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HEMATOPOIESIS
From Department of Hematopoietic Factors and Department
of Clinical Oncology, The Institute of Medical Science, University of
Tokyo, Minato-ku, Tokyo, Japan; Faculty of Bioscience and
Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; and
Division of Hematology, Department of Internal Medicine, School of
Medicine, Keio University, Tokyo, Japan.
In a search for key molecules that prevent murine M1 leukemia cells
from undergoing interleukin (IL)-6-induced differentiation into
macrophages, we isolated an antisense complementary DNA (cDNA) that
encodes full-length mouse MgcRac-GTPase-activating protein (GAP)
through functional cloning. Forced expression of this antisense cDNA
profoundly inhibited IL-6-induced differentiation of M1 cells into
macrophage lineages. We also isolated a full-length human MgcRacGAP
cDNA, which encodes an additional N-terminal polypeptide of 105 amino
acid residues compared with the previously published human MgcRacGAP.
In human HL-60 leukemic cells, overexpression of the full-length form
of human MgcRacGAP alone induced growth suppression and macrophage
differentiation associated with hypervacuolization and de novo
expression of the myelomonocytic marker CD14. Analyses using a
GAP-inactive mutant and 2 deletion mutants of MgcRacGAP indicated that
the GAP activity was dispensable, but the myosin-like domain and the
cysteine-rich domain were indispensable for growth suppression
and macrophage differentiation. The present results indicated that
MgcRacGAP plays key roles in controlling growth and differentiation of
hematopoietic cells through mechanisms other than regulating Rac GTPase activity.
(Blood. 2000;96:2116-2124) Rho-related guanosine triphosphate (GTP)-binding
proteins constitute a functionally distinct group within the small G
protein family, which includes Rho A, B, C, and G, Rac1 and Rac2,
Cdc42, and TC10.1 These proteins are 30% identical to Ras
and 50% to 60% identical to each other at the amino acid level. Rho
family members have been linked to a variety of cellular functions,
including changes in cytoskeletal dynamics (actin polymerization and
reorganization), gene expression (p38/Jun NH2-terminal
kinase and serum response factor activity), G1 cell cycle progression,
endocytosis, exocytosis, and superoxide production.2-5
GTPase-activating proteins (GAPs) for Rho GTPases constitute a class of
regulatory proteins that can bind GTP-bound active forms of small G
proteins and stimulate GTP hydrolysis.6,7 Harboring this
catalytic function, RhoGAPs negatively regulate Rho-mediated signals.
However, certain GAPs, including p120RasGAP, n-chimaerin,
and phospholipase C (a GAP for heterotrimeric G proteins)
simultaneously function as effectors downstream of the
GTPases.8-10 More specifically, n-chimaerin was
shown to cooperate with Rac1 and Cdc42 in inducing specific changes in
cytoskeletal morphology, ie, the formation of lamellipodia and
filopodia, respectively.8 This effect of
n-chimaerin requires G protein binding capacity and the
non-GAP N-terminal extension but not GAP activity.8 A
conventional myosin, myosin-IXb, harboring a chimaerin-like Rho/Rac GAP
domain in its tail colocalizes with F-actin in the cell periphery in
undifferentiated human HL-60 leukemia cells, while in differentiated
cells the localization becomes more cytoplasmic, with the highest
levels being seen in the perinuclear region.11
Regulation of the cytoskeleton by Rho family members seems to depend on
a cascade of events within the acto-myosin system.8 Thus,
the view is favored that RhoGAPs control a variety of cellular
functions through Rho family proteins as well as other
signaling molecules.
We report here functional cloning of murine MgcRacGAP (mMgcRacGAP) as a
differentiation-regulating gene of murine M1 leukemia cells.
Overexpression of the full-length human MgcRacGAP (hMgcRacGAP) suppressed cell growth and induced macrophage differentiation of HL-60
cells, indicating that MgcRacGAP is involved in the control of growth
and differentiation of hematopoietic cells.
