From the Howard Hughes Medical Institute and the
§ Herman B Wells Center for Pediatric Research, Department
of Pediatrics, Indiana University School of Medicine, Indianapolis,
Indiana 46202, and the ¶ Department of Biochemistry, University of
Tennessee at Memphis, Memphis, Tennessee 38163
Received for publication, November 20, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The Rho GTPase, Rac2, is expressed
only in hematopoietic cell lineages, suggesting a specific cellular
function in these cells. Genetic targeting studies in mice showed that
Rac2 is an essential regulator of neutrophil chemotaxis, L-selectin
capture and rolling, and superoxide production. Recently, a dominant
negative mutation of Rac2, D57N, has been reported to be
associated with a human phagocytic immunodeficiency. To
understand further the cellular phenotypes associated with this D57N
Rac2 mutant we examined its biochemical characteristics and functional
effects when expressed in primary murine bone marrow cells. When
compared with wild type (WT) Rac2, D57N Rac2 displayed ~10% GTP
binding ability resulting from a markedly enhanced rate of GTP
dissociation and did not respond to the guanine nucleotide exchange
factors. These results suggest that D57N Rac2 may act in a dominant
negative fashion in cells by sequestering endogenous guanine nucleotide
exchange factors. When expressed in hematopoietic cells, D57N Rac2
reduced endogenous activities of not only Rac2, but also Rac1 and
decreased cell expansion in vitro in the presence of growth
factors due to increased cell apoptosis. Unexpectedly, D57N expression
had no effect on proliferation. In contrast, expansion of cells
transduced with WT Rac2 and a dominant active mutant, Q61L, was
associated with significantly increased proliferation. Transplantation
of transduced bone marrow cells into lethally irradiated recipients showed that the percentage of D57N-containing peripheral blood cells
decreased markedly from 40% at 1 month to <5% by 3 months postinjection. Neutrophils derived in vitro from the
transduced progenitor cells containing D57N demonstrated markedly
impaired migration and O The Rho GTPase family, including Rho, Cdc42, and Rac, is a growing
subgroup of Ras proteins. A number of studies have shown that Rho
GTPases are involved in multiple cellular processes such as actin
polymerization and cytoskeleton rearrangement, regulation of gene
transcription, cell cycle progression, and cell survival. Rho GTPases
were initially found to be required for the regulation of actin
polymerization in eukaryotic cells. In these and subsequent studies it
has been demonstrated that Rho activates the assembly of actin-myosin
stress fibers and focal adhesions, Rac induces lamellipodial extension
and membrane ruffling, and Cdc42 promotes filopodia or microspike
formation (for review, see Refs. 3-6). To date, Rho GTPases have been
implicated in cell adhesion, cell motility, cytokinesis, and membrane
trafficking (7-9). When microinjected into fibroblasts, it has also
been reported that Rho, Rac, and Cdc42 stimulate cell cycle progression
through G1 and DNA synthesis (10); and expression of Rho,
Rac, and Cdc42 activates gene transcription via serum response factor
and nuclear factor The Rac subfamily of Rho GTPases has three highly homologous members,
Rac1, Rac2, and Rac3. Unlike Rac1 and Rac3, which are widely expressed,
Rac2 is found only in hematopoietic cells (14-17). In addition to its
reported roles in actin remodeling, the Rac proteins have been
implicated in the generation of O Like all other GTPases, Rac2 functions as a molecular switch by cycling
between an inactive GDP-bound form and an active GTP-bound form. A
number of proteins, such as guanine exchange factors
(GEFs)1 and GTPase-activating
proteins, regulate the ratio of GTP/GDP-bound forms which determines
the activities of Rac GTPases (8). Mutations that disrupt the cycling
of the two forms of GTPases have been identified in the guanine
nucleotide binding domain or the effector domain regions. These
mutations have been shown in Rac, Rho, and Cdc42, and in some cases are
they similar to mutations reported in Ras. Mutations at positions 12 (G12V) and 61 (Q61L) inhibit GTP hydrolysis to produce constitutively
active mutants (26), and a mutation at position 17 (T17N) destabilizes
the guanine nucleotide-binding site resulting in a dominant negative
mutant (27).
Recently, Ambruso et al. (2) and our laboratory (1)
identified a genetic mutation (D57N) in Rac2 GTPase which is associated with a human phagocyte immunodeficiency. The patient suffered severe
recurrent infections and defective neutrophil cellular functions
similar to those found in Rac2 To examine carefully the biochemical and biological consequences of
D57N Rac2 expression in hematopoietic cells further, we studied
recombinant protein in vitro and introduced this mutant into
murine hematopoietic stem and progenitor cells via retrovirus-mediated gene transfer. Biochemical analyses show that D57N Rac2 has
significantly decreased binding affinity for GTP resulting from a very
rapid dissociation rate of GTP but not GDP. The mutant protein is also nonresponsive to GEF binding, suggesting that D57N Rac2 acts in a
dominant negative fashion by sequestering endogenous GEFs from Rac-related Rho GTPases. To study the effects of D57N in hematopoietic cells, GST-effector pull-down assays were performed on transduced myeloid cells and demonstrated loss of both Rac1 and Rac2 activities. The biological consequences of expression of D57N include reduced cell
expansion of hematopoietic cells in vitro unexpectedly not because of reduced proliferation but associated with increased apoptosis. Long term engraftment in vivo after bone marrow
transplantation was also impaired significantly. As expected,
neutrophils derived from D57N-transduced myeloid progenitors displayed
reduced chemotaxis and superoxide generation. On the other hand,
overexpression of WT Rac2 or the constitutively activated mutant Q61L
was associated with increased proliferation of normal cells and
slightly reduced apoptosis. Thus, D57N Rac2 appears to affect the
activity of both Rac2 and Rac1. Although normal Rac2 expression and
function appear critical for the growth factor-induced survival,
overexpression of Rac2 is associated with increased proliferation. The
data imply that Rac proteins are critical regulators of both actin
function and cell survival/proliferation in hematopoietic cells.
Protein Expression and Purification--
The human D57N Rac2
mutant cDNA was cloned into the bacterial expression plasmid
pET-28a at EcoRI and XhoI sites; and WT human Rac2 cDNA was cloned into the pET-15b plasmid without the 11-amino acid T7 tag (Novagen, Milwaukee, WI). The Rac2 constructs were expressed in the Escherichia coli BL21 strain as
(His)6-tagged fusion proteins. Recombinant proteins were
purified through the His-binding resins as described in the
manufacturer's protocol. The isolated proteins were >90% pure judged
by Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis gels
(New England Biolabs, Beverly, MA).
The PAK1 p21 binding domain (PBD) and cDNA of TrioN were fused to
glutathione S-transferase (GST) in the pGEX bacterial
expression plasmids, respectively (Amersham Pharmacia Biotech).
Expressed proteins were isolated from glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech) following instructions for bulk GST
purification modules given by the manufacturer. The final protein
products were demonstrated to have >80% purity analyzed by
SDS-polyacrylamide gel electrophoresis. GST-PBD fusion protein was
stored in 25 mM Tris-HCl, pH 7.5, 0.2 M
dithiothreitol (DTT), 1 mM MgCl2, and 5%
glycerol at Guanine Nucleotide Binding and Exchange
Assays--
[
To measure GEF-stimulated change in nucleotide binding, 1 µg of
recombinant Rac2 proteins was preloaded with [3H]GDP and
exchanged for cold GTP in a buffer of 100 mM NaCl, 20 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 1 mM DTT, and 0.5 mM GTP with or without the
addition of GEF TrioN. The remaining bound [3H]GDP
(Amersham Pharmacia Biotech) was determined at five different time
points by filtration.
