From the Division of Hematopoietic Factors, The
Institute of Medical Science, University of Tokyo, Minato-ku Tokyo
108-8639, Japan and the § Department of Internal Medicine
II, Chiba University School of Medicine, Chiba 260-0856, Japan
Received for publication, August 10, 2000
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ABSTRACT |
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We have recently cloned a cDNA for a
full-length form of MgcRacGAP. Here we show using anti-MgcRacGAP
antibodies that, unlike other known GAPs for Rho family, MgcRacGAP
localized to the nucleus in interphase, accumulated to the mitotic
spindle in metaphase, and was condensed in the midbody during
cytokinesis. Overexpression of an N-terminal deletion mutant resulted
in the production of multinucleated cells in HeLa cells. This mutant
lost the ability to localize in the mitotic spindle and midbody.
MgcRacGAP was also found to bind A cell divides into two daughter cells through nuclear division
and cytokinesis. In cytokinesis, formation of an actomyosin-based contraction ring separates the cytoplasm to two daughter cells (1, 2).
A small GTPase RhoA localizes in the cleavage furrow during cytokinesis
and plays important roles in cytokinesis (3). In Xenopus
eggs, microinjection of a Rho-specific inhibitor C3, an exoenzyme from
Clostridium botulinum, prevents the progression of
cytokinesis (4). Other members of the Rho family of GTPases may also be
involved in cytokinesis. A human cell line expressing a constitutively
active mutant of Cdc42, as well as a Dictyostelium strain
lacking the RacE gene encoding a Rac/Cdc42 related protein, produced multinucleated cells as a result of impaired cytokinesis (5,
6). Recently, putative cytoplasmic targets of the Rho family of GTPases
have been identified, and some of which, including Gic, Bni1p, and
citron kinase, seem to be specifically involved in cytokinesis
(7-10).
The Rho family of small GTPases, represented by RhoA, Rac, and Cdc42,
act as molecular switches of diverse biological functions involving
remodeling of cytoplasmic actin and microtubules (11, 12). The
GTP-bound Rho protein is an active form, while the GDP-bound form is
inactive. Activation of the Rho family is promoted by guanine
nucleotide exchange factors
(GEFs),1 which catalyze the
replacement of bound GDP by GTP. The GTP-bound form of the Rho family
can specifically interact with their effectors or targets and transmit
signals to downstream molecules. On the other hand, they are
inactivated through hydrolysis of bound GTP to GDP by their intrinsic
GTPase activities, assisted by GTPase activating proteins (GAPs) (13).
Several GEFs are known to play roles in cytokinesis. For example,
Vav-3, a novel Vav family protein which has a GEF activity for RhoA,
RhoG, and Rac, has an important role in cytokinesis (14). Another GEF
for the Rho family, ECT2, is implicated in cytokinesis (15). In
Drosophila, Pbl, a homologue of mammalian ECT2, was found to
be required for initiation of cytokinesis (16). On the other hand,
which GAPs inactivate the Rho family of GTPases involved in cytokinesis
has been largely unknown.
In a search for molecules involved in macrophage differentiation, we
identified a murine cDNA for Rac/Cdc42-specific GAP, whose antisense
cDNA inhibited macrophage differentiation of mouse leukemic M1
cells induced by IL-6. Moreover its overexpression induced
differentiation of human leukemic HL-60 cells into macrophage. This
Rac/Cdc42-specific GAP is a homologue of a previously identified human
RacGAP, MgcRacGAP (17). However, our clone encoded an additional 106 amino acids at the N terminus that has a myosin-like sequence (18). In
fact, the human counterpart we identified had a similar myosin-like
domain, indicating that the previously reported cDNA for human
MgcRacGAP encoded a protein with an N-terminal deletion. Overexpression
of MgcRacGAP retarded proliferation and induced formation of
multinucleated cells in all cell lines examined. In addition, the
highest expression level of MgcRacGAP mRNA was observed at the
G2/M phase. These data prompted us to determine whether
MgcRacGAP regulates cell proliferation through the control of
cytokinesis. Immunohistochemical studies showed that MgcRacGAP colocalized with the mitotic spindle in metaphase, and was transferred to the midzone in anaphase and telophase, and moved to the midbody in
cytokinesis. Analysis of deletion mutants of MgcRacGAP showed that
overexpression of a myosin-like domain-deletion mutant or a GAP
activity defective mutant of MgcRacGAP halted cell division and led to
form multinucleated cells. We also showed that MgcRacGAP associated
with microtubules in vivo through the N-terminal myosin-like domain. These data indicated that MgcRacGAP plays key roles in the cell
cycle machinery, especially in the G2/M phase, through binding to microtubules.
