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HEMATOPOIESIS
From Howard Hughes Medical Institute;
Herman B Wells Center for Pediatric Research, Department of Pediatrics,
Department of Medicine, and Department of Anatomy, Indiana University
School of Medicine, Indianapolis, IN; and Division of Experimental
Hematology, Children's Hospital Research Foundation, Cincinnati, OH.
Rac GTPases regulate a wide variety of cellular processes
including actin cytoskeleton organization, gene expression, cell-cycle progression, and apoptosis. Here we report that the TRQQKRP
motif of Rac2 located near the C-terminus, a region of sequence
disparity among Rac proteins, is essential for complementation of Rac2
function in Rac2-deficient cells. Deletion of this sequence can also
intragenically suppress the dominant-negative Rac2D57N
mutation in a variety of functional assays. In Rac2-deficient cells, expression of TRQQKRP-deleted Rac2 protein is unable to completely rescue migration and nicotinamide adenine dinucleotide phosphate oxidase deficiencies previously described in these
cells. In fibroblasts, the Rac2D57N mutant phenotypes of
abnormal proliferation, cell morphology, and membrane ruffling are
suppressed by the TRQQKRP motif deletion. In myeloid hematopoietic
cells, the deletion of the TRQQKRP motif eliminates a
Rac2D57N-induced block in in vitro differentiation of
neutrophils not previously described with this mutant. Mechanistically,
deletion of the TRQQKRP motif results in diminished geranylgeranylation and delocalization of intracellular Rac2 protein. Taken together, these
results indicate that the TRQQKRP motif in Rac2 protein is required for
efficient prenylation and correct intracellular localization of Rac2
protein and is essential for Rac2 to mediate a variety of its biologic
functions. These data suggest that precise localization of Rac2 protein
in intracellular compartments and/or with other proteins/lipids is a
prerequisite for its diverse functions.
(Blood. 2002;100:1679-1688) The Rho GTPases belong to the Ras superfamily of
small GTP-binding proteins that have a molecular mass of 20 to 30 kDa.
Rho GTPases have been shown to play essential roles in a wide variety of cellular functions, such as the regulation of the actin
cytoskeleton, membrane trafficking, transcriptional regulation, oxidant
generation, cell growth control, chemotaxis, and cell
adhesion.1 To date, there are at least 16 members
identified in the mammalian Rho subfamily, including Rho, Rac, and
Cdc42. By cycling between inactive GDP- and active GTP-bound
conformations, the small GTPases function as critical relays in the
transduction of signals originating from cell-surface receptors. In
fibroblasts, Rac regulates actin reorganization to produce lamellipodia
and membrane ruffles, transcriptional activation, cell proliferation,
transformation, and apoptosis.2-7
Members of the Rho GTPases are subject to posttranslational
modifications of prenylation, proteolysis, and carboxylmethylation. Mammalian Rac and most other Rho GTPases are geranylgeranylated, and
prenylation occurs at a conserved cysteine in the carboxy terminal
motif of the sequence CAAL, where C is cysteine, A is an aliphatic
amino acid, and L is leucine. Subsequent to prenylation, the carboxy
terminal tripeptide (AAL) is removed by proteolysis, and the newly
generated carboxy terminal amino acid is methylated.8-10 Protein prenylation is thought to be crucial for targeting GTPases to
cellular membranes, protein-protein interactions, and
membrane-associated protein trafficking.11-13
In mammals, 3 isoforms of Rac proteins have been identified:
Rac1, Rac2, and Rac3. The 3 Rac isoforms share a high degree of amino
acid identity (> 89% overall) but differ substantially in tissue
distribution and levels of expression. Rac1 and Rac3 proteins are
widely expressed in different tissues, including cells of hematopoietic
origin, while the expression of rac2 gene is
restricted to cells of hematopoietic origin.14-17 In
addition, Rac2 differs from Rac1 and Rac3 in the primary sequence
