, RLC-GFP showed stress fiber
localization in interphase cells and a localization at the cleavage
furrow in mitotic cells. Therefore, we concluded that myosin containing
RLC-GFP retains the authentic properties of myosin. Nevertheless, to
ensure our findings further, RLC cDNA was cloned into a pTracer
vector containing a separate GFP expression cassette. The cells
transfected with this vector express RLC and GFP independently so that
non-tagged RLC associates with myosin, but the transfected cells can
still be identified by the GFP signal. Practically identical results were obtained using both approaches in both cell types for the motile
events examined in this study, i.e. surface capping and cell division.
We first demonstrate that deletion of RLC phosphorylation sites by
substitution of Ser-19/Thr-18 by Ala inhibits the induction of the
receptor cap structure. The antibody that recognizes Ser-19 phosphorylation of RLC was detected under the ConA cap in both non-transfected and wild type RLC-GFP transfected cells but not T18A/S19A RLC-GFP-transfected cells. These results clearly indicate that the myosin RLC phosphorylation at Ser-19 is critical for receptor
capping. Previously, it was reported that overall RLC phosphorylation
in whole cell extracts increases during the capping event (9) and that
actomyosin-containing structures are associated with cap formation
(5-11). The present results are consistent with these earlier findings
and provide direct evidence that myosin phosphorylation is required for
receptor capping in mammalian cells. It was reported previously that,
in Dictyostelium, ConA-induced capping was unaffected by
disruption of a calmodulin-independent MLCK (mlck-A)
gene (47), even though mlck-A increases RLC
phosphorylation of Dictyostelium myosin by treatment with
ConA. The discrepancy from the present results is likely to be due to a
difference in the regulation between the two myosins. Whereas RLC
phosphorylation is required for myosin motor activity in vertebrate
smooth muscle and non-muscle myosin, RLC phosphorylation does not
significantly augment the actin translocating activity of
Dictyostelium myosin (48).
It has been assumed that the activation of myosin contractile activity
is important for mitosis, especially during cytokinesis, since
actomyosin is enriched at the cleavage furrow and forms the contractile
ring that narrows during cell division (12-15). Although it is
accepted that actomyosin function is crucial for cell division, it is
unclear how actomyosin contractile activity is regulated during mitosis
in vertebrate cells. As vertebrate myosin motor activity is regulated
by RLC phosphorylation, it is reasonable to assume that actomyosin
function and thus myosin phosphorylation is temporally and spatially
regulated during mitosis, but the functional significance of serine 19 phosphorylation of myosin II RLC during cell division in mammalian
cells is not understood. It has been proposed that phosphorylation of
myosin at Ser-19 of RLC takes place in the equatorial contractile ring
and activates contraction of the cleavage furrow after the
dephosphorylation of RLC at the Cdc2 kinase sites (49), but the
significance of the phosphorylation at the Cdc2 kinase sites during
cell division is questioned (50). The present results show that the
cells expressing the unphosphorylatable form of RLC become
multinuclear, suggesting an abnormal cell dividing process, which is
consistent with this model. Microscopic observation of mitotic cells
revealed that there are two types of defects in the cells expressing
unphosphorylatable RLC. One is the formation of an abnormal cleavage
furrow followed by the failure to complete proper cytokinesis. The
other is the inhibition of transition from metaphase to anaphase. As
shown in Fig. 9, T18A/S19A RLC expressing cells showed a distorted
cleavage furrow. This result suggests that the contractile ring is not properly arranged at the equator of the cells. Previously, Wang and
co-workers (34) observed that NRK cells microinjected with constitutively active MLCK had a delayed onset of anaphase, but the
treatment did not alter the rate of progression of cytokinesis. It has
also been demonstrated (51) that the level of RLC phosphorylation at
Ser-19/Thr-18 increases at the cell stage just prior to entering cytokinesis, and the phosphorylation level is maintained during cell
division. Therefore, it would be plausible that myosin II phosphorylation is elevated before myosin is recruited into the contractile ring, and the disruption of myosin phosphorylation may
alter the formation of a normal contractile ring.