Immunostaining--
HeLa cells were plated on glass coverslips,
and the next day the cells were washed three times with ice-cold PBS
and fixed with 4% paraformaldehyde/PBS for 20 min at room temperature.
The cells were washed three times with ice-cold PBS followed by a 10-min incubation at room temperature in PBS containing 0.1% Nonidet P-40. The permeabilized cells were washed three times with ice-cold PBS. The antibodies, anti-mouse Cell Culture and Transfection--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 5% fetal calf
serum (FCS) and seeded into a 6-cm dish at 1 × 106/dish. On the following day, HeLa cells were transiently
transfected with plasmids encoding the wild type or the mutant forms of
MgcRacGAP using LipofectAMINE PLUS (Life Technologies, Inc.) according
to manufacturer's recommendations. Ba/F3 cells were grown in RPMI 1640 supplemented with 10% FCS and 2 ng/ml of mouse IL-3. HL-60 and Jurkat
cells were grown in RPMI 1640 supplemented with 10% FCS.
Preparation of Cell Extracts and
Immunoprecipitation--
Immunoprecipitation from the cell lysates was
performed as described previously (19). In brief, cells were washed
three times with ice-cold PBS, and lysed in TCSD buffer (50 mM Tris-HCl, pH 7.4, 1% CHAPS, 300 mM NaCl, 1 mM dithiothreitol, 10 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) on ice for 1 h, with occasional shaking. The lysate was
then centrifuged at 10,000 × g at 4 °C for 1 h. Equal aliquots of the supernatant fractions were incubated overnight
with appropriate antibodies and Protein A-Sepharose CL-4B beads
(Amersham Pharmacia Biotech) at 4 °C. After this incubation period,
Sepharose beads were washed five times with TCSD buffer. Proteins bound
on the beads were released by boiling them in the SDS sample buffer
(20) for 5 min, then loaded on a polyacrylamide gel for electrophoresis.
Synchronization of the Cell Cycle--
Synchronization of the
cell cycle was achieved by double thymidine block (21) with some
modifications. In brief, HeLa cells in the exponential growth phase
were exposed to 2.5 mM thymidine in Dulbecco's modified
Eagle's medium, 5% FCS for 12 h and then incubated in
fresh medium for 12 h. Cells were once again exposed to 2.5 mM thymidine for 12 h, after which the block was
released by replacing the medium with fresh Dulbecco's modified
Eagle's medium, 5% FCS. Flow cytometry was performed for analysis of
cell cycle as previously described. (22)
Preparation of the Anti-MgcRacGAP Antibodies--
To generate
the anti-MgcRacGAP antibodies, a fusion protein between glutathione
S-transferase (GST) and C terminus domain of MgcRacGAP was
produced in Escherichia coli. For preparation of
a polyclonal antibody against MgcRacGAP, C-terminal 275 amino acids was
purified and injected into rabbits. The antibody was purified from
whole serum using the C terminus of MgcRacGAP on an affinity column.
Expression Constructs and Retrovirus Vectors--
A Flag-tagged
MgcRacGAP was cloned into EcoRI and NotI sites of
a mammalian expression vector pME 18S (pME-MgcRacGAP) (23). The
deletion construct that defects the N-terminal myosin-like domain
(
A cDNA for MgcRacGAP was cloned into
EcoRI-NotI sites of a retrovirus vector
pMX-IRES-EGFP (24), upstream of IRES (internal ribosomal entry site)
sequence so that both MgcRacGAP and EGFP were expressed from a single
mRNA (pMX-MgcRacGAP-IRES-EGFP).
Retrovirus-mediated Gene Transfer--
High titer retroviruses
were produced from pMX-MgcRacGAP-IRES-EGFP, using a transient
retrovirus packaging cell line PLAT-E (25), as described previously
(26). We first established stable transfectants expressing the
ecotropic viral receptor (27, 28). For infection, HL-60 and Jurkat
cells transduced with the ecotropic viral receptor and Ba/F3 cells
(1 × 106) were incubated with 10 ml of the
retroviruses in the presence of hexadimethrine bromide (10 µg/ml)
(Sigma). Twenty-four hours after infection, cells were washed, and
refed with growth medium. Two days after infection, the infected cells
were washed twice with PBS, suspended in PBS containing 1% bovine
serum albumin, and then sorted based on EGFP expression by a FACS
Vantage (Becton Dickinson). The sorted cells were then expanded in a
growth medium.