located upstream of the C-terminal prenylation site. Rac2-deficient
mice display defects in neutrophil, stem cell, and mast cell functions, including superoxide production, chemotaxis, adhesion, degranulation, and F-actin generation, as well as abnormalities in host defense in
spite of continued expression of Rac1 in these
cells.18,19,43 These data suggest that the area of
sequence divergence may play a key role in the functions of Rac2. A
dominant-negative mutation (D57N) of Rac2 is associated with human
phagocytic immunodeficiency, suggesting that Rac2 also plays a critical
role in human phagocyte cells.20,21
The structure and most of the functional domains of Rho GTPases,
including GTP binding and hydrolysis domains, effector domains, an
insert domain, GEF interaction domains, and a prenylation site, are
well studied and characterized.22-24 However, the motif
located just upstream of the C-terminal prenylation site, an area of
sequence diversity among Rac proteins, has been investigated less
thoroughly. The domain near the C-terminal prenylation site found in
Rac1, the related Rho GTPase, Cdc42, and also in K-Ras proteins is
termed the polybasic domain, since it usually consists of 4 to 6 basic residues. The polybasic domain in Rac1 is composed of 6 consecutive basic residues, whereas 3 of the 6 residues in the corresponding motif
of Rac2, RQQKRP, are replaced with neutral amino acids. Rac1, through
its polybasic domain, has been shown to bind to and stimulate the
kinase activity of p21-activated kinase (PAK) more efficiently
than Rac2.25 Moreover, it has been shown that the
polybasic domain of Rac1 but not the TRQQKRP motif of Rac2 is important
for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase
activation in cell-free systems.26,27 The polybasic domain
of Rac1 also has been shown to be required for the oligomerization of
Rac1.28 However, it is still not clear to what extent the TRQQKRP motif in Rac2 is critical to the intracellular localization and
the functions of this protein.
In this study, we have shown that a TRQQKRP motif deletion mutant
of Rac2 cannot rescue phenotypic abnormalities present in Rac2-deficient primary myeloid cells. The same deletion, placed in the
dominant-negative mutant D57N Rac2, can intragenically suppress the
phenotypic abnormalities of this mutant in a variety of functional
assays. Furthermore, deletion of the TRQQKRP motif results in reduced
geranylgeranylation and the delocalization of intracellular Rac2
protein. Taken together, these results indicate that the TRQQKRP motif
is required for correct intracellular localization of Rac2 protein and
is essential for the biologic functions of Rac2, suggesting strongly
that precise localization of Rac2 protein in the intracellular
compartments and/or interactions of Rac2 with other proteins are
prerequisites for its diverse biologic functions.
Construction of retroviral vectors, transfections, and
infection
Phoenix-Eco and Phoenix-Ampho packaging cell lines (ATCC, Manassas, VA)
were transfected with various retroviral constructs using LipofectAMINE
reagent (GIBCO BRL Life Technologies, Rockville, MD). The titers of the
retroviral supernatants collected from the transfected Phoenix cells
were approximately 5 × 105 colony-forming units per mL
(CFU/mL). Bone marrow (BM) mononuclear cells were transduced twice on
fibronectin fragment CH-296 with the retroviral supernatants as
described previously.21,29 NIH3T3 cells were infected in
the presence of polybrene (5.3 µg/mL). The transduced
EGFP+ cells were isolated by fluorescence-activated
cell-sorter (FACS; FACStar Plus; Becton Dickinson, Mountain View, CA)
48 hours after the second infection. NIH3T3 cells were obtained from
American Type Culture Collection (ATCC; Manassas, VA). The cells were
cultured in Dulbecco modified Eagle medium (DMEM; GIBCO BRL
Life Technologies) supplemented with 10% fetal bovine serum (FBS;
HyClone, Logan, UT), 4 mM L-glutamine, 100 U/mL penicillin,
and 100 µg/mL streptomycin (P/S; GIBCO BRL Life Technologies).