Cell lines
Retrovirus vectors
Production of retroviruses and infection with them High-titer retroviruses carrying the hMgcRacGAP-IRES-EGFP were produced with a transient retrovirus packaging cell line BOSC23 12 as described previously.13 We first established a stable transfectant expressing the ecotropic virus receptor.14,15 Infection was performed as described.16 Briefly, cells were incubated with 10 mL of the retroviruses in the presence of 10-µg/mL hexadimethrine bromide (Sigma, St Louis, MO). Twenty-four hours after infection, cells were washed, refed with growth medium, and left for one more day before cell sorting with EGFP.Cell sorting and flow cytometry EGFP+ cells were sorted using a modification of the technique described earlier.17 Briefly, 2 days after virus infection, the infected cells were washed twice with phosphate buffered saline (PBS), suspended in PBS containing 1% bovine serum albumin (BSA), and then sorted based on EGFP expression on a FACSVantage (Becton Dickinson, San Jose, CA). The sorted cells (40 per well) were then expanded in growth medium. Flow cytometric analysis was carried out to quantify morphologic changes and to confirm the expression of EGFP in the HL-60 transfectants on a FACSCalibur flow cytometer (Becton Dickinson). The cells were also stained with a phycoerythrin (PE)-conjugated mouse antihuman CD14 monoclonal antibody (PharMingen, San Diego, CA) on ice for 30 minutes after blocking with 100-fold excess of mouse immunoglobulin (Ig) G, and analyzed on a FACSCalibur flow cytometer. PE-conjugated mouse IgG2a was used as an isotype-matched negative control.Immunoprecipitation and Western blotting Immunoprecipitation, gel electrophoresis, and immunoblotting were performed as described18 but with minor modifications. Exponentially growing cells were washed with PBS, lysed in lysis buffer (50-mmol/L Tris-HCl, pH 7.5; 150-mmol/L NaCl; 1% Triton X-100; 1-mmol/L ethylenediaminetetraacetic acid [EDTA]; 0.2-mmol/L Na3VO4; 2-mmol/L phenylmethylsulfonyl fluoride; 2-µg/mL leupeptin; 10-µg/mL aprotinin) (5 × 106 cells/mL), and incubated on ice for 30 minutes. Cell lysates were clarified by centrifugation for 15 minutes at 12 000g prior to incubation with the anti-Flag M2 monoclonal antibody (Eastman Kodak, Kingsport, TN) or the control whole-mouse IgG and protein A-Sepharose at 4°C overnight. The immunoprecipitates were washed 3 times with lysis buffer, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and electrophoretically transferred onto Immobilon filters (Millipore, Bedford, MA). After blocking in a solution containing 3% BSA, the filter was probed with the anti-Flag M2 monoclonal antibody. The filter-bound antibody was detected using the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).Northern blot analysis Poly(A)+ RNA was isolated from cells using FastTrack 2.0 kit (Invitrogen, San Diego, CA). Two micrograms of Poly(A)+ RNA was denatured in 50% formamide at 60°C, electrophoresed on a 1% agarose formaldehyde gel, and blotted to Hybond-N nylon filter (Amersham). The filter was incubated at 42°C in 50% formamide, 3 × Denhardt's reagent (0.06% polyvinylpyrrolidone, 0.06% BSA, 0.06% Ficoll), 5 × standard saline citrate (SSC; 1 × SSC = 0.15-mol/L NaCl, 0.015-mol/L trisodium citrate), 1% SDS, 200-µg/mL denatured salmon sperm DNA, and a 32P-labeled complementary DNA (cDNA) probe, prepared using Random Prime Kit (Stratagene, La Jolla, CA). After hybridization, the filter was washed in 0.1 × SSC, 0.1% SDS at room temperature, and autoradiographed. Reprobing was carried out after washing the filter in a stripping buffer of 10-mmol/L Tris-HCl (pH 7.5), 1-mmol/L EDTA, 0.1% SDS, and 0.3 × Denhardt's reagent at 90°C for 20 minutes.GTP hydrolysis assays The [ -32P] GTP-bound form of small GTPases was
prepared by incubating 10 pmol of protein with 74 kBq of
[-32P] GTP (1.11 TBq/mmol, NEN Life Science Products,
Boston, MA) in a 50-µL volume of 25-mmol/L Tris-HCl (pH 7.5),
5-mmol/L EDTA, 0.2-mmol/L MgCl2, 0.1-mg/ml BSA, 1-mmol/L
dithiotreitol, and 4-µmol/L GTP for 15 minutes at room temperature.