An interesting finding is that the overexpression of unphosphorylatable
RLC disrupted the movement of chromosomes from the metaphase plate,
suggesting that myosin plays an important role in the progression from
metaphase to anaphase and that myosin phosphorylation is involved in
properly performed karyokinesis. This view is supported by the previous
finding that microinjection of constitutively active MLCK delayed the
onset of anaphase (34). These results suggest that proper myosin
phosphorylation during mitosis is critical for normal transition from
metaphase to anaphase. Although the mechanism by which activation of
myosin function influences chromosome separation is unclear, it was
reported that anti-myosin IIA antibody stains the spindle poles between
prophase and metaphase (31). Recently, it was reported (16) that
anti-phospho-RLC polyclonal antibody stained the spindle poles,
although since this antibody also recognized other proteins it remained
uncertain whether or not phospho-RLC was present there. However, our
monoclonal anti-phospho-RLC antibody also recognized the spindle poles
during transition from prophase to metaphase, supporting the presence of phosphorylated myosin.2
The present findings, together with these observations, suggest that
myosin phosphorylation is involved not only in cytokinesis but also in
the separation of sister chromatids. It was previously found that
microinjection of anti-myosin II antibodies into starfish eggs
inhibited cytokinesis (32), but this also produced an abnormal mitotic
apparatus. These results suggest that anti-myosin II antibodies might
influence the mitotic apparatus. The expression of T20A/S21A RLC in
Drosophila RLC null germ line cells showed an increase in
multinuclear cells (52), resembling the results of the present study.
The Drosophila experiment detected abnormal cytokinesis, but
an abnormality of karyokinesis was not reported. The difference from
the present study could be due to the following: 1) a difference between mitosis during oogenesis and during the somatic cell cycle (present study); 2) a difference in the regulation of mitosis between
Drosophila and mammalian cells; and 3) a difference between the regulation of myosin II in mammalian cells and in
Drosophila (which is not well understood at a molecular
level). The myosin RLC null mutant of Dictyostelium showed a
deficiency in cytokinesis and produced multinuclear cells (48, 53).
However, the effect on karyokinesis is not well studied. It should be
noted that RLC null cells retain myosin ATPase activity (1/3 of the
wild type) and that RLC domain-deficient myosin of
Dictyostelium showed more than 50% of the motility activity
of the wild type myosin. These results suggest that
Dictyostelium RLC null myosin retains some motor activity.
Therefore, it is plausible that the inhibition of each biological
process related to myosin II activity depends on the extent of the
inhibition of myosin II function. Alternatively, the involvement of
myosin II in karyokinesis might be developed in mammalian cells but not
in lower eukaryotes. Recently, Simerly et al. (54) reported
that the injection of myosin IIA antibody disrupts the eccentric
positioning of the metaphase spindle during meiotic maturation,
probably through depletion of spindle-associated myosin IIA. The
present result together with earlier results suggest the involvement of
myosin II function in proper chromosomal segregation during mitosis.
We thank Dr. R. Adelstein for the gift of
anti-myosin IIB antibody. We also thank Dr. D. J. Schmidt for
reading the manuscript.
*
This work was supported by National Institutes of Health
Grants AR41653, HL56218, HL60831, and HL61426.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Physiology, University of Massachusetts Medical School, Worcester, MA 01655. Tel.: 508-856-1954; Fax: 508-856-4600; E-mail:
mitsuo.ikebe@umassmed.edu.
Published, JBC Papers in Press, August 15, 2000, DOI 10.1074/jbc.M003019200
2
S. Komatsu and M. Ikebe, unpublished observations.
The abbreviations used are:
RLC, regulatory
light chain of myosin;
CMV, cytomegalovirus;
Cy5, indodicarbocyanine;
ConA, concanavalin A;
DAPI, 4',6-diamidino-2-phenylindole;
GFP, green
fluorescent protein;
MLCK, myosin light chain kinase;
NRK, normal rat
kidney;
PKC, protein kinase C;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
TRITC, tetramethylrhodamine B
isothiocyanate.