Isolation, culture, and in vitro differentiation of bone
marrow cells
Quantitation of NADPH oxidase activity Reduction of nitroblue tetrazolium (NBT) as a measure of superoxide production was assayed as described with minor modifications.30 Briefly, the transduced and differentiated normal BM cells were added to 800 µL of 0.1% NBT (Sigma, St Louis, MO) in the presence of 1.6 × 10 6 M
phorbol myristate acetate (PMA). The cells were then incubated for 20 minutes at 37°C, pelleted, resuspended in 150 µL PBS, and cytocentrifuged onto a slide. After Safranin-O (Sigma)
counterstain, the cells were examined by light microscopy to
assay for dark purple deposits. The number of NBT+ cells
and the total number of cells per high-power field were counted in at
least 5 independent fields per sample, and the percentage of
NBT+ cells was calculated from > 300 cells. For
complementation study, a different protocol was used. Briefly,
transduced rac2/ BM cells
(2~5 × 104) were seeded onto chamber slides in 750 µL of Iscove modified Dulbecco medium (IMDM) containing P/S,
and incubated for 1 hour at 37°C to allow cells to adhere to the
slide. Subsequently, 200 µL of saturated NBT solution containing 10 µM N-formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma)
were added to the cells, and the cells were incubated for 20 minutes at
37°C. After the cells were washed with cold PBS, they were fixed with
methanol and stained with Safranin-O. The percentage of
NBT+ cells was determined, in duplicate, by evaluating a
total of 200 cells.
Chemotaxis Low-density BM cells isolated from rac2/ mice were transduced with vector, wt
Rac2, Rac2 CT, or Rac2M, and
GFP+ cells were sorted by FACS. The sorted cells were then
cultured for 7 to 10 days in IMDM supplemented with 10% FCS, 2% P/S,
100 ng/mL stem cell factor (SCF), 100 ng/mL megakaryocyte growth and development factor (MGDF), and 100 ng/mL granulocyte
colony-stimulating factor (G-CSF). Chemotaxis of the in vitro
differentiated neutrophils was assayed using a 48-well microchemotaxis
chamber (Neuro Probe, Cabin John, MD) as described
previously18 by counting 4 randomly chosen fields per well
using 1 µM chemoattractant fMLP.
Hematopoietic progenitor assays Transduced and sorted low-density BM cells were plated at 2 × 104 cells/mL in MethoCult M3231 methycellulose medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 100 ng/mL rSCF, 100 U/mL mIL-3, 100 U/mL hIL-6, 10 ng/mL human granulocyte colony-stimulating factor (hG-CSF, Amgen), 100 ng/mL human megakaryocyte growth and differentiation factor (MGDF, Amgen), and 4 U/mL human erythropoietin (Epo, Amgen). Cultures were incubated at 5% CO2 and 37°C for 11-12 days and then scored using an inverted microscope. For each experiment, triplicate plates were scored per data point. The colony sizes were visually estimated under the microscope.Scanning electron microscopy Transduced and in vitro differentiated BM cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for about 1 hour at room temperature followed by one week at 4°C. The cells were then allowed to settle by gravity overnight onto polylysine-coated coverslips at 4°C, cross-linked to polylysine, and postfixed for 30 minutes with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4). Subsequently, the samples were dehydrated in a graded series of ethanol, critical point dried, coated with 40 nm of Au/Pd with a SPUTTER coater (Hummer V, Anatech, Springfield, VA), and examined in an AMR 1000A scanning electron microscope (SEM) (Amray, Burlington, MA).Immunofluorescence Transduced NIH3T3 cells were cultured, serum starved for 20 hours, treated with platelet-derived growth factor (PDGF) (Pepro Tech, Rocky Hill, NJ) (5 ng/mL) for 8 minutes, and stained for actin filaments with rhodamine (TRITC) phalloidin as described.21 Images were visualized and recorded using a Bio-Rad confocal microscope. For localization of Flag-tagged Rac2 proteins, cells were treated with PDGF and stained with the purified anti-Flag M2 monoclonal antibody (Sigma) (10 µg/mL) for 90 minutes at room temperature followed by 40 minutes of incubation with affinity purified TRITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). The cells were then visualized on a Zeiss epifluorescent microscope and recorded with a Spot RT digital imaging system (Diagnostic Instrument, Sterling Heights, MI) as well as Bio-Rad confocal microscope (Hercules, CA).Western blotting and flow cytometric analysis The Western blot analysis of the transduced and FACS-sorted cells was performed as previously described using anti-Flag M2 monoclonal antibody (Sigma) and an anti-Rac