Exchange Factor Binding Assay--
Complex formation of
His-tagged WT Rac2 and D57N Rac2 with GST-TrioN was carried out as
described for the Dbl-G-protein interactions (31). Briefly, 1 µg of
purified (His)6-WT Rac2 or (His)6-D57N Rac2 in
a buffer containing 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 2 mM EDTA, 0.5% Triton 100, and 1 mM DTT was incubated with 2 µg of GST or GST-TrioN
immobilized on agarose beads for 30 min at 4 °C under constant
agitation. The coprecipitates were washed three times with the
incubation buffer and were subjected to 10% SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose for Western blot
analysis using anti-His polyclonal antibody (Amersham Pharmacia
Biotech). The immune complexes were visualized by chemiluminescence reagents.
Retroviral Vectors and Stable Virus Packaging Cell Lines--
WT
mouse Rac2 and mutant Rac2 (human D57N and Q61L) cDNAs were cloned
into a modified murine stem cell virus-based bicistronic vector
(MIEG3; (1)) at unique EcoRI and XhoI sites. The
resultant retroviral plasmids were introduced into a
Phoenix-ampho packaging cell line (American Type Culture
Collection) cultured in Iscove's modified Dulbecco's medium (Life
Technologies, Inc.) with 10% fetal calf serum (Hyclone Laboratories,
Logan, UT), 2% penicillin and streptomycin, 1% glutamine, using
LipofectAMINE (Life Technologies, Inc.). The viral supernatant derived
from the transfected Phoenix-ampho cells was used to establish
stable GP+E86-derived producer clones for MIEG3, WT Rac2, D57N, and
Q61L Rac2 as described in Williams et al. (1). The titers of
viral supernatants collected from different stable E86 clones were
determined by fluorescence-activated cell sorting (FACS; Becton
Dickinson, Mountain View, CA) analysis using the method reported by
Felts et al. (48). High titer E86 clones
(>1 × 105/ml) were used to collect viral supernatant
to infect mouse bone marrow cells.
Mouse Low Density Bone Marrow (LDBM) Isolation and Viral
Transduction--
Whole bone marrow was collected from WT and
Rac2
Virus-mediated LDBM transduction was performed as described previously
(32). Briefly, 2 × 106 cells were infected by viral
supernatant overnight on CH296 (Tokara Shuzo Co., Japan)-coated
six-well plates. The infected cells were kept in culture with complete
RPMI medium for 48 h. Cells were subsequently analyzed and sorted
for green fluorescence using a FACScan or FACStar Plus (Becton Dickinson).
Western Blot and Affinity Precipitation of Active (GTP-bound)
GTPases Using GST-PBD Fusion Protein--
Transduced and enhanced
green florescence protein positive (EGFP+) BM cells were
immunoblotted using mouse antibodies for Rac2 (1:5,000, a gift from Dr.
Gary Bokoch, Scripps Institute, La Jolla, CA), Rac1 (23A8, 1:2,000,
Upstate Biotechnology, Lake Olacid, NY), Cdc42 (sc87G, 1:2,000, Santa
Cruz Biotechnology, Santa Cruz, CA), and total p38 (1:2,000, New
England Biolabs).
For GST-effector pulldown assays, in vitro transduced and
cultured bone marrow cells (cytokine-stimulated, 1 × 106 cells/assay) were incubated with 2 × lysis buffer
(50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 200 mM NaCl, 2% Nonidet P-40, 10%
glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, and 2 µg/ml aprotinin, all from Roche, Indianapolis, IN)
on ice. The lysates were clarified by low speed centrifugation for 5 min at 4 °C. For in vitro guanine nucleotide binding,
cell lysates were incubated for 15 min at 30 °C in the presence of
10 mM EDTA and 100 µM GTP
The crude or guanine nucleotide-loaded cell lysates (100 µl) were
added to 200 µl of binding buffer (25 mM Tris-HCl, pH
7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, and 0.5% Nonidet P-40), 10 µg of PAK1 PBD-GST
recombinant protein, and 5 µl of glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech). The binding reaction was incubated for
1 h at 4 °C and then washed twice with washing buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, and 0.5%
Nonidet P-40) and 3 times with washing buffer without detergent. The
bead pellets were finally resuspended in 15 µl of Laemmli sample
buffer. Each sample was analyzed on 12% SDS-polyacrylamide gel
electrophoresis and blotted by specific antibodies for Rac1 (1:2,000) and Cdc42 (1:2,000). The secondary antibodies were
horseradish peroxidase-conjugated (1:2,500, New England Biolabs). The
immunoblots were detected by New England Biolabs Luminol kit and Kodak
Biomax film.
Cell Growth, Cell Proliferation, and Cell Apoptosis
Assays--
After cell sorting, infected BM cells were >95%
EGFP+. For liquid culture, 1 × 105 cells
(expressing MIEG3, WT Rac2, D57N, and Q61L, respectively) were seeded
on a 24-well tissue culture plate in complete RPMI medium. Cells were
enumerated by a hemocytometer every 2 days. For colony-forming assays,
1 × 104/ml cells were plated in triplicate in
methylcellulose (Stem Cell Technology, Vancouver, Canada) supplemented
with 100 ng/ml hG-CSF, 100 ng/ml MGDF, 100 ng/ml SCF, and 10 ng/ml
murine interleukin-3 (PeproTech, Rocky Hill, NJ) and incubated for 7 days at 37 °C in 5% CO2. Colonies were enumerated and
scored for size under an inverted microscope at day 7.
To examine cell proliferation, 5 × 104 cells/well in
200 µl of complete RPMI were plated on the 96-well plate (six wells
for each sample). Cells were incubated with 1 µCi
[3H]thymidine (Amersham Pharmacia Biotech) for 6 h
at 37 °C in 5% CO2 before being harvested on a cell
harvester (Packard Instrument, Meriden, CT). The retained radioactivity
on the filter was counted by the scintillation counter
(Beckman/Coulter, Fulleton, CA) as described (33).
To determine the frequency of apoptotic cells, 1 × 105 cells (in vitro cultured in the complete
RPMI medium) in 100 µl of binding buffer (10 mM
HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM
CaCl2) were stained with either 1 µg of propidium iodide
(PI; Calbiochem)/100 µl of cells for 10 min on ice in dark, or 3 µl
of annexin V-biotin/100 µl of cells for 30 min on ice followed by 2 µl of streptavidin-PE (all from PharMingen, San Diego, CA) for 30 min
on ice in the dark. Cells were washed twice in 1 × PBS and were
analyzed by flow cytometry on FACScan. For TUNEL assay, 2 × 106 cells were fixed in 200 µl of a freshly prepared
paraformaldehyde solution (2% in PBS, pH 7.4) for 1 h at room
temperature and then washed in 1 × PBS once and resuspended in
permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate)
for 2 min on ice. The prefixed cells were labeled with an in
situ cell death detection kit, TMR red (Roche), and analyzed by
flow cytometry on FACScan.
Myeloid Cell Chemotaxis and Superoxide Production
Assays--
Transduced BM cells were cultured in the complete RPMI
medium with cytokines for 7 days, and 105 cells were then
stained for myeloid cell lineage markers Gr-1 (1 µl of PE-Ly6G/100
µl of cells, PharMingen) and Mac1 (0.2 µl of PE-CD11b/100 µl of
cells, PharMingen). More than 90% of the cells showed both
Gr-1+ and Mac1+ on FACS analysis.
Chemotaxis of in vitro differentiated myeloid cells
(Gr-1+, Mac1+, and GFP+) was
performed using a modified Boyden chamber (Neuro Probe, Inc., Cabin
John, MD) as described in Roberts et al. (22).
10
Superoxide production in the transduced and differentiated myeloid
cells was determined by the nitro blue tetrazolium (NBT) test as
described previously (34). Cells were stimulated by either 100 ng/ml
phorbol 12-myristate 13-acetate (PMA; PharMingen) or
10 In Vivo Transplantation and Engraftment
Studies--
Virus-transfected LDBM cells were analyzed for their
EGFP expression before transplantation back into mice. WT
C57BL/6 mice (8-10 weeks old, Jackson Laboratory, Bar Harbor, ME) were
lethally irradiated (1,100 Rads split dose) using a 154Cs
irradiator (Novdian International, Canada) as transplantation recipients. Transfected donor cells were resuspended in PBS with 2%
bovine serum albumin (Roche) at a concentration of 2 × 106/100 µl, and a total of 2 × 106
cells was injected into the lateral tail vein of each irradiated mouse.