GTP hydrolysis was initiated by raising MgCl2 and GTP to a
final concentration of 20 mmol/L and 200 µmol/L, respectively, and
was stopped after 3 minutes by adding 2 mL of ice-cold 50-mmol/L
Tris-HCl (pH 8), 35-mmol/L MgCl2, 1-mmol/L
dithiotreitol, and 150-mmol/L NaCl. Amounts of [-32P]
GTP bound to GTPases were determined by radioactivity counting after
rapid vacuum filtration of the samples on BA 85 nitrocellulose (Schleicher and Schuell, Dassel, Germany). Typically, 100% of [-32P] GTP bound to GTPases is around 25 000 cpm in
our hands. The GAP assay was conducted in the presence of the GAP
domain (20 pmol) of hMgcRacGAP or a GAP inactive mutant of hMgcRacGAP
(R385A*MgcRacGAP) during the step of GTP hydrolysis. GTP exchange
reactions during the GTP hydrolysis step were examined using
[ -32P] GTP-preloaded GTPases under similar conditions
and were not significantly affected by the presence of hMgcRacGAP.
Mutagenesis and production of retroviruses The mutation R385A was introduced by overlap-extension polymerase chain reaction (PCR) mutagenesis.19 DNA encoding the conserved arginine residue was amplified by PCR using primer pairs as follows:Upstream primer: 5'-AGCTGCGGAATTCCGGAATT-3' Mutagenic primer, sense: 5'-AGGCCTGTATGCGATCTCTGGCT-3' Downstream primer: 5'-TTCACCAACAGCTTGGTACAT-3' Mutagenic primer, antisense: 5'-AGCCAGAGATCGCATACAGGCCT-3'. PCR products were joined and amplified by the upstream primer and the
downstream primer and cloned into EcoRI-BstXI
sites of pMX-MgcRacGAP-IRES-EGFP. Mutagenic sequence was confirmed by automated sequencing by an ABI PRISM 310 Genetic Analyzer
(Perkin-Elmer, Branchburg, NJ). To construct a mutant MgcRacGAP lacking
the myosin-like domain (
Expression of an antisense DNA encoding MgcRacGAP partially blocked IL-6-induced differentiation of M1 cells To identify key molecules that regulate cell differentiation into macrophages, we generated a cDNA library in a retrovirus vector pMXneo20 from a subclone of mouse myeloid leukemia M1 cells (termed MD1 cells) expressing a constitutively active STAT5A mutant.21 This particular mutant retained the potential to proliferate while differentiating into various stages along the monocytic differentiation pathway and giving rise to a heterogeneous population of blast, intermediate, and mature monocytic cells through the production of interleukin-6 (IL-6) (T. Kawashima et al, unpublished data). We searched for a gene for this particular phenotype by retrovirus-mediated functional screening of the MD-1-derived cDNA library; M1 cells infected with the cDNA library were screened for clones resistant to IL-6-induced terminal differentiation in the medium containing IL-6 (100 ng/mL) and G418 selection reagents (600 µg/mL) as described.16,22 After screening of 1.5 × 105 independent cDNA clones, we happened to isolate an antisense cDNA encoding the complete coding sequence of mMgcRacGAP23 from an M1 clone that became
resistant to IL-6.
To determine if the expression of this antisense cDNA
(asMgcRacGAP) indeed made M1 cells resistant to
IL-6-induced macrophage differentiation, we subcloned it into a
bicistronic retrovirus vector pMX-IRES-EGFP to construct
pMX-asMgcRacGAP-IRES-EGFP, and we reintroduced it into M1
cells via retrovirus infection. As a control, pMX-IRES-EGFP was also
introduced into M1 cells. After transduction of M1 cells with these
vectors, GFP+ cells were sorted on a fluorescence-activated
cell sorter (FACS) as described in "Materials and methods" and were
cultured for 4 days in the presence or absence of 5-ng/mL IL-6. Flow
cytometric analysis was performed to quantify morphologic changes after
the culture. The increase in cell size and the granulosity of cytoplasm were evaluated based on increases in forward scatter and side scatter,
respectively, in M1 cells expressing either the antisense mMgcRacGAP or
the control vector after IL-6 treatment (Figure 1A,C). Only 15.2% of M1 cells transduced
with the antisense mMgcRacGAP showed a shift from region R1 to region
R2, a hallmark of macrophage differentiation after treatment with IL-6,
while 56.9% and 47.6% of the parental M1 cells and M1 cells
transduced with the control vector showed similar shifts, respectively.