Effects of the Regulatory Light Chain Phosphorylation of Myosin
II on Mitosis and Cytokinesis of Mammalian Cells*
,, Medical and
Biological Laboratories, Ina, Nagano 396, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol), and 20 mM
Tris-HCl, pH 7.5. The reaction solutions were incubated for 15 min at
25 °C in the presence of various concentrations of isolated RLC-GFP
(mol of RLC-GFP/mol of myosin). Phosphorylated myosin were subjected to
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction (data
not shown). Furthermore, the 47-kDa proteins were also recognized by
anti-RLC antibody (Fig. 1B, lanes 1 and 2). These
results showed that the 47-kDa peptides were the RLC-GFP chimeric
protein. The apparent molecular mass of 47 kDa is consistent with the
sum of the molecular mass of each component, i.e. 20 kDa for
RLC and 27 kDa for GFP. To confirm the deletion of phosphorylation sites in the mutant RLC-GFP, the chimeric protein was incubated with
MLCK in the presence of calmodulin and Ca2+, and the
phosphorylation was detected by immunostaining with anti-phospho-RLC
monoclonal antibody. Although the wild type RLC-GFP chimera was readily
phosphorylated by MLCK (Fig. 1B, lane 4), anti-phospho-RLC antibody did not recognize the mutant RLC-GFP (Fig.
1B, lane 6), indicating that the mutant RLC-GFP cannot be phosphorylated by MLCK. To check the possibility that the mutation created new phosphorylation sites, T18A/S19A RLC-GFP was incubated with
MLCK/Ca2+/CaM and [-32P]ATP, and the
phosphorylation was examined by autoradiography. There was no
phosphorylation detected (not shown). As Fig. 1C shows,
anti-phospho-RLC antibody only recognizes the RLC phosphorylated by
MLCK but not unphosphorylated RLC or RLC phosphorylated by PKC. It was
also revealed that the antibody recognizes both singly and doubly
MLCK-phosphorylated RLC.
View larger version (24K):
[in a new window]
Fig. 1.
Expression of RLC-GFP chimeric protein.
A, Western blot of RLC-GFP with anti-GFP antibody
(Ab). After 8 h of
isopropyl-1-thio- -D-galactopyranoside induction,
partially purified proteins were subjected to SDS-polyacrylamide gel
electrophoresis. Immunoblot analysis with anti-GFP polyclonal antibody
was carried out for wild type RLC-GFP (Wt, lane
1) and T18A/S19A RLC-GFP (AA, lane 2).
B, Western blots of RLC-GFP with anti-RLC antibody and
anti-phosphorylation site antibody. Anti-phospho-RLC antibody reacted
only with wild type RLC-GFP phosphorylated by MLCK (lane 4)
but not with either unphosphorylated wild type RLC-GFP (lane
3) or T18A/S19A RLC-GFP (lanes 5 and 6, with
or without MLCK), whereas anti-RLC antibody reacted with both wild type
and T18A/S19A RLC-GFP (lanes 1 and 2).
C, specificity of anti-phospho-RLC antibody.
Unphosphorylated or phosphorylated RLC either by MLCK or PKC were
separated by urea/glycerol gel. Lanes 1 and 5,
unphosphorylated RLC; lanes 2 and 6,
mono-phosphorylated RLC by MLCK; lanes 3 and 7,
di-phosphorylated RLC by MLCK; lanes 4 and 8,
mono- and di-phosphorylated RLC by PKC. Lanes 1-4 were
stained with Coomassie Blue (CBB), and lanes 5-8
were immunostained with anti-phospho-RLC antibody. Note that
anti-phospho-RLC antibody reacted with both mono- and di-phosphorylated
RLC by MLCK but not by PKC.
ATPase activities of RLC-GFP myosin II
View larger version (25K):
[in a new window]
Fig. 2.
Localization of RLC-GFP in COS-7
cells. COS-7 cells were transfected with either wild type RLC-GFP
(A and E) or T18A/S19A RLC-GFP (C and
G). A, C, E, and G are GFP signals.
B and D are staining with anti-myosin IIB
polyclonal antibody. F and H are stained with
Texas Red-conjugated phalloidin. A and B,
C and D, E and F, or
G and H are the images obtained from the same
cells, respectively. Both wild type and T18A/S19A RLC-GFP were
localized with myosin and actin stress fibers. Bar, 10 µm.
View larger version (44K):
[in a new window]
Fig. 3.
Effects of RLC phosphorylation on
capping of cell surface receptors. COS-7 cells transfected with
either wild type (A-C) or T18A/S19A RLC-GFP
(D-F) were treated with TRITC-conjugated ConA to induce cap
formation. The transfected cells were stained with anti-GFP antibody
followed by FITC-conjugated 2nd antibody and with anti-phospho-RLC
antibody followed by Cy5-conjugated 2nd antibody. A and
D, B and E, and C and
F, are FITC, TRITC and Cy5 images, respectively.