Four or five mice were used for each vector construct, and three mice
were used as irradiated nontransplanted controls. For engraftment
studies, tail vein blood samples (100 µl) were withdrawn from each
transplanted mouse. Peripheral blood cells were incubated in red blood
cell lysis buffer (Puregene, Minneapolis, MN) for 20 min on ice. Cells
were then washed twice and resuspended in 1 ml of PBS and 0.2% bovine
serum albumin. The percentages of EGFP+ cells of each
sample were analyzed by flow cytometry on FACScan (Becton Dickinson).
Sequestration of GEF by the D57N Rac2 Mutant--
The G169A
(aspartic acid to asparagine, D57N) mutation in Rho GTPase Rac2 was
originally identified in blood cells from a human patient and was
associated with severe phagocyte immunodeficiency (1, 2). Along with
D57N, similar mutations such as D57Y and D57A have been reported at the
same residue in the Ras proto-oncogene. These Ras mutants have
decreased GTP binding activity (35). As shown in Williams et
al. (1), we found that the D57N Rac2 mutant can bind GDP normally,
but it binds to GTP at only 10% of the normal level. Biochemically the
D57N Rac2·[
In vivo, Rac2 GTPase functions as a molecular switch through
its transition between inactive GDP-bound and active GTP-bound forms.
The exchange of GDP for GTP is catalyzed by GEFs. We further examined
the D57N Rac2 exchange rate of bound [3H]GDP. When
preloaded with [3H]GDP and in the presence of excess GTP
(for details, see "Experimental Procedures"), WT Rac2 exchanged
30% of [3H]GDP to GTP within 15 min at room temperature,
and D57N Rac2 exchanged at a similar rate. In the presence of the GEF
TrioN (36), WT Rac2 significantly increased the rate of GDP/GTP
exchange such that 90% became GTP-bound in 15 min (Fig.
2A). In contrast, D57N Rac2 did
not respond to TrioN. By immunoblotting the His-tagged GTPases,
GST-TrioN was found to coprecipitate equally well with both WT Rac2 and
D57N Rac2 (Fig. 2B). Because D57N Rac2 can still physically
bind to TrioN, the lack of responsiveness to GEF is most likely the
result of its poor GTP binding activity with consequent impaired
turnover rates. Thus, these biochemical data suggest that D57N Rac2 may
act in a dominant negative fashion by sequestering endogenous GEFs from
other Rho GTPases in cells.
Expression of Rac2 Mutant in Hematopoietic Cells--
To examine
more carefully the biological effects of mutant Rac2 GTPases on a
variety of hematopoietic cell-related events, we introduced D57N, along
with WT Rac2 and a dominant active Rac2 mutant, Q61L, into
hematopoietic stem and progenitor cells. As shown in Fig.
3A, the WT Rac2, D57N Rac2, or
Q61L Rac2 cDNA was ligated into retroviral vector MIEG3. An
internal ribosome re-entry site is inserted between Rac2 and EGFP
sequences to quantitate the expression of both genes in individual
cells using the intensity of GFP staining assayed by flow cytometry.
Stable GP+E86 retrovirus packaging cell lines expressing MIEG3 (vector
only), WT Rac2, D57N Rac2, and Q61L Rac2 were established (for details,
see "Experimental Procedures"). The integrity of integrated
provirus in cells infected with virus from each producer line was
demonstrated by Southern blot using probes for Rac2 cDNA and EGFP
(data not shown).
High titer E86 viral supernatant generated from producer clones were
used to transduce LDBM cells derived from either WT C57BL/6 or
Rac2 Ectopic Expression of Rac2 Alters Rac Activation in BM
Cells--
To determine whether Q61L and D57N Rac2 mutants affect
endogenous GTPase activity in BM cells, we examined the level of active GTP-bound GTPases in transduced and GFP+ bone marrow cells
using the PAK1 PBD pulldown assay (38). As shown in Fig.
4, we used Rac2-deficient BM cells in
addition to WT to examine whether D57N Rac2 affects other GTPases. In
cells transduced with D57N, GTP-bound Rac2 is not detectable
(lane 3, upper panel, Rac2 antibody). Expression
of D57N Rac2 also prevents the binding of GTP to endogenous Rac1
(lanes 3 and 7, lower panel, Rac1
antibody). There is no significant reduction of active, GTP-bound Cdc42
in transduced WT, but we inconsistently saw small reductions in
GTP-bound Cdc42 in Rac2
As shown in Fig. 4, Q61L Rac2 expressed in both WT and Rac2 Expression of Rac2 Mutants Affects BM Cell Growth--
Rac2
To confirm these in vitro observations and to determine the
physiological relevance of these observations in reconstituting hematopoietic stem cells, we transplanted transduced BM cells into
lethally irradiated mice. Mice were subsequently bled monthly after the
BM injection, and the level of GFP+ cells in the peripheral
blood was determined. As shown in Fig. 6B, the percentage of D57N
GFP+ cells dropped from more than 25% to less than 10% in
1 month and decreased to less than 5% by 3 months postinjection. In
contrast to the increased expansion of cells seen in vitro,
the engraftment of WT Rac2 (Fig. 6A) and Q61L Rac2 (data not
shown) compared with empty vector MIEG3 (data not shown) was relatively
stable during this same 3-month interval. These data suggest that the
expression of dominant negative D57N mutant Rac2 in bone marrow cells
is also associated with reduced reconstitution of stem/progenitor cells
in vivo.
Rac2 Mutants Affect BM Cell Survival and/or Cell
Proliferation--
Rac proteins have been shown to be critical for
cell proliferation by regulating G1 cell progression in
Swiss 3T3 fibroblasts (39). In addition, Rac2 specifically has been
shown to be critical in growth factor-induced survival in mast cells
(25). To determine whether the D57N Rac2-induced expansion defect in
bone marrow cells is caused by abnormal cell proliferation, we examined
DNA synthesis of the cells using [3H]thymidine
incorporation. As reported previously for Rac2-deficient mast cells,
Rac2
Because reduction of BM cell expansion and colony formation by D57N
Rac2 is not associated with the abnormal cell cycle progression, we
next examined the effect of Rac2 on programmed cell death in these
cells. Cultured GFP+-transduced BM cells stained for
annexin V-PE or PI were analyzed by flow cytometry. After 10 days of
culture, loss of Rac2 activity in BM cells was associated with
increased cell death in the presence of SCF, MGDF, and G-CSF. 30% of
Rac2 Myeloid Cell Function Is Affected by Expression of Rac2
Mutants--
Rac2 has previously been shown to be required for normal
neutrophil chemotaxis, rolling via L-selectin and superoxide production both in vitro and in vivo in mice (22). In
addition, the D57N mutant Rac2 was associated with abnormal neutrophil
migration, rolling, and superoxide generation in a human patient with
recurrent infections (1). The effect of Q61L Rac2 expression on normal neutrophil function has not been studied previously. To understand better the physiological effects of expression of these two different Rac2 mutants in myeloid cell function, we induced myeloid
differentiation of transduced and GFP+-sorted BM cells
in vitro. After 10 days of culture (7 days after transduction) in the presence of SCF, MGDF, and G-CSF cytokines, greater than 90% of the cells were Mac1+ and
Gr-1+. These cells were subsequently analyzed for cell
migration using a modified Boyden chamber assay. Expression of D57N
Rac2 completely inhibited cell migration, particularly in Rac2
Rac GTPases play an essential role in generating superoxide via NADPH
oxidase in a cell-free system in vitro. Both Rac1 and Rac2
have been demonstrated to be involved in assembly and activation of the
NADPH-dependent respiratory burst oxidase (40, 41). In
cultured and in vitro differentiated BM neutrophils, D57N
mutant Rac2 completely abolishes superoxide production, particularly in
Rac2 Compensatory Function of Rac Proteins--
All three members of
the Rac subfamily are highly conserved in their primary sequences. Rac1
and Rac2 have overlapping expression in the hematopoietic cell
lineages. Unlike Rac1, which is ubiquitously expressed, Rac2 is
expressed primarily in the hematopoietic cells (42). Previous
gene-targeting studies demonstrated defective migration and F-actin
generation by freshly explanted BM neutrophils from Rac2 Rho GTPase members of the Ras superfamily have been shown to
control actin cytoskeleton organization in eukaryotic cells (9, 43).