GST Fusion Proteins--
The myosin-like domain (Myo; amino
acids 1-124), the cystein-rich domain (Cys; amino acids 280-340), and
three blocks conserved in RhoGAPs (GAPD; amino acids 345-620) were
derived from MgcRacGAP and each domain was cloned into a pGEX-2T vector
(Amersham Pharmacia Biotech). The transformed bacteria (DH5 Enforced Expression of MgcRacGAP-induced Growth Suppression and
Formation of Multinucleated Cells in Hematopoietic Cells--
Using
retrovirus-mediated expression screening, we identified a full-length
cDNA for MgcRacGAP in the antisense configuration based on the
ability to inhibit IL-6 induced differentiation of M1 cells.
Overexpression of MgcRacGAP induced macrophage differentiation in HL-60
(18). We also found that expression of MgcRacGAP partially inhibited
proliferation in the cell line HL-60, M1, Ba/F3, and TF-1 (data not
shown). Thus, MgcRacGAP seemed to regulate differentiation and
proliferation of hematopoietic cells.
To determine the mechanisms by which MgcRacGAP regulates cell
proliferation, HL-60, M1, Ba/F3, Jurkat, and HeLa cells were transduced
with a bicistronic retrovirus vector pMX-IRES-EGFP carrying a
full-length cDNA for MgcRacGAP, where EGFP expression guarantees
the expression of exogenous MgcRacGAP. EGFP-positive cells transduced
with these viruses were sorted by FACS 2 days after infection.
EGFP-positive cells transduced with a control pMX-IRES-EGFP vector were
similarly sorted and served as a negative control.
Overexpression of MgcRacGAP retarded proliferation of all cell lines
tested in this study. Unexpectedly, morphological analysis of Jurkat
and Ba/F3 cells overexpressing MgcRacGAP detected a significant number
of multinucleated giant cells (Fig. 1). A
small number of multinucleated cells were also found in HL-60 and HeLa cells overexpressing this GAP (data not shown). These results suggested
that MgcRacGAP was playing some roles in cytokinesis. To determine
whether MgcRacGAP overexpression blocked the normal progression of
mitosis, we performed cell cycle analysis in HL-60 overexpressing
MgcRacGAP. However, no remarkable change was observed when compared
with control cells, except for marginal increase of the cells in
G2/M phase, which may have been caused by the presence of
multinucleated cells (data not shown).
Expression of MgcRacGAP Is Cell
Cycle-dependent--
Because MgcRacGAP was implicated in
cytokinesis, we investigated if the expression levels of MgcRacGAP
change throughout the cell cycle. To this end, HeLa cells were
synchronized at the G1/S transition by double thymidine
block, and followed through to completion of mitosis. After release
from the G1/S transition, the cells were analyzed for
expression of MgcRacGAP mRNA. The DNA content at each time point
was analyzed by flow cytometry (Fig.
2a). The mRNA levels for
MgcRacGAP were gradually increased as the cells progressed through S
phase, and reached the maximum at 9-12 h after the release,
corresponding to the G2/M phase (Fig. 2b). Thus
expression of MgcRacGAP mRNA was cell cycle-dependent, and peaked during the G2/M phase. These observations,
together with the findings that overexpression of MgcRacGAP induced
formation of multinucleated cells, suggested that MgcRacGAP functions
during mitosis, in particular during cytokinesis.
MgcRacGAP Co-localized with the Mitotic Spindle and Midbody in M
Phase--
To further examine the function of this protein during the
cell cycle, rabbit polyclonal antibodies against a recombinant protein
corresponding to the C-terminal half of MgcRacGAP was prepared, as
described under "Experimental Procedures." This antibody specifically recognized an 85-kDa protein (Fig.
3a).
To observe the subcellular distribution of the endogenous MgcRacGAP,
HeLa cells were fixed, and MgcRacGAP was immunostained using the
antibodies. In interphase, MgcRacGAP was mainly localized in the
nucleus although no Rho family proteins have been reported to localize
in the nucleus. MgcRacGAP was also detected in the cytoplasm as a
reticular pattern, along with microtubules (Fig. 3b, panel
A).