Similarly, morphologically differentiated cells were evident in the
control M1 cells but not in the antisense mMgcRacGAP-transduced M1
cells after treatment with IL-6 (Figure 1B), indicating that expression
of antisense mMgcRacGAP profoundly inhibited the IL-6-induced
differentiation of M1 cells. On the other hand, the growth rate of M1
cells was not affected by the expression of the antisense MgcRacGAP
(data not shown).
Identification of the full-length form of hMgcRacGAP cDNA To isolate cDNA clones encoding the complete coding sequences of both murine and human MgcRacGAP, we screened libraries derived from the murine M1 cell line and the human TF-1 erythroleukemic cell line. The isolated cDNAs for 2919 base pairs of the mMgcRacGAP and 3050 base pairs of the hMgcRacGAP were then sequenced. We found that the open reading frame of the hMgcRacGAP cDNA encoded a putative protein of 632 amino acids, which is longer than the previously reported sequence of 527 amino acids by N-terminal 105 amino acids.24 The corresponding cDNA and the protein sequence of the previously reported hMgcRacGAP were not found in the FASTA/BLAST program of the National Center for Biotechnology Information or the DNA Databank of Japan/European Molecular Biology Laboratory/GenBank nucleotide sequence database. In addition, an analysis of other mRNA sources derived from Jurkat cells and phytohemagglutinin (PHA)-activated human T-cell blasts by reverse transcription PCR indicated that these cells expressed the full-length hMgcRacGAP cDNA, which was identical to the cDNA reported in the present paper (data not shown). Thus, it was shown that the hMgcRacGAP cDNA reported here is the full-length form of this RacGAP (accession No. AB030251). The surrounding sequence of the putative initiation codon ATG for the full-length form of hMgcRacGAP well matched to the Kozak consensus sequence,25,26 and a calculated molecular mass of this full-length form is 71 000 kd. Murine MgcRacGAP and the full-length form of hMgcRacGAP are 84% identical and 97% similar to each other at the amino acid level (Figure 2A). Database searches revealed the similarity between the N-terminal region of the full-length hMgcRacGAP (amino acids 41-124) and myosins, mainly tropomyosins (Figure 2B). In this paper, we call the full-length form of this molecule hMgcRacGAP.
Expression of the hMgcRacGAP mRNA Northern blot analysis revealed that a single hMgcRacGAP transcript of 3.3 kilobases (kb) was expressed in most tissues tested (Figure 3), with expression being high in thymus and placenta and low in spleen and peripheral blood leukocytes. It was noteworthy that hMgcRacGAP was highly expressed in tissues such as thymus and placenta containing immature hematopoietic cells but not in nonproliferating peripheral blood leukocytes. We also confirmed that mMgcRacGAP mRNA was expressed in murine fetal liver cells and in hematopoietic cell lines including Ba/F3 cells, CTLL-2 cells, and DA-1 cells (data not shown).
Enforced expression of hMgcRacGAP induced macrophage differentiation in the human myeloid leukemia HL-60 cells We attempted to elucidate the role of MgcRacGAP in macrophage differentiation using murine myeloid leukemia M1 cells and human myeloid leukemia HL-60 cells. The mMgcRacGAP cDNA and the Flag-tagged hMgcRacGAP cDNA were transduced into M1 cells and HL-60 cells, respectively, using the retrovirus vector pMX-IRES-EGFP. GFP+ M1 and HL-60 cells transduced with these viruses were sorted on FACS 2 days after infection. GFP+ M1 and HL-60 cells transduced with a control pMX-IRES-EGFP vector were similarly sorted and served as negative controls.Overexpression of either mMgcRacGAP or hMgcRacGAP in M1 cells induced
no significant differentiation but did moderately inhibit the growth of
M1 cells (data not shown). Overexpression of mMgcRacGAP or hMgcRacGAP
also retarded proliferation of a murine proB cell line Ba/F3. On the
other hand, enforced expression of hMgcRacGAP in HL-60 cells profoundly
suppressed growth (Figure 4A) and induced differentiation (Figure 4B-D) of the cells. Differentiation of HL-60
cells into macrophages was evaluated by FACS, expression of a monocyte
marker CD14, and the morphology. First, the increase in cell size and
granulosity of the cytoplasm were evaluated by increases in forward
scatter and side scatter, respectively. As shown in Figure 4B,
expression of hMgcRacGAP induced a shift from region R1 to region R2 in
65.8% of the transduced HL-60 cells, while expression of the control
vector pMX-IRES-EGFP did not induce a significant shift. Second,
expression of a myelomonocytic marker CD14 was induced in HL-60 cells
expressing hMgcRacGAP but not in control HL-60 cells (Figure 4C).