monoclonal antibody (BD Transduction Laboratories, San Diego, CA).31 FACS analysis was used to determine the expression of specific cell-surface markers on the transduced, sorted, and in vitro differentiated BM cells. Briefly, the cells were first incubated with 10% normal rat serum to block the nonspecific binding sites then incubated with phycoerythrin-labeled isotype control antibody or monoclonal antibodies against c-Kit, Sca-1, Mac-1, or Gr-1 (Pharmingen, San Diego, CA) (~8-38 µg/mL). The labeled cells were analyzed with a FACScan flow cytometer (Becton Dickinson). In addition, FACS analysis was performed to estimate the percentage of neutrophils present in the EGFP+ and differentiated BM cells based on size (forward scatter).Subcellular fractionation Transduced and sorted NIH3T3 cells were grown to 90% confluence and scraped in hypotonic homogenization buffer (15 mM Tris, pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 0.25M sucrose) supplemented with protease inhibitor mixture. Cells were homogenized using tight-fitting Dounce homogenizers (Wheaton Science Products, Millville, NJ) (50 strokes), and the homogenates were clarified by centrifugation at × 1000g for 10 minutes at 4°C to remove unbroken cells and nuclei. The resulting supernatants were centrifuged at × 100 000g for 90 minutes to yield cytosol (S100) and total membrane (P100) fractions. Equivalent proportions of S100 and P100 fractions (cell equivalents) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with anti-Flag M2 antibody as described above.Prenylation of Rac2 proteins Prenylation of wild-type and mutant Rac2 proteins was assessed by determining the amount of label, derived from [3H] mevalonolactone, incorporated into the Rac2 proteins.32,33 Briefly, at 60 hours after seeding, the transduced and sorted NIH3T3 cells were incubated in Dulbecco modified Eagle medium containing 40 µM mevastatin (Calbiochem, San Diego, CA) for 4 hours. This was followed by a 12-hour incubation at 37°C with (R, S)-[3H] mevalonolactone (78 µCi/mL [2.886 MBq/mL], 24 Ci/mmol [8.88 × 1011 Bq/mmol]; NEN Life Science Products, Boston, MA) in the presence of 40 µM mevastatin. The cells were solubilized in 1% Triton X-100 lysis buffer containing protease inhibitor mixture and immunoprecipitated with anti-Flag M2 affinity gel (Sigma). The immunoprecipitates were analyzed by SDS-PAGE, and the gels were fluorographed with EN3HANCE (NEN Life Sciences Products).
To examine whether the TRQQKRP motif located near the C-terminus
of Rac2 is crucial for biologic function, we first generated a Rac2
mutant in which the TRQQKRP motif is deleted (Rac2
Rac stimulates growth factor-induced membrane ruffling in fibroblast
cells, whereas dominant-negative mutants of Rac proteins block this
response.1,34 To assess whether the TRQQKRP motif is
required for Rac2 to stimulate membrane ruffling, we examined the actin
cytoskeleton reorganization by TRITC-phalloidin staining in NIH3T3
cells transduced with each respective retrovirus. As noted previously,
PDGF clearly induced actin reorganization and membrane ruffles at the
cell periphery of a cell transduced with a control vector (Figure
3A, at arrowheads and the insert)
compared to unstimulated cells (Figure 3B), whereas expression of
Rac2D57N severely reduced membrane ruffling at the cell
periphery (Figure 3C). The majority of Rac2D57N-expressing
cells exhibited irregular actin cytoskeleton organization in their
cytoplasm, characterized by punctate phalloidin staining over the
perinuclear region and irregular stress fibers, which existed in the
presence or absence of PDGF stimulation (Figure 3C-D). Importantly, the
membrane ruffling at the cell periphery was largely restored by
expression of either Rac2D57N
Rac proteins have been shown to be essential for activation of the
NADPH oxidase enzyme complex that produces
superoxide,49,50 and we have previously demonstrated that
expression of Rac2D57N inhibits superoxide production
induced by some agonists in normal human neutrophils.21 To
assess NADPH oxidase function in neutrophils derived from transduced
and differentiated wild-type BM cells expressing Rac mutants, an NBT
test was performed. The results shown in Table
1 indicated that the number of
NBT+ cells in Rac2D57N cultures was
significantly reduced as compared to that in vector control. In
contrast, expression of either Rac2D57N
Previous studies from our laboratory have demonstrated significant
negative effects of D57N Rac2 on hematopoietic cell growth in vitro and
reconstitution in vivo.35 To determine whether the TRQQKRP
motif is required for this effect of Rac2D57N, we expressed