A-C show wild type RLC-GFP-transfected cells. RLC-GFP
localizes with the ConA cap (indicated by arrowheads in
A), and anti-phospho-RLC antibody stains under the cap
(indicated by arrowhead in C). Cap formation is
inhibited (E) in T18A/S19A RLC-GFP expressing cell
(D). Cells were treated with ConA (E) and stained
with anti-phospho-RLC antibody (F). Indirect immunostaining
of the cells revealed that phosphorylated RLC accumulated under the
ConA cap. Bar, 10 µm.
Effect of RLC phosphorylation on ConA capping structure
View larger version (16K):
[in a new window]
Fig. 4.
An excess amount of RLC-GFP does not inhibit
MLCK-induced myosin phosphorylation. Myosin II was phosphorylated
by MLCK in the presence of various concentrations of isolated RLC-GFP.
Wild type RLC-GFP (Wt, lanes 2-4) or (T18A,S19A
RLC-GFP, AA, lanes 5-7) were added to the
reaction mixture at the indicated times (mol of RLC-GFP/mol of myosin).
Phosphorylated myosin was subjected to SDS-polyacrylamide gel
electrophoresis followed by autoradiography.
View larger version (61K):
[in a new window]
Fig. 5.
Localization of RLC-GFP containing myosin and
phosphorylated myosin during cytokinesis. A and
C and B and D show GFP localization
and phospho-RLC staining of COS-7 cells in early telophase and
cytokinesis, respectively. GFP signal (A and C
indicated by arrows) and phospho-RLC signal (B
and D, indicated by arrowheads) are concentrated
at the cleavage furrows of the dividing cells. GFP signals are enhanced
by anti-GFP antibody. Bar, 5 µm.
View larger version (17K):
[in a new window]
Fig. 6.
Effects of mutation in MLCK phosphorylation
sites on cytokinesis in COS-7 cells. A, production of
multinucleate cells in T18A/S19A RLC overexpressing cells. COS-7 cells
were transfected with either wild type (panels 1 and
3) or T18A/S19A RLC-GFP (panels 2 and
4). 72 h after transfection, the cells were fixed and
stained with DAPI (panels 3 and 4). B,
percentage of multinucleate cells expressing either wild type
(open column) or T18A/S19A RLC-GFP
([TS18,19AA], closed column). 72 h after
the transfection, the number of nuclei per cell were determined by
staining with Hoechst 33342 in living cells. More than 200 individual
cells were counted from three independently transfected cells. The
values shown are means ± S.E. from three independent experiments.
Bar, 10 µm
View larger version (20K):
[in a new window]
Fig. 7.
Increase in multinucleated cells by
expression of non GFP-tagged T18A/S19A RLC. COS-7 cells
(A) and NRK cells (B) were transfected with
pTracer-CMV vector containing either wild type RLC cDNA or
T18A/S19A ([TS18,19AA]) RLC cDNA. The number of nuclei
per cell was determined by staining with Hoechst 33342 in living cells
at 72 h after the transfection. The values shown are means ± S.E. from at least three independent experiments.
View larger version (93K):
[in a new window]
Fig. 8.
Time lapse series of phase images in mitotic
cells overexpressing non-GFP-tagged wild type RLC. 72 h after
the transfection with pTracer-RLC construct, both COS-7 cells
(A-E) and NRK cells (F-J) expressing wild type
RLC were observed by video microscopy. Observation began 30 min after
the release from nocodazole arrest. Transfected cells were identified
by GFP fluorescence signal (I and II). Images
were recorded during metaphase (A and F),
anaphase onset (B and G), anaphase (C
and H), telophase (D and I), and
cytokinesis (E and J). The arrowheads
(A-D and F-I) indicate the chromosomes,
and the asterisks (E and J) indicate
the cleavage furrows. Overexpression of wild type RLC in both cells did
not affect normal morphology at metaphase, anaphase, telophase,
and during cytokinesis.
Effect of the disruption of RLC phosphorylation on cell division
View larger version (150K):
[in a new window]
Fig. 9.