MgcRacGAP spread throughout the cytoplasm in prometaphase and
accumulated in the mitotic spindle in metaphase (Fig. 3b, panels B and C). In anaphase and telophase, MgcRacGAP formed a
distinct fine band extending across the midzone (Fig. 3b, panels
D-E). As the cell progressed to cytokinesis, MgcRacGAP became more
sharply concentrated in the midbody (Fig. 3b, panel F). To
exclude the possibility that the antibodies we used in this study
cross-reacted with some protein other than MgcRacGAP, we introduced
Flag-tagged MgcRacGAP, followed by immunodetection with an antibody
against Flag (M2 monoclonal antibody). In this experiment, Flag-tagged MgcRacGAP was also detected in the midbody of dividing cells, thus
confirming that anti-MgcRacGAP antibodies specifically recognize MgcRacGAP in immunostaining (Fig.
4).
Expression of a Myosin-like Domain-defective Mutant
(
Next we co-transfected pME-EGFP with a vector harboring the wild type
or each mutant of MgcRacGAP into HeLa cells. pME 18S (23) was used as a
vector control. The morphology of the EGFP-positive cells was assessed
72 h after transfection. When the wild type and mutant MgcRacGAPs
were overexpressed in HeLa cells, some populations of the cells became
multinuclear. Interestingly, expression of the The N-terminal Myosin-like Domain Is Required for Co-localization
with Microtubules--
Analysis of deletion mutants indicated that the
myosin-like domain and the GAP activity of MgcRacGAP were both required
for completion of cytokinesis. Next, to identify the molecular
mechanisms by which MgcRacGAP Associates with
To confirm the association between MgcRacGAP and The N-terminal Myosin-like Domain of MgcRacGAP Is Required for Its
Binding to Tubulins--
To determine which domain of MgcRacGAP is
required for its association with tubulins, we transiently transfected
deletion mutants of MgcRacGAP that were Flag-tagged (Fig.
4a) into 293T cells. Two days after transfection, the cell
lysates were prepared and expression of each mutant was examined with
the anti-Flag antibody (Fig.
7a, upper panel).
Immunoprecipitation was performed with the monoclonal antibody against
In this study, we have shown that MgcRacGAP plays critical roles
in cytokinesis, which is mediated by its localization to the central
spindle and the midbody through binding to microtubules in the late M
phase. Originally, we cloned the cDNA for MgcRacGAP through
functional expression cloning which inhibited IL-6 induced macrophage
differentiation of M1 cells, when expressed in the antisense
configuration (18). On the other hand, overexpression of MgcRacGAP
retarded cell growth and induced a significant number of multinucleated
cells in all cell lines tested including HL-60, M1, Ba/F3, Jurkat, and
HeLa cells (Fig. 1 and data not shown). These data implicated that
MgcRacGAP has some roles in cytokinesis. In addition expression levels
of the mRNA for MgcRacGAP increased by 3-4-fold in the
G2/M phase (Fig. 2). These results are consistent with a
previous study by Wooltorton et al. (29). They showed that
expression of MgcRacGAP in 3T3-L1 cells and
C2C12 cells were growth-regulated. We thus
assumed from these findings that MgcRacGAP controls cell proliferation
through regulating the progression of the G2/M phase.
However, how MgcRacGAP retarded cell proliferation remains to be determined.
Immunohistochemical studies revealed that in interphase, MgcRacGAP
localized in the nucleus and in the cytoplasm along with microtubules,
then redistributed to the central spindle in anaphase and to the
midbody in late telophase (Fig. 3b). This distribution implied the possibility that MgcRacGAP interact with microtubules. In
fact, we found by co-immunoprecipitation experiments that MgcRacGAP was
associated with It was previously reported that tubulin mediates nuclear translocation
of the glucocorticoid receptor and the vitamin D receptor (31, 32). It
was also proposed that c-Myc translocates from the nucleus to the
cytoplasm along with tubulin (33). It is therefore possible that
tubulin binding allows MgcRacGAP to use the microtubule network for its
translocation in the mitotic phase. Taken together MgcRacGAP apparently
plays important roles in cytokinesis through interaction with and
movement on microtubules. Tubulin and microtubules have been shown to
interact with regulatory components of the cell cycle apparatus as well
as cellular oncogene products. For example, tubulin was shown to
associate with neurofibromin to inhibit the Ras GAP activity (34). In
an analogy, the association of tubulin with MgcRacGAP may regulate the
GAP activity for Rac and Cdc42 in the mitotic structure and then
control the cytokinesis.