Similar to Ras, by cycling between inactive GDP-bound and active
GTP-bound states, Rho GTPases are key regulators of a wide spectrum of
cellular functions in eukaryotic cells, including actin polymerization,
membrane trafficking, gene transcription, and cell cycle progression
(3). The regulation of Rho GTPase activity is achieved through
protein-protein interactions with GEFs, GTPase-activating proteins, and
guanine nucleotide dissociation inhibitors (44). In general, GEFs
function to stimulate GDP/GTP exchange thus activating the Rho GTPase,
whereas GTPase-activating proteins stimulate intrinsic GTPase activity,
leading to decreased signaling. Guanine nucleotide dissociation
inhibitors interfere with both GTP hydrolysis and GDP/GTP exchange and
may function additionally in modulating subcellular localization
patterns of the GTPases (8). The mammalian Rho-like GTPases consist of several distinct proteins, including the Rac subfamily (45). Among
three identified Rac proteins, all of which share very high sequence
homology (Rac1 and Rac2 are 92% homologous, and Rac2 and Rac3 are 91%
homologous), Rac2 is expressed only in hematopoietic cells (42).
We have generated Rac2-deficient mice by homologous recombination (22).
Mast cells and neutrophils from Rac2 The described D57N mutation occurs in a sequence conserved in all small
GTPases and is located in one of the guanine nucleotide binding
domains, suggesting a potentially important role in stabilizing the
nucleotide binding complexes. Asp-57 has been suggested, in GTP-bound
form, to coordinate the oxygen atom from the The cellular phenotypes associated with Rac2 deficiency in mice and
dominant negative D57N Rac2 in man have been characterized mainly in
phagocytic cells, and they include defective migration, defective
capture, and rolling on the L-selectin ligand, glycam-1, and reduced
superoxide generation in response to some agonist of the phagocytic
oxidase pathway (1). As shown here, expression of D57N Rac2 in primary
mouse hematopoietic cells was associated with markedly impaired
migration and superoxide generation. However, recent data derived from
other lineages deficient in Rac2, including lymphocytes (46) and mast
cells (25), taken together with the biochemical data reported here
would suggest the potential for multilineage abnormalities as a result
of expression of D57N Rac2. As shown here, expression of D57N Rac2
compared with either WT Rac2 or the activated Q61L Rac2 was associated
with markedly reduced expansion of transduced cells in vitro
and a significant lack of engraftment in vivo. Impaired
expansion in vitro in large part was caused by increased
apoptosis even in the presence of growth factors that act as survival
factors for primitive hematopoietic cells. In Rac2-deficient mast
cells, increased apoptosis has been demonstrated to be associated with
defective activation of Akt by phosphatidylinositol 3-kinase as well as
reduced expression of the antiapoptotic protein Bcl-XL and
enhanced expression of the proapoptotic protein BAD (25).
Interestingly, there are no cell cycle progression changes as assayed
by thymidine incorporation demonstrated in the absence of WT Rac2 or
the presence of D57N Rac2, whereas increased proliferation is apparent
when WT Rac2 or Q61L Rac2 is overexpressed. Thus previous data
implicating Rac function in cell cycle progression using transduction
of activated or dominant negative mutants of Rac in fibroblast cells
may not accurately reflect the role of this GTPase in normal cell cycle events, and the D57N Rac2 phenotype in humans requires more careful analysis in the future to determine the full extent of the effect in
all blood cells.
A surprising but consistent finding in the studies reported here is the
loss of phenotypic abnormalities of migration and superoxide generation
in Rac2-deficient neutrophils after extensive in vitro
growth and differentiation in growth factors. These same cells
demonstrated a significant induction of Rac1 expression and activity
compared with cells freshly isolated from Rac2-deficient mice. These
data suggest that factors regulating expression of Rac1 in
vivo are overcome after stimulation in vitro and that Rac1, when highly overexpressed, can subserve at least some functions in blood cells normally performed by Rac2. The nature of the regulatory control of Rac1 (and Rac2) expression and its protein activity is
currently unknown. In addition, the basis of the specificity of Rac2
function in blood cells, given the sequence similarity of the Rac
proteins, will be an important issue to be addressed in the future.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (11, 12). Studies of downstream signaling
pathways have shown that Rac and Cdc42 are distinguished from Rho by
activating c-Jun kinase, also known as stress-activated protein
kinase (10). In addition, cross-talk between Rho proteins has
also been observed. Cdc42 acts as a stronger activator of Rac, and
activated Rac can activate or inhibit Rho depending on the cells
involved (3, 13). Very little is known about how Rho GTPases
differentially orchestrate cellular processes and signaling pathways in
different mammalian cell lineages.
/
mast cells also demonstrate a significant
decrease in growth factor-dependent survival. Studies of
kinase signaling pathways indicate that Rac2 function is required for
activation of Akt downstream of phosphatidylinositol 3-kinase (25). In addition, Rac2 appears to be critical for the appropriate expression of
Bcl2 family members Bcl-XL and BAD in these cells. Although ectopic expression of activated Rac mutants in fibroblasts has been demonstrated to stimulate cell cycle progression through G1 and subsequent DNA synthesis (10), no significant cell
proliferation defect has been found in Rac2
/
neutrophils or mast
cells (25).
/
mice. The patient's genotype wild
type (WT) Rac2/D57N Rac2 (2) was associated with normal Rac2 message
levels (1) and normal cDNA sequences in 50% of cloned cDNAs
(1). Neutrophils derived from normal human bone marrow progenitor cells
transduced by the cloned D57N Rac2 cDNA mimic the patient's
neutrophil phenotypes of decreased cell migration and superoxide
production (28). All of these data suggest that the D57N protein had a
dominant negative effect on WT Rac2 protein. In addition, expression of
the D57N mutant in NIH/3T3 cells, which express Rac1 but not Rac2
endogenously, resulted in dominant negative phenotypic changes in these
cells, such as diminished growth, abnormal cell shape, and reduced
membrane ruffling in response to PDGF. These data imply that the D57N
mutant may affect the function of other Rac GTPases and that the
patient's phenotype could be caused by dysfunction not only of Rac2,
but also of Rac1, function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
-35S]GTP and [3H]GDP
(PerkinElmer Life Sciences) were preloaded onto 1-2 µg of
recombinant Rac2 proteins (WT and D57N) in the presence of 100 mM NaCl, 20 mM Tris-HCl, pH 7.6, 2 mM EDTA, and 1 mM DTT for 20 min at room
temperature. The labeled Rac2 protein complexes were stabilized by
adding MgCl2 to a final concentration of 5 mM.
Binding of guanine nucleotides to Rac2 proteins was determined by
counting radioactivity bound to filters as described previously (29,
30). Dissociation assays were performed by adding activation solution
(20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM DTT, 1 mM GTP, and 5 mM
MgCl2) to the preloaded reaction mixture (26), and the
remaining bound radioactivity was examined at different time points by
filtration (Midwest Scientific, Valley Park, MO).
/
mice 48 h after treatment with 5-fluorouracil (150 mg/kg
of body weight; American Pharmaceutical Partners, Los Angeles, CA) and
was resuspended in RPMI medium (Life Technologies, Inc.). LDBM cells
were isolated from fresh BM by centrifugation on Histopaque-1083
(Sigma) gradient for 30 min at 1,500 rpm at room temperature. Cells at
the interface were collected and washed twice with RPMI and then
counted on a hemocytometer. 20 million LDBM cells were plated on a
10-cm non-tissue culture Petri dish and prestimulated in RPMI medium supplemented with 10% fetal calf serum, 2% penicillin and
streptomycin, cytokines (100 ng/ml hG-CSF, 100 ng/ml MGDF, 100 ng/ml
SCF, all from Amgen, Thousand Oaks, CA) (complete medium), for 48 h at 37 °C in 5% CO2.