Interference of chromosome separation and
abnormal cell division in T18A/S19A RLC expressing cells. NRK
cells expressing T18A/S19A RLC were monitored from metaphase
(t = 30 min) after the release of mitotic arrest
(A-C and D-H). In some cells,
interference of chromosome separation was observed (A-C:
A, t = 30 min; B,
t = 50 min; and C, t = 90 min), causing inhibition of cytokinesis progression. In other cases,
abnormal cell division was observed (D-H).
Distortion of the cleavage furrow appeared (G) (indicated by
arrow) which resulted in failure to complete cell division
(H). The arrowheads (A-E) indicate
the chromosomes- and the arrows (G) indicate the
cleavage furrows.
Formation of abnormal cleavage furrow
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Wilson, A. K.,
Gorgas, G.,
Claypool, W. D.,
and de Lanerolle, P.
(1991)
J. Cell Biol.
114,
277-283
2.
Valerius, N. H.,
Stendahl, O.,
Hartwig, J. H.,
and Stossel, T. P.
(1981)
Cell
24,
195-202
3.
Zigmond, S. H.,
and Hirsch, J. G.
(1972)
Exp. Cell Res.
73,
383-393
4.
Korn, E. D.
(1978)
Proc. Natl. Acad. Sci. U. S. A.
75,
588-599
5.
Schreiner, G.,
Fujiwara, K.,
Pollard, T. D.,
and Unanue, E.
(1977)
J. Exp. Med.
145,
1393-1398
6.
Bourguignon, L. Y. W.,
and Singer, S. J.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5031-5035
7.
Bourguignon, L. Y. W.,
Tokuyasu, K. T.,
and Singer, S. J.
(1978)
J. Cell. Physiol.
95,
239-257
8.
Bourguignon, L. Y. W.
(1980)
Cell Biol. Int. Rep.
4,
541-547
9.
Bourguignon, L. Y.,
Nagpal, M. L.,
and Hsing, Y. C.
(1981)
J. Cell Biol.
91,
889-894
10.
Flanagan, J.,
and Koch, G. L. E.
(1978)
Nature
273,
278-281
11.
Condeelis, J.
(1979)
J. Cell Biol.
80,
751-758
12.
Fujiwara, K.,
and Pollard, T. D.
(1976)
J. Cell Biol.
71,
848-875
13.
Aubin, J. E. K.,
Weber,
and Osborn, M.
(1979)
Exp. Cell Res.
124,
93-109
14.
Maupin, P.,
and Pollard, T. D.
(1986)
J. Ultrastruct. Mol. Struct. Res.
94,
92-103
15.
Sanger, J. M.,
Mittal, B.,
Dome, J. S.,
and Sanger, J. W.
(1989)
Cell Motil. Cytoskeleton
14,
201-219
16.
Matsumura, F.,
Ono, S.,
Yamakita, Y.,
Totsukawa, G.,
and Yamashiro, S.
(1998)
J. Cell Biol.
140,
119-129
17.
Mabuchi, I.
(1986)
Int. Rev. Cytol.
101,
175-213
18.
Salmon, E. D.
(1989)
Curr. Opin. Cell Biol.
1,
541-547
19.
Sellers, J. R.
(1991)
Curr. Opin. Cell Biol.
3,
98-104
20.
Trybus, K. M.
(1991)
Cell Motil. Cytoskeleton
18,
81-85
21.
Tan, J. L.,
Ravid, S.,
and Spudich, J. A.
(1992)
Annu. Rev. Biochem.
61,
721-759
22.
Hartshorne, D. J.
(1987)
in
Physiology of the Gastrointestinal Tract
(Johnson, L. R., ed), 2nd Ed.
, pp. 423-482, Raven Press, Ltd., New York
23.
Sellers, J. R.,
and Adelstein, R. S.
(1987)
The Enzymes
, Vol. 18
, pp. 381-418, Academic Press, New York
24.
Kamm, K. E.,
and Stull, J. T.
(1989)
Annu. Rev. Physiol.
51,
299-313
25.
Edelman, A. M.,
Lin, W. H.,
Osterhout, D. J.,
Benett, M. K.,
Kennedy, M. B.,
and Krebs, E. G.
(1990)
Mol. Cell. Biochem.
97,
87-98
26.
Amano, M.,
Ito, K.,
Kimura, Y.,
Fukata, K.,
Chihara, T.,
Nakano, Y.,
Matsuura, Y.,
and Kaibuchi, K.
(1996)
J. Biol. Chem.
271,
20246-20249
27.