The association of MgcRacGAP with During the preparation of this manuscript, Jantsch-Plunger et
al. (38) published an article which contains a genetic,
biochemical, and cell biological analysis of the CYK-4 protein from
Caenorhabditis elegans. Many features of MgcRacGAP that we
present here using the mammalian system are similar to those reported
by these authors in the nematode system. CYK-4 localizes to the central
spindle and persists at cell division remnants. They have also shown
that central spindle assembly was defective in the cyk-4
(t1689ts) allele that carries a mutation at the N terminus. This is
probably because the N-terminal myosin-like domain is required for its correct localization and association with microtubules and is indispensable for the cytokinesis in an analogy with MgcRacGAP shown in
the present paper. Jantsch-Plunger et al. speculated that RhoA is the target of CYK-4 GAP activity for cytokinesis. In fact,
RhoA was reported to be essential for cytokinesis and to be localized
to the midbody (10, 39-41). CYK-4 activates GTP hydrolysis not only by
Rac1 and Cdc42 but also by RhoA, albeit to a weaker extent with a much
slower time course. Toure et al. (17) also showed that
MgcRacGAP had 30 times less GAP activity on Rho compared with Rac and
Cdc42. However, in contrast to these reports, we did not detect any GAP
activity of MgcRacGAP on RhoA (18). Thus, although our data showed that
the GAP activity of MgcRacGAP is required for cytokinesis (Fig.
5b), it is not clear at present which small G protein is a
target of MgcRacGAP in the central spindle and the midbody. While in
lower eukaryotes, a human homologue of Rac has been shown to be
involved in cytokinesis (6, 42). However, there is no report that
implicated Rac in cytokinesis in mammals. Concerning Cdc42, expression
of a constitutively active Cdc42 in a Xenopus egg and a
human cell line induced formation of multinucleated cells (5, 41).
These data indicated that Cdc42 is involved in cytokinesis. However,
there is no data showing that Cdc42 localizes to the mitotic spindle or
the midbody in mammals. Although further studies will be required to
define the target of MgcRacGAP in the mitotic spindle, one possible
explanation is that there is an unknown GTPase, which is involved in
cytokinesis and localized to the central spindle and the midbody. It is
also possible that MgcRacGAP changes its specificity toward Rho with some modification when it localizes to the central spindle and the
midbody. In fact, we have some preliminary data suggesting that
MgcRacGAP is phosphorylated in the midbody. Recently various midbody-localized protein kinases have been isolated in vertebrate cells, including AIM-1 (43), AUR 1 (44), citron kinase (10), ERK/MKK
(45), and Plk (46). Some of these kinases may function as regulators of
MgcRacGAP localization and/or GAP activities, and these possibilities
are now under investigation.
In summary, we have shown that MgcRacGAP associates with microtubules
and plays important roles in cytokinesis. Recently there have been
several notable developments in the field of cytokinesis. However, a
molecular understanding of cytokinesis still remains elusive. We
believe that further work on MgcRacGAP will provide some new insights
into cytokinesis. But, how overexpression of MgcRacGAP induced
macrophage differentiation of HL-60 cells, and how antisense MgcRacGAP
inhibited IL-6-induced differentiation of M1 cells is still open to question.
-,
-, and
-tubulins through
its N-terminal myosin-like domain. These results indicate that
MgcRacGAP dynamically moves during cell cycle progression probably
through binding to tubulins and plays critical roles in cytokinesis.
Furthermore, using a GAP-inactive mutant, we have shown that the GAP
activity of MgcRacGAP is required for cytokinesis, suggesting that
inactivation of the Rho family of GTPases may be required for normal
progression of cytokinesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin (Sigma clone No. DM 1A),
mouse anti-Flag (Sigma clone No. M2), and rabbit anti-Flag (Zymed Laboratories Inc.) were diluted in PBS
containing 3% bovine serum albumin, placed as a drop on the
coverslips, and incubated for 1 h at 37 °C. The coverslips were
washed six times with PBS and covered with a solution containing
fluorescein isothiocyanate-conjugated goat anti-rabbit Ig (Wako),
rhodamine-conjugated goat anti-mouse Ig (Sigma), and DAPI
(4',6-diamino-2-phenylindole) at 1 µg/ml for 30 min in the dark at
37 °C. The coverslips were then washed six times with PBS. After the
final wash, the coverslips were mounted with glycerin containing
para-phenylenediamine at 10 mg/ml and viewed with a
fluorescence microscope IX70 (Olympus) equipped with SenSys/OL cold CCD
camera (Olympus) and IP-Lab software (Signal Analytics Co.).