Rac2D57N
One of the major functions of Rac proteins is the organization of the
actin cytoskeleton, which contributes to cell morphology and shape. To
test whether expression of Rac2D57N causes abnormal surface
morphology in myeloid cells and whether the deletion of the TRQQKRP
motif can suppress the Rac2D57N morphological phenotypes,
we examined transduced myeloid cells expressing the control vector,
Rac2D57N, and Rac2D57N
In addition to morphological characterization, expression of specific
cell-surface antigens of hematopoietic progenitor cells, including
c-Kit (CD117) and Sca-1 (Ly-6A/E), and of the lineage-specific and
differentiation-associated surface antigens, such as Gr-1 (Ly-6G) and
Mac-1 (CD11b), was examined in the transduced BM cells cultured for 15 days in SCF, IL-3, and IL-6. FACS analyses demonstrated that the
majority of the cells in vector control cultures expressed Gr-1 (67%)
or Mac-1 (68%), whereas fewer cells expressed c-Kit (36%) or Sca-1
(31%), in agreement with the histological analysis. In contrast,
significantly fewer cells in Rac2D57N culture expressed
Gr-1 (47%) or Mac-1 (47%), whereas the majority were positive for
c-Kit (60%) or Sca-1 (70%), suggesting an increase in the number of
immature myeloid cells. Moreover, the level of Sca-1 expression in
Rac2D57N cells was increased as compared to that in vector
control, Rac2D57N
To better assess the effect of Rac2D57N on myeloid
differentiation as well as the impact of deletion of TRQQKRP motif on
the effect of Rac2D57N in this aspect, the transduced and
sorted BM cells were assayed for colony forming capacity in standard
methylcellulose assays. As expected, bone marrow transduced with the
empty vector exhibited both granulocyte-macrophage (GM) and mast cell
colony morphologies (Figure 7A-B). In
contrast, the morphology of the colonies generated from the
Rac2D57N-expressing progenitors was severely compacted
regardless of the colony sizes, suggesting reduced cell migration
within the colony (Figure 7C-D). Moreover, the number of myeloid
progenitor colonies in Rac2D57N culture was significantly
increased as compared to that in control cell cultures (Figure 7I). In
contrast, the colony morphology and number of colonies generated from
either the Rac2D57N
To further confirm the results obtained using Rac2D57N
Data presented thus far demonstrate that deletion of TRQQKRP motif can
intragenically suppress a variety of phenotypes associated with
expression of Rac2D57N and that the TRQQKRP deletion mutant
cannot fully complement the rac2
To further investigate whether the membrane association of Rac2
proteins was affected by deletion of TRQQKRP motif and to validate the
localization results, we performed subcellular fractionations of NIH3T3
cells expressing EGFP alone, Rac2
To determine whether this change in membrane localization is the result
of defective prenylation of Rac2 proteins due to the TRQQKRP motif
deletion, the transduced cells expressing EGFP alone, Rac2
Rho GTPase members of the Ras superfamily have been shown to control actin cytoskeleton organization as well as signaling pathways and gene transcription in eukaryotic cells.1,38 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.39 The mammalian Rho-like GTPases consist of several distinct proteins, including the Rac subfamily.40 Among 3 identified Rac proteins, all of which share very high sequence homology, Rac2 is expressed only in hematopoietic cells,41 whereas Rac1 and Rac3 are widely expressed. We have previously generated Rac2-deficient mice by homologous recombination18 and demonstrated phenotypic abnormalities in multiple blood lineages derived from these mice, including neutrophils, mast cells, lymphocytes, and stem/progenitor cells.18,19,42,43 These phenotypic abnormalities occur in spite of continued expression of Rac1 (and presumably Rac3), suggesting that Rac2 subserves distinct functions in blood cells in spite of the high degree of sequence homology between the Rac proteins. Previous studies have suggested that Rac1 and Rac2 vary in efficiency of interactions with proteins of the NADPH oxidase complex26,27 and differ in binding affinities with certain effector proteins.25 In all of these studies, the region spanning the polybasic domain of Rac1 has been implicated. More recently, Zhang et al have demonstrated that oligomerization of Rac1 is mediated by this domain.28 This region is also critical for function of other GTPases, such as Cdc42, and represents the region of greatest sequence divergence among the Rac proteins. In Rac2, the sequence analogous to the polybasic domain