S or 1 mM GDP. The loading was stopped by the addition of
MgCl2 to 30 mM (30).
6 mol/liter
formyl-methionyl-leucyl-phenylalanine (fMLP, Sigma) was used as the
chemoattractant. The numbers of migrating cells were counted on the
filter in six random 400 × microscope fields.
6 mol/liter fMLP. The percentage of
NBT+ cells was determined by evaluating 200 cells in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-35S]GTP complex is much less
stable compared with that of WT Rac2. As shown in Fig.
1, WT Rac2 has a comparable intrinsic
dissociation rate for both [3H]GDP and
[
-35S]GTP such that ~30% GDP or GTP has been
dissociated in 15 min. D57N Rac2, on the other hand, retained a similar
rate of GDP dissociation, but it dissociates GTP significantly faster
than WT Rac2, reaching about 75% in 2 min.
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Fig. 1.
Intrinsic GTP/GDP dissociation of D57N Rac2
mutant. Recombinant WT Rac2 (solid symbols) and D57N
Rac2 (open symbols) proteins were preloaded with either
[ -35S]GTP (circles) or
[3H]GDP (triangles). Guanine nucleotide
dissociation was initiated by adding the radionucleotide-loaded
proteins to the activation buffer, and the remaining bound
radioactivity was examined at different time points: 0, 2, 5, 10, and
15 min. The amount of bound nucleotide at t = 0 min was
set to 100%. The data are presented as the percentage of bound
[
-35S]GTP or [3H]GDP remaining. The
results shown are representative of three experiments.
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Fig. 2.
GDP/GTP exchange of D57N Rac2 mutant
stimulated by guanine nucleotide exchange factor TrioN.
Panel A, 2 µg of recombinant WT Rac2
(circles) and D57N Rac2 (triangles) were
preloaded with [3H]GDP and exchanged for cold GTP in the
absence (solid symbols) or presence (open
symbols) of 0.5 µg of TrioN. The remaining bound
[3H]GDP was determined at 0, 2, 5, 10, and 15 min after
GTP exchange. The amount of bound nucleotide at t = 0 min was set to 100%. The data are presented as the percentage of bound
[3H]GDP remaining. The results shown are representative
of three experiments. Panel B, binding of guanine
nucleotide exchange factor, TrioN, to WT and D57N Rac2. Purified
recombinant His-tagged WT Rac2 and D57N Rac2 proteins were assayed for
direct binding to the GST-TrioN fusion protein. Equal amounts of
proteins were used for each lane. Protein was detected on the
immunoblot by anti-His-tag antibody. GST alone was used as control in
lane 1. GST-TrioN binds to both WT Rac2 (lane 2)
and D57N Rac2 mutant (lane 3) at similar levels. His-Rac2
D57N (lane 3) runs higher because of an 11-residue T7 tag
fused to its N terminus. The results shown are representative of three
experiments.
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Fig. 3.
Retrovirus-mediated transduction of mouse
bone marrow cells with Rac2 retrovirus vectors. Panel
A, MIEG3-based retroviral vector was used as a backbone for
the Rac2 transgenes. Murine cDNAs encoding WT Flag-tagged Rac2 or
mutant Rac2 (D57N and Q61L) was cloned into MIEG3 in front of the
internal ribosome re-entry site and EGFP reporter gene. LTR,
long terminal repeats; IRES, internal ribosome entry site.
Panel B, immunoblot analyses of transduced and
EGFP+ mouse bone marrow cells using antibodies to Rac1,
Rac2, Cdc42, and p38 (mitogen-activated protein kinase, as a loading
control) proteins. Anti-Rac2 antiserum has weak cross-reaction with
Rac1 as seen in lane 2 (MIEG3). The size of the murine WT
Rac2 (lane 3) protein is slightly higher because of the Flag
tag.
/
mice (22) in the presence of cytokines (SCF, MGDF, and G-CSF)
using a standard infection protocol (37). The transduced bone marrow
cells were sorted for GFP+ (Table
I), and GFP+ cells were used for
further biochemical and cellular analyses. We consistently saw only a
low level of gene transfer in cells infected with Q61L. As shown in
Fig. 3B, immunoblot of the transduced and GFP+
Rac2
/
BM cells confirmed the ectopic expression of WT Rac2 and Rac2
mutants. WT BM cells transduced with the empty vector (MIEG3) were also
used as a control of normal endogenous GTPase expression levels
(lane 1). The Rac2 antibody has weak cross-reaction with
another protein (likely Rac1 as shown in Ref. 22) in lysates from
Rac2
/
BM cells (lane 2). In the transduced and
GFP+ cells, the expression level of Q61L (lane
5) is lower than expression of either WT or D57N Rac2 (lanes
3 and 4). This may be because of the lower viral
transduction rate (6-8%, Table I) of Q61L Rac2 vector, resulting in
lower proviral copy number cell in transduced cells. Southern blot
analysis supports this interpretation (data not shown). As noted
previously (1, 25), the WT Rac2 protein encoded by WT Rac2 is slightly
larger (lane 3) because of the Flag tag. Expression of Q61L
was associated with a decreased endogenous Rac1 protein level
(lane 5 versus lane 2), but expression of dominant negative
D57N or loss of Rac2 activity in Rac2
/
cells was associated with
increased endogenous Rac1 (lanes 2 and 4 versus lane 1, and see below). Expression of WT
Rac2 or D57N had little effect on the level of Cdc42 protein expression
in the transduced bone marrow cells (lanes 1 and
3, 4 versus lane 2),
whereas a very slight reduction of Cdc42 was seen in Rac2
/
cells
expressing Q61L (lane 5 versus lane
2).
GFP expression in vector-transduced bone marrow cells
/
mice were transduced with
GFP-expressing retroviral vectors, MIEG3, WT Rac2, D57N, and Q61L. 2 days after transduction, cells were analyzed for green fluorescence by
flow cytometry. Data are presented as the percentages of GFP+
cells and are representative of three experiments.
/
cells (data not shown). Thus, as predicted
by the biochemical data presented above, D57N Rac2, when expressed via
retrovirus vector, has a dominant negative effect not only on Rac2, but
also on other Rac GTPases in BM cells.
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Fig. 4.
GTPase activities in the transduced bone
marrow cells. Both WT and Rac2 /
mouse BM cells expressing
transgenes MIEG3, WT Rac2, D57N, and Q61L (dominant active mutant Rac2)
were used for the PAK1 PBD GST effector pull-down assay. GTP-bound
proteins were analyzed by immunoblot using antibodies for Rac2
(upper panel) and Rac1 (lower panel). Flag-tagged
WT Rac2 protein bands were slightly higher (lanes 2 and
6, upper panel). The results shown are
representative of four independent experiments.
/
cells
binds to GTP tightly (lanes 4 and 8, upper
panel, Rac2 antibody) (26), and expression of this mutant is
associated with a reduction of the endogenous active (GTP-bound) Rac1
(lane 4 versus 1 and lane 8 versus 5, lower panel, Rac1 antibody), but not GTP-bound Cdc42 (data not shown). Transgenic expression of WT Rac2
increases the amount of active Rac2 in transduced cells (lanes
2 and 6, upper panel), and this enhanced
Rac2 activity results in a decrease in active Rac1 (lanes 2 and 6, lower panel). In bone marrow cells
genetically deficient in Rac2, there is an apparent compensatory
increase in GTP-bound Rac1 (lane 5 versus 1, lower panel, and see below). Results identical
to those seen in Fig. 4 have been observed when cells transduced with
each virus are stimulated with either PMA or fMLP (data not shown).
Taken together, these results suggest that ectopic expression of either WT or mutant Rac2 can lead to changes in endogenous Rac GTPases activation.
/
mast cells display decreased cell growth and colony formation because
of increased apoptosis even in the presence of growth factors (25).