Myo-MgcRacGAP) was generated by PCR with a 5' primer that contained
an EcoRI site (5'-GAAAGAATTCCGAGAGATGCTCATGTGTGA-3'), and
the 3' primer that is located downstream of the BstXI site in MgcRacGAP (5'-TTCACCAACAGCTTGGTACAT-3'). The resulting PCR fragment
was digested with EcoRI and BstXI, and subcloned
into pME-MgcRacGAP (pME-
Myo-MgcRacGAP). A mutant MgcRacGAP lacking the cysteine-rich domain (pME-
Cys-MgcRacGAP) was generated by overlapping extension, using PCR. GTPase activating activity defective mutant (R386A*-MgcRacGAP) was generated by overlapping extension PCR
mutagenesis. PCR was carried out using a high fidelity DNA polymerase
Pyrobest (Takara). The PCR amplified sequence was confirmed by an
automated sequencing using an ABI PRISM 310 Genetic analyzer (PerkinElmer Life Sciences).
) were
grown, incubated with
isopropyl-1-thio-
-D-galactopyranoside (1 mM), lysed, and the GST fusion proteins were purified
according to the protocol of the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of MgcRacGAP-induced
multinucleated cells. Ba/F3 and HL-60 cells were transduced
with a retrovirus vector pMX-MgcRacGAP-IRES-EGFP. Ba/F3 and HL-60 cells
were centrifuged onto glass slides and stained with May-Grunwald Giemsa
solution.
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Fig. 2.
Cell cycle-dependent changes in
the expression levels of the mRNA for MgcRacGAP. Exponentially
growing HeLa cells were synchronized at the G1/S transition
by double thymidine block. Cells were harvested at indicated times for
fluorescence-activated cell sorter analysis (FACS) and RNA isolation.
a, DNA contents of synchronized HeLa cells. FACS analysis
was performed on HeLa cells harvested at 0, 1.5, 3, 6, 9, and 12 h
after release. b, Northern blot of synchronized HeLa cells
probed with a 32P-labeled MgcRacGAP cDNA. Equal amounts
of total RNA (20 µg) were loaded in each lane and probed with a
32P-labeled MgcRacGAP cDNA. Reprobing was carried out
using 32P-labeled human glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) cDNA as a loading control. The
mRNA levels were quantified by radioactivity using a Fujix BAS2000
bioimage analyzer (Fuji Photo Film Co., Tokyo, Japan). Signal
intensities of MgcRacGAP were normalized by the
glyceraldehyde-3-phosphate dehydrogenase mRNA level, and were shown
in the lower column.
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Fig. 3.
MgcRacGAP localizes to mitotic
structures. a, specificity of the affinity-purified
anti-MgcRacGAP antibody. Total cell lysates of HeLa cells (lane
1) and purified GST-MgcRacGAP C terminus fusion protein
(lane 2) were separated by SDS-polyacrylamide gel
electrophoresis, blotted onto a nitrocellulose membrane, and incubated
with anti-MgcRacGAP antibody. b, MgcRacGAP localizes to the
central spindle and the midzone in cytokinesis. HeLa cells at various
stages of mitosis were stained with the anti-MgcRacGAP polyclonal
antibody (left column), anti- -tubulin mAb (middle
column), and 4,6-diamidine-2-phenylindole (DAPI) for
DNA staining (right column); A, interphase;
B, prometaphase; C, metaphase; D,
anaphase; E, telophase; F, cytokinesis.
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Fig. 4.
Localization of the wild type and each mutant
form of MgcRacGAP in HeLa cell. HeLa cells were transfected with
cDNA encoding Flag-tagged wild type and mutant forms of MgcRacGAP.
After 48 h, cells were stained with anti- -tubulin monoclonal
antibody (red) and rabbit anti-Flag polyclonal antibody
(green).
Myo-MgcRacGAP) or a GAP Activity-defective Mutant
R386A*MgcRacGAP Resulted in Formation of Multinucleated
Cells--
MgcRacGAP contains three conserved domains, including an
N-terminal myosin-like coiled-coil domain, a cysteine-rich domain, and
a C-terminal GAP-conserved domain. To determine which domain of
MgcRacGAP was important for the control of cytokinesis, we constructed
three mutants of MgcRacGAP;
Myo-MgcRacGAP lacking the myosin-like
domain,
Cys-MgcRacGAP lacking the cysteine-rich domain, and
R386A*MgcRacGAP with a GAP-inactivating mutation (Fig. 5a). All of these mutants were
Flag-tagged at the C terminus, and were introduced into a mammalian
expression vector pME 18S (23). Expression of these mutant proteins was
confirmed by transient expression in 293T cells, through lipofection.