Morphologic changes were also noticed in HL-60 cells transduced with
hMgcRacGAP; most HL-60 cells became larger with morphologic changes of
monocytic differentiation and showed reduced nuclear/cytoplasmic ratios
and notable hypervacuolation in the cytoplasm after transduction of
hMgcRacGAP (Figure 4D). These results clearly demonstrated that
overexpression of hMgcRacGAP alone induced differentiation of HL-60
cells into macrophages.
Stable expression of hMgcRacGAP transgene in HL-60 and M1 transfectants
was confirmed by the Northern blot and the Western blot analysis
(Figure 5A,B). In Northern blot analysis
of HL-60 cells carrying the hMgcRacGAP transgene (Figure 5A, lane 3),
we detected retroviral transcripts migrating at approximately 4.7 kb
and 4.0 kb, which were presumably genomic and subgenomic forms of the
transcripts, respectively, derived from the retrovirus vector pMX
harboring splicing donor and acceptor sites as well as the endogenous
hMgcRacGAP mRNA at approximately 3.3 kb. Western blot analysis
demonstrated that M1 cells transduced with the Flag-tagged hMgcRacGAP
transgene expressed the recombinant protein of approximately 80 kd
(Figure 5B).
Expression of endogenous MgcRacGAP was down-regulated during IL-6- and TPA-induced macrophage differentiation of M1 and HL-60 cells, respectively To determine whether the expression level of endogenous MgcRacGAP was altered during macrophage differentiation, M1 and HL-60 cells were stimulated with IL-6 (50 ng/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (16 nmol/L), respectively, and at various time points we isolated poly(A)+ RNA. Intriguingly, Northern blot analysis of these samples showed that the expression of endogenous MgcRacGAP mRNA dramatically decreased along with macrophage differentiation of both M1 and HL-60 cells (Figure 6A,B).
The mutant MgcRacGAP in which the conserved arginine was replaced with an alanine exhibited no GTPase activity A conserved arginine residue found in the first homology box of GAP domains of Rho-GAP family was also conserved in MgcRacGAP (Figure 7A), and structural studies suggested that this arginine residue was a key catalytic residue.27,28 Indeed, it was reported that mutation (or deletion) of this conserved arginine residue disrupted GAP activities of n-chimaerin and Cdc42-GAP without affecting their binding activities to GTP-bound Rho GTPase.29,30 Therefore, to generate a GAP-inactive mutant of hMgcRacGAP, we replaced the conserved arginine (Arg385) of hMgcRacGAP with an alanine (R385A*MgcRacGAP). We expressed the mutant GAP domain of R385A*MgcRacGAP as GST fusion products in vitro and confirmed that the GAP activity of hMgcRacGAP toward Rac1 and Cdc42 was inactivated as a result of this mutation (Figure 7B).
GAP activity was dispensable, but the myosin-like domain and the cysteine-rich domain were indispensable for hMgcRacGAP-induced growth suppression and differentiation To determine if the GAP activity of hMgcRacGAP is crucial and which domain of this RacGAP is important for the growth suppression and macrophage differentiation in HL-60 cells, we used the GAP activity-negative mutant (R385A*MgcRacGAP) and 2 deletion mutants, lacking the N-terminal myosin-like domain or the cysteine-rich domain ( Myo-MgcRacGAP or Cys-MgcRacGAP, respectively) (Figure 8).