Komatsu, S.,
and Hosoya, H.
(1996)
Biochem. Cell Biol.
223,
741-745
28.
Nishikawa, M.,
Sellers, J. R.,
Adelstein, R. S.,
and Hidaka, H.
(1984)
J. Biol. Chem.
259,
8808-8814
29.
Bengur, A. R.,
Robinson, E. A.,
Appella, E.,
and Sellers, J. R.
(1987)
J. Biol. Chem.
262,
7613-7617
30.
Ikebe, M.,
Hartshorne, D. J.,
and Elzinga, M.
(1987)
J. Biol. Chem.
15,
9569-9573
31.
Kelley, C. A.,
Sellers, J. R.,
Gard, D. L.,
Bui, D.,
Adelstein, R. S.,
and Baines, I. C.
(1996)
J. Cell Biol.
134,
675-687
32.
Mabuchi, I.,
and Okuno, M.
(1977)
J. Cell Biol.
74,
251-263
33.
De Lozanne, A.,
and Spudich, J. A.
(1987)
Science
236,
1086-1091
34.
Fishkind, D. J.,
Cao, L. G.,
and Wang, Y. L.
(1991)
J. Cell Biol.
114,
967-975
35.
Ikebe, M.,
and Hartshorne, D. J.
(1985)
J. Biol. Chem.
260,
13146-13153
36.
Spudich, J. A.,
and Watt, S.
(1971)
J. Biol. Chem.
246,
4866-4871
37.
Chien, Y. H.,
and Dawid, I. B.
(1984)
Mol. Cell. Biol.
4,
507-513
38.
Ikebe, M.,
Reardon, S.,
Schwonek, J. P.,
Sanders, C. R., II,
and Ikebe, R.
(1994)
J. Biol. Chem.
269,
28165-28172
39.
Ikebe, M.,
Kambara, T.,
Stafford, W. F.,
Sata, M.,
Katayama, E.,
and Ikebe, R.
(1998)
J. Biol. Chem.
273,
17702-17707
40.
Yano, T.,
Taura, C.,
Shibata, M.,
Hirono, Y.,
Ando, S.,
Kusubata, M.,
Takahashi, T.,
and Inagaki, M.
(1991)
Biochem. Cell Biol.
175,
1144-1151
41.
Kamisoyama, H.,
Araki, Y.,
and Ikebe, M.
(1994)
Biochemistry
33,
840-847
42.
Yano, K.,
Araki, Y.,
Hales, S. J.,
Tanaka, M.,
and Ikebe, M.
(1993)
Biochemistry
32,
12054-12061
43.
Laemmli, U. K.
(1970)
Nature
227,
680-685
44.
Highashihara, M.,
Frado, L. L.,
Craig, R.,
and Ikebe, M.
(1989)
J. Biol. Chem.
264,
5218-5225
45.
Perrie, W. T.,
and Perry, S. V.
(1970)
Biochem. J.
119,
31-38
46.
Carrington, W. A.,
Lynch, R. M.,
Moore, E. D.,
Isenberg, G.,
Fogarty, K. E.,
and Fay, F. S.
(1995)
Science
268,
1483-1487
47.
Smith, J. L.,
Silveira, L. A.,
and Spudich, J. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12321-12326
48.
Ostrow, B. D.,
Chen, P.,
and Chisholm, R. L.
(1994)
J. Cell Biol.
127,
1945-1955
49.
Pollard, T. D.,
Satterwhite, L.,
Cisek, L.,
Corden, J.,
Sato, M.,
and Maupin, P.
(1990)
Ann. N. Y. Acad. Sci.
582,
120-130
50.
Shuster, C. B.,
and Burgess, D. R.
(1999)
J. Cell Biol.
146,
981-992
51.
Yamakita, Y.,
Yamashiro, S.,
and Matsumura, F.
(1994)
J. Cell Biol.
124,
129-137
52.
Jordan, P.,
and Karess, R.
(1997)
J. Cell Biol.
139,
1805-1819
53.
Chen, P.,
Ostrow, B. D.,
Tafuri, S. R.,
and Chisholm, R. L.
(1994)
J. Cell Biol.
127,
1933-1944
54.
Simerly, C.,
Nowak, G.,
de Lanerolle, P.,
and Schatten, G.