These mutant proteins as well as the wild type protein were expressed
at similar levels in 293T cells (Fig. 5b).
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Fig. 5.
Effects of expression of wild type and mutant
MgcRacGAP on cytokinesis. a, the structures of the wild
type, mutant R386A*MgcRacGAP and deletion mutants of MgcRacGAP.
b, expression of the wild type and the mutant forms of
MgcRacGAP. The 293T cells were transiently transfected with Flag-tagged
MgcRacGAP constructs as indicated. Forty-eight hours after
transfection, cells were collected and lysed by TCSD buffer. Total cell
lysates were subjected to immunoblotting with a rabbit anti-Flag
polyclonal antibody. c, expression of Myo-MgcRacGAP and
R386A*MgcRacGAP induced abortive cytokinesis. MgcRacGAP (wild type),
Myo-MgcRacGAP,
Cys-MgcRacGAP, R386A*MgcRacGAP, or pME18S as a
vector control was co-transfected with a reporter construct pME EGFP
into HeLa cells. EGFP-expressing cells were visualized by green
fluorescence (upper panels). DNA was stained with DAPI
(middle panels). GFP expressing multinucleated cells were
counted under an immunofluorescence microscopy 72 h after
transfection. Data represent average of three independent experiments.
DAPI, 4,6-diamidine-2-phenylindole. d, formation
of multinucleated cells by overexpression of wild type and mutant
McgRacGAPs.
Myo-MgcRacGAP and
R386A*MgcRacGAP produced multinucleated cells more frequently (32 and
28% of the EGFP positive cells, respectively) than that of the wild
type and
Cys-MgcRacGAP did (10.5 and 8.5%, respectively) (Fig.
5b). These data demonstrated that the GAP activity and the
N-terminal myosin-like domain of MgcRacGAP were required for
cytokinesis. It has to be noticed that this procedure may increase the
frequency of the cells with lower expression levels of the transduced
wild type or mutant MgcRacGAP, which may lead to underestimation of the
frequencies of multinucleated cells after 72 h.
Myo-MgcRacGAP and R386A*MgcRacGAP hampered
cytokinesis, we expressed each mutant of MgcRacGAP in HeLa cells and
immunostained using the anti-Flag antibody. Both mutant proteins
entered the nucleus during interphase, as did the wild type of
MgcRacGAP. However, unlike other mutants in cytokinesis,
Myo-MgcRacGAP was not detected in the midbody of dividing cells
(Fig. 4). These results demonstrated that the N-terminal myosin-like
coiled-coil domain was indispensable for localization to the midbody
during cytokinesis, and also indicated that completion of cytokinesis requires the GAP activity of MgcRacGAP.
-,
-, and
-Tubulin--
Immunohistochemical studies suggested that MgcRacGAP
associates with microtubules and plays important roles in cytokinesis. To determine whether MgcRacGAP associates with microtubules in vivo, we performed co-immunoprecipitation using cell lysates from HeLa cells with the anti-MgcRacGAP antibodies followed by Western blotting with monoclonal antibodies against
-,
-, and
-tubulins. As shown in Fig.
6a, all tubulin isoforms were
co-precipitated with MgcRacGAP from the cell lysates, indicating that
MgcRacGAP associates with microtubules in vivo (Fig.
6a).
View larger version (49K):
[in a new window]
Fig. 6.
Association of MgcRacGAP with
-,
-, and
-tubulins in vivo. a,
co-immunoprecipitation (IP) of tubulins with MgcRacGAP. Cell
extracts of interphase and mitotic HeLa cells synchronized with double
thymidine block were subjected to immunoprecipitation with an
anti-MgcRacGAP antibody. The immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. The membrane was probed by monoclonal
antibodies against
-,
-, and
-tubulins. Reprobing was carried
out by antibodies against MgcRacGAP. b,
coimmunoprecipitation of MgcRacGAP with tubulins. Immunoprecipitations
were performed with anti-
-,
-, and
-tubulin mAbs and
precipitates were separated by SDS-polyacrylamide gel electrophoresis,
followed by blotting with an anti-MgcRacGAP antibody.
-,
-, and
-tubulins, reciprocal immunoprecipitations were also performed. MgcRacGAP was detected in the precipitates of
-,
-, and
-tubulins (Fig. 6b). In these experiments,
immunoprecipitation experiments were carried out with a lysis buffer
containing detergent and high concentrations of salt as described under
"Experimental Procedures" to avoid nonspecific binding between
MgcRacGAP and tubulins as much as possible.