Each mutant was introduced into HL-60 cells via retrovirus
infection using the retrovirus vector pMX-IRES-EGFP. GFP+
cells with comparable fluorescence intensities were then sorted. As
shown in Figure 9A, growth of HL-60 cells
was suppressed after transduction of the GAP activity-negative mutant
of hMgcRacGAP to an extent similar to that seen with HL-60 cells
transduced with the wild-type hMgcRacGAP, indicating that the GAP
activity of hMgcRacGAP is dispensable for the hMgcRacGAP-induced growth suppression of HL-60 cells. On the other hand,
To determine effects of these mutations in inducing macrophage
differentiation, we performed May-Grunwald-Giemsa staining of cytospin
preparations of the sorted cells (Figure 9C). Large cells, which
differentiated along the monocytic differentiation pathway with
distinctive hypervacuolation in the cytoplasm, were observed in HL-60
cells transduced with R385A*MgcRacGAP as well as those transduced with
the wild-type hMgcRacGAP, thereby demonstrating that the GAP activity
of hMgcRacGAP was not required for its activity to induce macrophage
differentiation of HL-60 cells. However, well-differentiated cells were
not observed in the population of HL-60 cells expressing
In this paper, we functionally cloned an antisense cDNA for mMgcRacGAP, which profoundly inhibited IL-6-induced differentiation of M1 cells. Although overexpression of sense cDNA for MgcRacGAP induced no detectable differentiation of M1 cells, it induced macrophage differentiation of human acute leukemic HL-60 cells. Moreover, overexpression of MgcRacGAP led to growth suppression of both M1 and HL-60 cells. These results indicated that MgcRacGAP was involved in control of growth and differentiation in both M1 and HL-60 cells. We also found that the GAP activity of hMgcRacGAP was dispensable for hMgcRacGAP-induced growth suppression and differentiation into macrophages. Rac/Cdc42 small G proteins were implicated in cytoskeletal
organization; membrane ruffling; production of superoxide,
phagocytosis, and chemotaxis; as well as regulation of cell
cycle.2,31-34 Rac/Cdc42 GAPs include Bcr, n-,
Interestingly, this potential of hMgcRacGAP to induce differentiation does not require its GAP activity but does require the N-terminal myosin-like domain and the cysteine-rich domain, as demonstrated by the experiments using a series of mutants. This result is reminiscent of that reported for n-chimaerin, which cooperates with Rac1 and Cdc42 in stimulating the formation of lamellipodia and filopodia, respectively; these functions of n-chimaerin require the G protein binding capacity and the non-GAP N-terminal extension but not GAP activity.8 Together, it is suggested that MgcRacGAP not only negatively regulates Rac-mediated signals through their catalytic functions, which stimulate GTP hydrolysis after binding to activated (or GTP-bound) forms of Rac GTPases, but also functions as downstream effectors of Rac proteins as a Rac-binding protein. The myosin-like domain of myosin-IXb and the cysteine-rich domain of n-chimaerin are required for interaction with actin filaments and phospholipids, respectively.11,35 Taken together, it seems likely that MgcRacGAP suppresses growth and differentiation through its multiple domains, which would interact with multiple signaling pathways but not through its GAP activity. An N-terminus-truncated molecule of the hMgcRacGAP was previously
isolated as a Rac-binding protein in a 2-hybrid
experiment.24 Human MgcRacGAP was reported to be highly
expressed in male germ cell and was implicated in spermatogenesis.
However, the reported sequence corresponded to the deletion mutant
Another group has recently cloned a cDNA for mMgcRacGAP under the name of band25.36 This was cloned using the differential display techniques as a cDNA whose expression well correlated with cell growth. In addition, it was shown that expression of the band25 decreased along with terminal differentiation into myocytes of a murine myogenic cell line C2C12. Thus, the expression level of MgcRacGAP was apparently parallel to the rate of cell proliferation. In a similar context, we found that the expression level of hMgcRacGAP was high in thymus and placenta, which contained a number of proliferating cells, but was extremely low in peripheral blood leukocytes, most of which were terminally differentiated and lost proliferative activities. In addition, expression of endogenous MgcRacGAP dramatically decreased in HL-60 and M1 cells when induced to terminally differentiate into macrophages by TPA and IL-6, respectively (Figure 6), again is correlated with cell proliferation. Then, the important question is why overexpression of MgcRacGAP
suppressed cell growth and induced differentiation of HL-60 cells.