(1998)
Mol. Biol. Cell
9,
2509-2525
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W.-m. Zhao and G. Fang Anillin Is a Substrate of Anaphase-promoting Complex/Cyclosome (APC/C) That Controls Spatial Contractility of Myosin during Late Cytokinesis J. Biol. Chem., September 30, 2005; 280(39): 33516 - 33524. [Abstract] [Full Text] [PDF]
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 W.-m. Zhao and G. Fang MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis PNAS, September 13, 2005; 102(37): 13158 - 13163. [Abstract] [Full Text] [PDF]
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M. Kanada, A. Nagasaki, and T. Q.P. Uyeda Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells Mol. Biol. Cell, August 1, 2005; 16(8): 3865 - 3872. [Abstract] [Full Text] [PDF]
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 D. Giner, P. Neco, M. d. M. Frances, I. Lopez, S. Viniegra, and L. M. Gutierrez Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells J. Cell Sci., July 1, 2005; 118(13): 2871 - 2880. [Abstract] [Full Text] [PDF]
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Y. Koga and M. Ikebe p116Rip Decreases Myosin II Phosphorylation by Activating Myosin Light Chain Phosphatase and by Inactivating RhoA J. Biol. Chem., February 11, 2005; 280(6): 4983 - 4991. [Abstract] [Full Text] [PDF]
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P. Neco, D. Giner, S. Viniegra, R. Borges, A. Villarroel, and L. M. Gutierrez New Roles of Myosin II during Vesicle Transport and Fusion in Chromaffin Cells J. Biol. Chem., June 25, 2004; 279(26): 27450 - 27457. [Abstract] [Full Text] [PDF]
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S. Komatsu and M. Ikebe ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts J. Cell Biol., April 26, 2004; 165(2): 243 - 254. [Abstract] [Full Text] [PDF]
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N. G. Dulyaninova, Y. V. Patskovsky, and A. R. Bresnick The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest J. Cell Sci., March 15, 2004; 117(8): 1481 - 1493. [Abstract] [Full Text] [PDF]
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 T. Chitapanarux, S. L. Chen, H. Lee, A. C. Melton, and H. F. Yee Jr. C-type natriuretic peptide induces human colonic myofibroblast relaxation Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G31 - G36. [Abstract] [Full Text] [PDF]
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J.-C. Kuo, J.-R. Lin, J. M. Staddon, H. Hosoya, and R.-H. Chen Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase J. Cell Sci., December 1, 2003; 116(23): 4777 - 4790. [Abstract] [Full Text] [PDF]
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 J. J. LoTurco, M. R. Sarkisian, L. Cosker, and J. Bai Citron Kinase is a Regulator of Mitosis and Neurogenic Cytokinesis in the Neocortical Ventricular Zone Cereb Cortex, June 1, 2003; 13(6): 588 - 591. [Abstract] [Full Text] [PDF]
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B.-H. Chen, J. T. C. Tzen, A. R. Bresnick, and H.-C. Chen Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells J. Biol. Chem., September 6, 2002; 277(37): 33857 - 33863. [Abstract] [Full Text] [PDF]
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 S. Komatsu, K. Miyazaki, R. A. Tuft, and M. Ikebe Translocation of telokin by cGMP signaling in smooth muscle cells Am J Physiol Cell Physiol, September 1, 2002; 283(3): C752 - C761. [Abstract] [Full Text] [PDF]
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 D. A. Guertin, S. Trautmann, and D. McCollum Cytokinesis in Eukaryotes Microbiol. Mol. Biol. Rev., June 1, 2002; 66(2): 155 - 178. [Abstract] [Full Text] [PDF]
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N. Niiro and M. Ikebe Zipper-interacting Protein Kinase Induces Ca2+-free Smooth Muscle Contraction via Myosin Light Chain Phosphorylation J. Biol. Chem., July 27, 2001; 276(31): 29567 - 29574. [Abstract] [Full Text] [PDF]
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K. Miyazaki, T. Yano, D. J. Schmidt, T. Tokui, M. Shibata, L. M. Lifshitz, S. Kimura, R. A. Tuft, and M. Ikebe Rho-dependent Agonist-induced Spatio-temporal Change in Myosin Phosphorylation in Smooth Muscle Cells J. Biol. Chem., January 4, 2002; 277(1): 725 - 734. [Abstract] [Full Text] [PDF]
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