-tubulin. The wild type MgcRacGAP,
Cys-MgcRacGAP, and
R386A*MgcRacGAP were detected in
-tubulin immunoprecipitates,
whereas no
Myo-MgcRacGAP mutant was detected (Fig. 7a, lower
panel), demonstrating that MgcRacGAP binds tubulin through its
myosin-like domain. Duplicate immunoprecipitation was performed with
antibodies against
- and
-tubulin, with essentially the same
results (data not shown). To further confirm this result, GST fusion
proteins containing each domain of MgcRacGAP were incubated with HeLa
cell lysates, and the precipitates were analyzed for the presence of
-,
-, and
-tubulins. As shown in Fig. 7b, all tubulin isoforms were precipitated by GST-Myo (myosin-like domain), but
not by the other domain constructs. These results indicated that the
N-terminal myosin-like domain mediates the association of MgcRacGAP and
tubulin.
View larger version (21K):
[in a new window]
Fig. 7.
N-terminal myosin-like domain mediates the
association with microtubules. a, N-terminal
myosin-like domain was required for its binding to tubulins. 293T cells
were transiently transfected with Flag-tagged MgcRacGAP constructs as
indicated. Forty-eight hours after transfection, cells were collected
and lysed by TCSD buffer. Total cell lysates (upper panel)
or immunoprecipitates with anti- -tubulin mAb from transfected cells
(lower panel) were subjected to immunoblotting with a rabbit
anti-Flag polyclonal antibody. b, GST pull-down assay of
distinct domains of MgcRacGAP. The GST fusion proteins containing each
of three distinct domains of MgcRacGAP were incubated for 4 h at
4 °C with HeLa cell lysates and the precipitated proteins were
separated by SDS-polyacrylamide gel electrophoresis followed by
transfer to a nitrocellulose membrane. The membrane was probed by
-,
-, and
-tubulin monoclonal antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-tubulin. Immunostaining showed that the
N-terminal myosin-like domain of MgcRacGAP was required for its
localization to the midbody of dividing cells (Fig. 5).
Immunoprecipitation studies revealed that the myosin-like domain is
responsible for its association with tubulin (Fig. 7). The myosin-like
domain of MgcRacGAP contains a coiled-coil structure (30), and tubulin also does. Therefore, it is predicted that interaction of these two
coiled-coil domains mediates binding of MgcRacGAP to tubulins. Moreover, the overexpression of
Myo-MgcRacGAP led to failure of
cytokinesis, which was demonstrated by a markedly increased number of
multinucleated cells. However, transfection of the wild type MgcRacGAP
also causes abortive cytokinesis albeit at a low level. We assume from
these data that expression of this protein at physiological levels
and/or changes of the expression level through these cell cycles are
important for normal progression of cytokinesis.
- and
-tubulins prompted us to
examine whether MgcRacGAP also binds to
-tubulin, and we found that
MgcRacGAP associated with
-tubulin as well.
-Tubulin has been
identified in the centrosomes (35) and the midbody (36) during the
mitotic phase. Observation with a confocal laser scanning microscope
and in situ extraction immunostaining revealed that
MgcRacGAP also localized to centrosomes in the mitotic
phase.2 Disruption of
-tubulin during anaphase by injection of either antibodies or
antisense RNA led to failure of midbody formation and consequently a
failure of cytokinesis (36, 37), indicating that the nucleation of new
microtubules may be important for the formation or maintenance of the
midbody. Whether or not MgcRacGAP has a role in the microtubule
nucleation or midbody formation and/or maintenance remains to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank A. Kaneko for excellent sorting on FACS and M. Ohara for providing language assistance.
![]() |
FOOTNOTES |
---|
* This work was also supported by grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare of Japan. The Department of Hematopoietic factors was supported by Chugai Pharmaceutical Company Ltd.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. Tel.: 81-3-5449-5758; Fax: 81-3-5449-5453; E-mail: kitamura@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M007252200
2 K. Hirose, T. Kawashima, I. Iwamoto, T. Nosaka, and T. Kitamura, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein; IL-6, interleukinn-6; PBS, phosphate-buffered saline: FCS, fetal calf serum; GST, glutathione S-transferase; PCR, polymerase chain reaction; IRES, internal ribosomal entry site; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; EGFP, enhanced green fluorescence protein.
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