Molecular mechanisms for this phenomenon remain to be clarified, but
there are 2 possibilities. The first possibility is that MgcRacGAP is a
protein primarily involved in cell proliferation and that its
overexpression negatively regulates or disrupts the normal control of
cell growth, thereby inducing differentiation of the cells as a
secondary event. In fact, our results as well as those of others
indicated that MgcRacGAP was rather associated with proliferation. This
hypothesis also explains why expression of endogenous MgcRacGAP became
undetectable along with terminal differentiation of M1 or HL-60 cells.
What, then, is the role of MgcRacGAP in cell proliferation? Of
particular interest, expression of a deletion mutant Finally, the fact that the expression of MgcRacGAP is associated with proliferation and inversely correlates with differentiation in 3 different cell types, including hematopoietic cells, preadipocytes, and myogenic cells, suggests a rather common mechanism by which MgcRacGAP controls cellular proliferation and differentiation.
We thank Dr Tatsutoshi Nakahata and Dr Tsuneo A. Takahashi for the FACS machines and Mariko Ohara and Dr Masato Nakafuku for critical reading of the manuscript.
Submitted December 23, 1999; accepted May 25, 2000.
Supported by the Chugai Pharmaceutical Company Ltd and grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Ministry of Health and Welfare of Japan.
The nucleotide sequence data for the human MgcRacGAP cDNA reported here will appear in the DNA Databank of Japan/European Molecular Biology Laboratory/GenBank nucleotide sequence database with accession No. AB030251.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Toshio Kitamura, Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: kitamura{at}ims.u-tokyo.ac.jp.
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© 2000 by The American Society of Hematology.
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  T. Kawashima, Y. C. Bao, Y. Minoshima, Y. Nomura, T. Hatori, T. Hori, T. Fukagawa, T. Fukada, N. Takahashi, T. Nosaka, et al. A Rac GTPase-Activating Protein, MgcRacGAP, Is a Nuclear Localizing Signal-Containing Nuclear Chaperone in the Activation of STAT Transcription Factors Mol. Cell. Biol., April 1, 2009; 29(7): 1796 - 1813. [Abstract] [Full Text] [PDF]
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 J. C. Canman, L. Lewellyn, K. Laband, S. J. Smerdon, A. Desai, B. Bowerman, and K. Oegema Inhibition of Rac by the GAP Activity of Centralspindlin Is Essential for Cytokinesis Science, December 5, 2008; 322(5907): 1543 - 1546. [Abstract] [Full Text] [PDF]
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T. Kawashima, Y. C. Bao, Y. Nomura, Y. Moon, Y. Tonozuka, Y. Minoshima, T. Hatori, A. Tsuchiya, M. Kiyono, T. Nosaka, et al. Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors J. Cell Biol., December 18, 2006; 175(6): 937 - 946. [Abstract] [Full Text] [PDF]
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A. Y. N. Goldstein, Y.-N. Jan, and L. Luo Function and regulation of Tumbleweed (RacGAP50C) in neuroblast proliferation and neuronal morphogenesis PNAS, March 8, 2005; 102(10): 3834 - 3839. [Abstract] [Full Text] [PDF]
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F. Oceguera-Yanez, K. Kimura, S. Yasuda, C. Higashida, T. Kitamura, Y. Hiraoka, T. Haraguchi, and S. Narumiya Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis J. Cell Biol., January 17, 2005; 168(2): 221 - 232. [Abstract] [Full Text] [PDF]
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K. Hirose, T. Kawashima, I. Iwamoto, T. Nosaka, and T. Kitamura MgcRacGAP Is Involved in Cytokinesis through Associating with Mitotic Spindle and Midbody J. Biol. Chem., February 16, 2001; 276(8): 5821 - 5828. [Abstract] [Full Text] [PDF]
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A. Toure, L. Morin, C. Pineau, F. Becq, O. Dorseuil, and G. Gacon Tat1, a Novel Sulfate Transporter Specifically Expressed in Human Male Germ Cells and Potentially Linked to RhoGTPase Signaling J. Biol. Chem., June 1, 2001; 276(23): 20309 - 20315. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
2-,