Because hematopoietic cell and colony growth studies were not reported
in the patient from which the human D57N mutant was identified and
cloned, we next examined the effect of D57N expression on transduced BM
cell growth in vitro. Post-sort BM cells (both WT and
Rac2
/
backgrounds) were confirmed > 95% EGFP+ by
flow cytometry. 1 × 105 GFP+ cells were
plated in the liquid medium containing SCF, MGDF, and G-CSF cytokines
and enumerated every 2 days. Rac2
/
BM cells expanded slowly
relative to WT cells (~32% of WT cell numbers after 1 week in
culture; data not shown). This growth deficiency could be completely
rescued by ectopic expression of either WT Rac2 or dominant active
mutant Q61L Rac2 (data not shown). However, WT cells failed to expand
after transduction and expression of the dominant negative mutant D57N
Rac2 (Fig. 5A). Similar results were observed when clonogenic progenitor cells were examined in methylcellulose colony assays in the presence of the same
cytokine combination (Fig. 5B). This reduction was even
greater in Rac2
/
BM cells (Fig. 5B), showing a gene
dosage effect of GTPases on cell growth and suggesting that both Rac2
and other Rho GTPases (at least Rac1) are required for BM cell
expansion. Interestingly, overexpression of WT Rac2 or Q61L Rac2 in BM
cells increased growth of WT cells significantly compared with cells
transduced with the control vector, MIEG3 (~4-fold increase in WT
cells, Fig. 5A; 10-fold increase in Rac2
/
cells, data
not shown). Also, WT Rac2 and Q61L Rac2 in myeloid progenitor cells
greatly increased colony numbers and colony size, suggesting that
increased GTPase activity enhances expansion of normal cells.
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Fig. 5.
Growth of transduced bone marrow cells
expressing WT and mutant Rac2 transgenes. Mouse WT LDBM cells were
infected with E86 viral supernatant containing retrovirus MIEG3 (vector
alone), WT Rac2, and mutants of Rac2 (D57N and Q61L) (for details, see
"Experimental Procedures"). Transduced cells were sorted for EGFP
and cultured in liquid RPMI medium (panel A) or methyl
cellulose (panel B) in the presence of G-CSF, SCF, and MGDF
(100 ng/µl each). Panel A, for liquid culture, 1 × 105 sorted cells were seeded in the 24-well tissue culture
plate on day 5 after transduction. Cell numbers were counted on every
other until day 13. *, p < 0.001 MIEG3
versus D57N; **, p < 0.01 WT Rac2
versus MIEG3. Panel B, for the colony-forming
assay, 1 × 104/ml transduced and GFP+
cells (both WT and Rac2 /
backgrounds) were plated in methyl
cellulose and incubated for 7 days. Colony numbers and sizes were
determined under an inverted microscope. Colony size was scored from
large (open bars) to small (leftside hatched
bars). Data are presented as the mean ± S.D. of three
independent experiments.
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Fig. 6.
In vivo engraftment of the
transduced hematopoietic stem and progenitor cells. WT mouse bone
marrow cells were transduced with retrovirus vectors expressing WT Rac2
(panel A) and D57N Rac2 (panel B) and
transplanted into the lethally irradiated mouse recipients. The level
of GFP+ cells in the peripheral blood of these mice was
determined each month by flow cytometry. Each line
represents a transplanted animal.
/
bone marrow cells displayed normal DNA synthesis in response
to SCF, MGDF, and G-CSF (data not shown). Expression of the dominant
negative D57N mutant Rac2 had no demonstrable effect on DNA synthesis
in transduced WT BM cells. In contrast, Q61L mutant Rac2 as well as WT
Rac2 significantly increased DNA synthesis compared with untransduced
cell or cells transduced with the empty vector (MIEG3) (Fig.
7A). Similar results were also
found in the transduced Rac2
/
cells (data not shown). These observations suggest that Rac2 GTPase is not essential for cell proliferation in BM cells, although overexpression of Rac2 can stimulate DNA synthesis.
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Fig. 7.
Effect of Rac2 mutants on DNA synthesis and
cell apoptosis. WT BM cells expressing empty vector (MIEG3),
WT Rac2, and mutants of Rac2 (D57N and Q61L) were stimulated in the
presence of G-CSF, SCF, and MGDF (100 ng/ml each). Untransduced BM
cells were also used as the control. Panel A, cell
proliferation. [3H]Thymidine was added for 6 h at
37 °C. 5 × 104 cells were then harvested on the
filter, and -emissions were measured. Data are presented as the
mean ± S.D. of six independent samples. Panel B, cell
apoptosis. 2 × 105 cells were collected at different
time points after transduction and stained with PE-conjugated annexin
V. *, p < 0.003 D57N versus MIEG3.
Panel C, TUNEL assay. 2 × 106 cells were
collected at 7 days after transduction and stained with the in
situ cell death detection kit, TMR red (Roche). The percentage
apoptosis was determined by FACS analysis. Data are presented as the
mean ± S.D. of three experiments. *, p < 0.003 D57N versus MIEG3. **, p < 0.05 MIEG3
versus WT Rac2 (or Q61L).
/
cells were annexin V+ compared with 15% of WT
cells (p < 0.01), whereas the percentage of
PI+ was 36% of Rac2
/
cells versus 17% of
WT cells (p < 0.01, data not shown). Expression of WT
Rac2 reversed this increased cell death in Rac2
/
BM cells (data not
shown). In contrast, expression of D57N Rac2 dramatically increased
cell apoptosis in both transduced WT and Rac2
/
cells, with up to
90% annexin V+ (Fig. 7B) and PI+
(data not shown) cells at 13 days after transduction. The Q61L mutant
slightly, but not significantly, reduced the percent annexin V+ cells (Fig. 7B). In addition, D57N Rac2
significantly induced DNA fragmentation in transduced cells detected by
TUNEL assay (14.8% for D57N versus 6.0% for MIEG3,
p = 0.003, Fig. 7C). Interestingly, WT Rac2
and Q61L significantly reduced apoptosis as analyzed by TUNEL assay
(Fig. 7C). Thus in BM cells, dominant negative D57N mutant
Rac2 reduces cell survival but has no measurable effect on cell
proliferation, whereas the dominant active Q61L mutant or
overexpression of WT Rac2 increases cell proliferation and may reduce
apoptosis slightly.
/
cells (Fig. 8), again suggesting a gene
dosage effect. Because, as shown above, D57N Rac2 biochemically blocks
Rac1 GTPase activity (Fig. 4), these data support a compensatory role
for Rac1 in cultured BM cells (details discussed below). Expression of
WT Rac2 or the dominant active Q61L mutant slightly increases
chemotaxis. Interestingly in contrast with freshly isolated cells, we
observed less of a difference in cell migration between the in
vitro differentiated WT and Rac2
/
BM neutrophils, although
this difference was still significant (p < 0.05) (Fig.
8). This is possibly due to increased Rac1 activity noted after
extensive in vitro culture in stimulating growth factors
(see below).
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Fig. 8.
Chemotaxis of transduced and in
vitro differentiated bone marrow cells. Mouse bone
marrow cells (both WT and Rac2 /
genetic backgrounds) expressing
empty vector (MIEG3), WT Rac2, and mutants of Rac2 (D57N and Q61L) were
stimulated to induce myeloid cell differentiation in the presence of
G-CSF, SCF, and MGDF (100 ng/ml each) for 7 days. At the time of assay,
>90% of cells were Mac1+/Gr-1+. Chemotaxis of
these cells was examined using a modified Boyden chamber as described
under "Experimental Proceduress." 1 µM fMLP was used
as the chemoattractant. The number of migrating cells on the filter was
determined by counting five random 400× microscope fields. Data are
expressed as the mean number of cells/field ± S.D. *,
p < 0.01 MIEG3 versus WT Rac2 and Q61L
Rac2.
/
cells, using either fMLP (Fig.
