MgcRacGAP Is Involved in Cytokinesis through Associating with Mitotic Spindle and Midbody*

Koichi HiroseDagger §, Toshiyuki KawashimaDagger , Itsuo Iwamoto§, Tetsuya NosakaDagger , and Toshio KitamuraDagger

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -, beta -, and gamma -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

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.).

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 (Delta 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-Delta Myo-MgcRacGAP). A mutant MgcRacGAP lacking the cysteine-rich domain (pME-Delta 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).

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 (DH5alpha ) were grown, incubated with isopropyl-1-thio-beta -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

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).



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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.

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.



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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.

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).



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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-alpha -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.

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).



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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-alpha -tubulin monoclonal antibody (red) and rabbit anti-Flag polyclonal antibody (green).

Expression of a Myosin-like Domain-defective Mutant (Delta 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; Delta Myo-MgcRacGAP lacking the myosin-like domain, Delta 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 Delta Myo-MgcRacGAP and R386A*MgcRacGAP induced abortive cytokinesis. MgcRacGAP (wild type), Delta Myo-MgcRacGAP, Delta 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.

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 Delta 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 Delta 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.

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 Delta 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, Delta 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.

MgcRacGAP Associates with alpha -, beta -, and gamma -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 alpha -, beta -, and gamma -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).



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Fig. 6.   Association of MgcRacGAP with alpha -, beta -, and gamma -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 alpha -, beta -, and gamma -tubulins. Reprobing was carried out by antibodies against MgcRacGAP. b, coimmunoprecipitation of MgcRacGAP with tubulins. Immunoprecipitations were performed with anti-alpha -, beta -, and gamma -tubulin mAbs and precipitates were separated by SDS-polyacrylamide gel electrophoresis, followed by blotting with an anti-MgcRacGAP antibody.

To confirm the association between MgcRacGAP and alpha -, beta -, and gamma -tubulins, reciprocal immunoprecipitations were also performed. MgcRacGAP was detected in the precipitates of alpha -, beta -, and gamma -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.

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 alpha -tubulin. The wild type MgcRacGAP, Delta Cys-MgcRacGAP, and R386A*MgcRacGAP were detected in alpha -tubulin immunoprecipitates, whereas no Delta 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 beta - and gamma -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 alpha -, beta -, and gamma -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.



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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-alpha -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 alpha -, beta -, and gamma -tubulin monoclonal antibodies.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha - and beta -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 Delta 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.

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 alpha - and beta -tubulins prompted us to examine whether MgcRacGAP also binds to gamma -tubulin, and we found that MgcRacGAP associated with gamma -tubulin as well. gamma -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 gamma -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.

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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