9A) or PMA (Fig. 9B) as
an agonist. However, ectopic expression of WT Rac2 and Q61L Rac2 in
in vitro derived BM neutrophils had no significant affect on
superoxide production (Fig. 9). Thus, in contrast to the effects on
cell proliferation, increased expression of functional Rac protein does
not appear to enhance oxidase activity.
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Fig. 9.
NADPH oxidase activity of transduced and
in vitro differentiated myeloid cells. WT and
Rac2 /
BM cells were transduced and express empty vector (MIEG3), WT
Rac2, and mutants of Rac2 (D57N and Q61L) and were differentiated into
myeloid cells as in Fig. 8. NADPH oxidase activity of these cells was
measured by the NBT test, as described under "Experimental
Procedures." Cells were stimulated with either 1 µM
fMLP (panel A) or 100 ng/ml PMA (panel B). The
percentage of NBT+ cells on the slide was determined by
counting 200 cells in triplicate. Data are expressed as the mean ± S.D. of three experiments. *, p < 0.005 MIEG3
versus D57N in WT cells; **, p < 0.001 MIEG3 versus D57N in Rac2
/
cells.
/
mice
(22), suggesting that Rac2 has essential roles in these cellular
events. In contrast, as noted above, after 10 days of in
vitro culture and differentiation in the presence of SCF, MGDF,
and G-CSF, Rac2
/
BM-derived neutrophils showed a reduced phenotype
with respect to chemotaxis compared with WT cells after stimulation
with 1 µM fMLP (Fig. 8). To determine if this change in
cell migration phenotype was related to Rac activity, we quantified the
active (GTP-bound) Rac1 in fresh and in vitro cultured
Rac2
/
cells. Both total Rac1 protein and active (GTP-bound) Rac1
are up-regulated in cultured Rac2
/
cells (lanes 4, Fig.
10) but not in freshly prepared Rac2
/
cells (lanes 2, Fig. 10). As shown in Fig. 4 (lower
panel, lane 5), Rac1 activity as measured using the PBD
GST pull-down assay (38) in cultured Rac2
/
cells increased more
than 5-fold compared with in vitro cultured WT cells
(lane 1). The excess activity of Rac1 was also associated
with restoration of defective superoxide production (measured by the
number of NBT+ cells) in Rac2-deficient neutrophils in
response to PMA or fMLP after in vitro culture (Fig. 9).
Taken together, these data suggest that up-regulation of Rac1 occurs
with in vitro culture of Rac2
/
progenitor cells but not
in vivo, and this increase in Rac1 activity can partially
rescue Rac2-deficient phenotypes.
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Fig. 10.
Expression and activity of endogenous Rac1
GTPase in Rac2 /
BM
cells. Low density BM cells were extracted from the littermates of
WT and Rac2
/
mice. Fresh BM cells were either assayed directly or
cultured in vitro in a complete RPMI medium (see
"Experimental Procedures") for 7 days and then used for immunoblots
and PAK1 PBD GST effector pull-down assay. Whole cell lysate
(panel A) and GTP-bound proteins (panel B) were
analyzed by immunoblot using antibodies for Rac1 and total p38. The
results shown are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice have a distinct
phenotype, characterized by abnormal actin-based functions, such as
adhesion, migration, L-selectin function, phagocytosis and
degranulation, and abnormal cell survival, with increased apoptosis
after growth factor stimulation (22, 25). Rac2 also appears to be
critical for T helper 1 lymphocyte differentiation (46). In addition,
our laboratory (1) and Ambruso et al. (2) have recently
identified a mutation of human Rac2 (G169A; aspartic acid to asparagine
at position 57 (D57N)), associated with a phagocytic immunodeficiency,
demonstrating that Rac2 also plays a critical role in human blood
cells. The abnormalities in Rac2
/
hematopoietic cells occur despite
continued expression of the highly homologous Rac1 in these cells.
Differences in Rac1 and Rac2 proteins are not apparent in their known
effector domains, rather they localize mainly the carboxyl terminus
(14-17). To date, the relationship between Rac2 and Rac1
function and the basis of any specificity of Rac2 function in
hematopoietic cells are largely unknown. In addition, the role of Rac2
in pluripotent hematopoietic stem and progenitor cells and other
hematopoietic lineages remain unclear but may be critical for stem cell
migration.2
-phosphate of GTP
through its hydrogen-bonded binding of Mg2+ ion (47). As
seen previously in D57A, D57N and D57Y mutations of Ras (for review,
see Ref. 45), we have shown that D57N Rac2 demonstrates an impaired
binding to GTP but normal binding to GDP. Because active GTP-bound Rho
GTPases trigger downstream signaling pathways, the enhanced rate of GTP
dissociation of this mutant resulting in accumulation of inactive
GDP-bound form explains the loss of function of D57N protein but not
the dominant negative nature of this mutant. Because binding of GEFs
stimulates efficient GDP/GTP exchange, we examined the ability of
TrioN, a Dbl homology family member GEF specific for Rac, to bind D57N
Rac2 and stimulate its guanine nucleotide exchange. Our in
vitro biochemical analysis of recombinant expressed protein shows
that D57N Rac2 does not respond catalytically to TrioN despite
maintaining the ability to bind to TrioN. These biochemical data
strongly suggest that D57N Rac2 may act in a dominant negative fashion
by sequestering GEFs from Rac2 and other related GTPases, particularly
Rac1. Further supporting this biochemical data, when D57N Rac2 is
expressed in primary murine hematopoietic cells, clear inhibition of
not only Rac2 but also Rac1 activity was demonstrated. These data are
consistent with previous data demonstrating phenotypic changes in
NIH/3T3 cells, which express Rac1 but not Rac2, after expression of
D57N Rac2 (1).
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ACKNOWLEDGEMENTS |
---|
We thank Eva Meunier and Sharon Smoot for excellent administrative assistance. We thank Dr. Mary Dinauer and members of our laboratory for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM60523 (to Y. Z.).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: Howard Hughes
Medical Institute, 1044 West Walnut St., Rm. 402, Indianapolis, IN
46202-5225. Tel.: 317-274-8960; Fax: 317-274-8679; E-mail: dwilliam@iupui.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M010445200
2 Yang, F. C., Atkinson, S. J., Gu, Y., Borneo, J. B., Roberts, A. W., Zheng, Y., Pennington, J., and Williams, D. A. (2001) Proc. Natl. Acad. Sci. U. S. A., in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
GEF(s). guanine
nucleotide exchange factor, WT, wild type;
GST, glutathione
S-transferase;
PBD, p21 binding domain;
DTT, dithiothreitol;
FACS, fluorescence-activated cell sorting;
BM, bone marrow;
LDBM, low
density bone marrow;
hG-CSF, human granulocyte
colony-stimulating factor;
MGDF, megakaryocyte growth and development
factor;
SCF, stem cell factor;
EGFP+, enhanced green fluorescence protein-positive;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
PI, propidium iodide;
PE, phosphatidylethanolamine;
TUNEL, terminal nucleotidyl
transferase;
fMLP, formyl-methionyl-leucyl-phenylalanine;
NBT, nitro
blue tetrazolium;
PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Williams, D. A.,
Tao, W.,
Yang, F. C.,
Kim, C.,
Gu, Y.,
Mansfield, P.,
Levine, J. E.,
Petryniak, B.,
Derrrow, C. W.,
Harris, C.,
Jia, B.,
Zheng, Y.,
Ambruso, D. R.,
Lowe, J. B.,
Atkinson, S. J.,
Dinauer, M. C.,
and Boxer, L.
(2000)
Blood
96,
1646-1654 |
2. |
Ambruso, D. R.,
Knall, C.,
Abell, A. N.,
Panepinto, J.,
Kurkchubasche, A.,
Thurman, G.,
Gonzalez-Aller, C.,
Hiester, A.,
deBoer, M.,
Harbeck, R. J.,
Oyer, R.,
Johnson, G. L.,
and Roos, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4654-4659 |
3. |
MacKay, D. J.,
and Hall, A.
(1998)
J. Biol. Chem.
273,
20685-20688 |
4. |
Allen, W. E.,
Jones, G. E.,
Pollard, J. W.,
and Ridley, A. J.
(1997)
J. Cell Sci.
110,
707-720 |
5. | Bengtsson, T., Sarndahl, E., Stendahl, O., and Andersson, T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2921-2925[Abstract] |
6. |
Cox, D.,
Chang, P.,
Zhang, Q.,
Reddy, P. G.,
Bokoch, G. M.,
and Greenberg, S.
(1997)
J. Exp. Med.
186,
1487-1494 |
7. | Narumiya, S., Ishizaki, T., and Watanabe, N. (1997) FEBS Lett. 410, 68-72[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Van Aelst, L.,
and D'Souza-Schorey, C.
(1997)
Genes Dev.
11,
2295-2322 |
9. |
Hall, A.
(1998)
Science
279,
509-514 |
10. | Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270-1272[Medline] [Order article via Infotrieve] |
11. | Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve] |
12. | Sulciner, D. J., Irani, K., Yu, Z. X., Ferrans, V. J., Goldschmidt-Clermont, P., and Finkel, T. (1996) Mol. Cell. Biol. 16, 7115-7121[Abstract] |
13. | Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[Medline] [Order article via Infotrieve] |
14. |
Didsbury, J.,
Weber, R. F.,
Bokoch, G. M.,
Evans, T.,
and Synderman, R.
(1989)
J. Biol. Chem.
264,
16378-16382 |
15. | Shirsat, N. V., Pignolo, R. J., Kreider, B. L., and Rovera, G. (1990) Oncogene 5, 769-772[Medline] [Order article via Infotrieve] |
16. | Moll, J., Sansig, G., Fattori, E., and van der Putten, H. (1991) Oncogene 6, 863-866[Medline] [Order article via Infotrieve] |
17. |
Haataja, L.,
Groffen, J.,
and Heisterkamp, N.
(1997)
J. Biol. Chem.
272,
20384-20388 |
18. | Segal, A. W., and Abo, A. (1993) Trends Biochem. Sci 18, 43-47[CrossRef][Medline] [Order article via Infotrieve] |
19. | Abo, A., Pick, E., Hall, A., Totty, N., Teahan, C. G., and Segal, A. W. (1991) Nature 353, 668-670[CrossRef][Medline] [Order article via Infotrieve] |
20. | Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., and Bokoch, G. M. (1991) Science 254, 1512-1515[Medline] [Order article via Infotrieve] |
21. |
Abo, A.,
Boyhan, A.,
West, I.,
Thrasher, A. J.,
and Segal, A. W.
(1992)
J. Biol. Chem.
267,
16767-16770 |
22. | Roberts, A. W., Kim, C., Zhen, L., Lowe, J. B., Kapur, R., Petryniak, B., Spaetti, A., Pollock, J. D., Borneo, J. B., Bradford, G. B., Atkinson, S. J., Dinauer, M. C., and Williams, D. A. (1999) Immunity 10, 183-196[Medline] [Order article via Infotrieve] |
23. |
Heyworth, P. G.,
Bohl, B. P.,
Bokoch, G. M.,
and Curnutte, J. T.
(1994)
J. Biol. Chem.
269,
30749-30752 |
24. |
Dorseuil, O.,
Reibel, L.,
Bokoch, G. M.,
Camonis, J.,
and Gacon, G.
(1996)
J. Biol. Chem.
271,
83-88 |
25. | Yang, F. C., Kapur, R., King, A. J., Tao, W., Kim, C., Borneo, J., Breese, R., Marshall, M., Dinauer, M. C., and Williams, D. A. (2000) Immunity 12, 557-568[Medline] [Order article via Infotrieve] |
26. | Xu, X., Wang, Y., Barry, D. C., Chanock, S. J., and Bokoch, G. M. (1997) Biochemistry 36, 626-632[CrossRef][Medline] [Order article via Infotrieve] |
27. | Menard, L., Tomhave, E., Casey, P. J., Uhing, R. J., Snyderman, R., and Didsbury, J. R. (1992) Eur. J. Biochem. 206, 537-546[Abstract] |
28. | Williams, D. A., and Smith, F. O. (2000) Hum. Gene Ther. 11, 2059-2066[CrossRef][Medline] [Order article via Infotrieve] |
29. | Chuang, T. H., Xu, X., Quilliam, L. A., and Bokoch, G. M. (1994) Biochem. J. 303, 761-767[Medline] [Order article via Infotrieve] |
30. |
Knaus, U. G.,
Heyworth, P. G.,
Kinsella, B. T.,
Curnutte, J. T.,
and Bokoch, G. M.
(1992)
J. Biol. Chem.
267,
23575-23582 |
31. |
Zhu, K.,
Debreceni, B.,
Li, R.,
and Zheng, Y.
(2000)
J. Biol. Chem.
275,
25993-26001 |
32. | Hanenberg, H., Hashino, K., Konishi, H., Hock, R. A., Kato, I., and Williams, D. A. (1997) Hum. Gene Ther. 8, 2193-2206[Medline] [Order article via Infotrieve] |
33. | Yee, N. S., Paek, I., and Besmer, P. (1994) J. Exp. Med. 179, 1777-1787[Abstract] |
34. |
Bjorgvinsdottir, H.,
Ding, L.,
Pech, N.,
Gifford, M.,
Li, L. L.,
and Dinauer, M. C.
(1997)
Blood
89,
41-48 |
35. | Jung, V., Wei, W., Ballester, R., Camonis, J., Mi, S., Van Aelst, L., Wigler, M., and Broek, D. (1994) Mol. Cell. Biol. 14, 3707-3718[Abstract] |
36. |
Seipel, K.,
Medley, Q. G.,
Kedersha, N. L.,
Zhang, X. A.,
O'Brien, S. P.,
Serra-Pages, C.,
Hemler, M. E.,
and Streuli, M.
(1999)
J. Cell Sci.
112,
1825-1834 |
37. | Hanenberg, H., Xiao, X. L., Dilloo, D., Hashino, K., Kato, I., and Williams, D. A. (1996) Nat. Med. 2, 876-882[Medline] [Order article via Infotrieve] |
38. |
Benard, V.,
Bohl, B. P.,
and Bokoch, G. M.
(1999)
J. Biol. Chem.
274,
13198-13204 |
39. | Lamarche, N., Tapon, N., Stowers, L., Burbelo, P. D., Aspenstrom, P., Bridges, T., Chant, J., and Hall, A. (1996) Cell 87, 519-529[Medline] [Order article via Infotrieve] |
40. |
Kreck, M. L.,
Uhlinger, D. J.,
Tyagi, S. R.,
Inge, K. L.,
and Lambeth, J. D.
(1994)
J. Biol. Chem.
269,
4161-4168 |
41. |
Xu, X.,
Barry, D. C.,
Settleman, J.,
Schwartz, M. A.,
and Bokoch, G. M.
(1994)
J. Biol. Chem.
269,
23569-23574 |
42. | Reibel, L., Dorseuil, O., Stancou, R., Bertoglio, J., and Gacon, G. (1991) Biochem. Biophys. Res. Commun. 175, 451-458[Medline] [Order article via Infotrieve] |
43. |
Scita, G.,
Tenca, P.,
Frittoli, E.,
Tocchetti, A.,
Innocenti, M.,
Giardina, G.,
and Di Fiore, P. P.
(2000)
EMBO J.
19,
2393-2398 |
44. |
Bokoch, G. M.,
and Der, C. J.
(1993)
FASEB J.
7,
750-759 |
45. | Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Li, B., Yu, H.,
Zheng, W.,
Voll, R.,
Na, S.,
Roberts, A. W.,
Williams, D. A.,
Davis, R. J.,
Ghosh, S.,
and Flavell, R. A.
(2000)
Science
288,
2219-2222 |
47. | Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J., and Wittinghofer, A. (1989) Nature 341, 209-214[CrossRef][Medline] [Order article via Infotrieve] |
48. | Felts, K., Bauer, J. C., and Vaillancourt, P. (1999) strategies 12, 74-77 |