of Rac1 is interrupted by several neutral amino acids. This TRQQKRP motif located near the C-terminus of Rac2 has not been previously studied in detail. Here we have used primary cells deficient in Rac2 to demonstrate that the TRQQKRP motif is essential for normal functioning on Rac2 in chemotaxis and superoxide generation. In addition, deleting this sequence can intragenically suppress a variety of dominant-negative phenotypes of Rac2D57N including abnormal superoxide production, abnormal cytoskeletal organization and function, and cell growth. Finally, the TRQQKRP motif of Rac2 is required for efficient prenylation and for correct intracellular localization of Rac2 protein. These data strongly suggest that precise localization of Rac2 proteins in various intracellular compartments and/or proper interactions with other proteins are prerequisites for the diverse functions of Rac2. In addition, data presented here suggest that Rac proteins are involved in normal myeloid cell differentiation, although the specific Rac protein involved is not yet clear. It is surprising that deletion of the TRQQKRP sequence with retention of the CSLL sequence (representing the CAAL motif) completely abrogates the functions of Rac2D57N proteins and results in impaired prenylation and delocalization of Rac2 proteins. These data differ from studies of Ras, where deletion of the hypervariable sequence with retention of the CaaX motif does not affect prenylation. Ras proteins deleted of the hypervariable sequence possess transforming ability.44-47 The implication of these data are that with regard to the function of sequences near the conserved CaaX box, each member of the Ras superfamily is unique and that sequence-dependent functions established for one member may not be readily applicable to other proteins within the same superfamily. At present it remains unclear how deletion of the TRQQKRP sequence impairs the prenylation of Rac2 proteins. It is possible that for Rac2, both the CXXL and TRQQKRP motifs constitute the biologic substrate for type I geranylgeranyl protein transferases (GGTase I). Thus, deletion of TRQQKRP motif structurally or spatially changes that region of the Rac2 proteins so that it becomes a poor substrate for GGTase I. It is also possible that deletion of TRQQKRP alters the location of Rac2 proteins within the cell so that the mutants are physically unavailable to GGTase I. Although the TRQQKRP motif of Rac2, similar to the polybasic domain contained in Rac1, Cdc42, and K-ras, is critical for normal functioning of the protein, this motif appears to function in a different fashion than these other polybasic domains. First, the polybasic domain of these other proteins has been thought to facilitate plasma membrane binding and enable cell transformation via ionic interactions with a negatively charged domain of a target protein and/or negatively charged membrane phospholipids. The polybasic domain in Rac1 or K-ras consists of 6 consecutive basic residues, whereas 3 of the 6 residues in the TRQQKRP motif of Rac2 are replaced with neutral amino acids, which reduces the ionic charges significantly. Second, all 6 lysines of the K-ras polybasic domain may be required for efficient transforming activity, since K-ras mutants containing even 2 Lys to Gln substitutions (2Q) have been shown to drastically reduce transforming activity. Moreover, 3Q and 4Q K-ras mutants had a significant decrease in membrane association.32,48 Third, superoxide anion production in Rac-dependent cell-free systems has been shown to be inhibited by the Rac1(178-188) peptide that spans the polybasic domain, but not by the corresponding Rac2(178-188) peptide.26,27 Finally, the TRQQKRP motif of Rac2 and the polybasic domain of Rac1 have been shown to possess different binding affinities and kinase-stimulating activities toward one of the effector proteins, PAK.25 More recently, Michaelson et al37 have demonstrated that the sequences upstream of the CAAL motive in Rac1 are sufficient to direct subcellular localization to the plasma membrane and endomembranes in live cells. Data presented in this paper demonstrate that the sequences contained within this motif play important roles in the function of Rac2 in hematopoietic cells and are required for efficient prenylation and for correct localization of Rac2 proteins. These results may explain, in part, the specificity of Rac2 functions in these cells. Such specificity would imply that the TRQQKRP motif is critical in specifying protein-lipid and/or protein-protein interactions and subcellular localization. Therefore, some specificity of Rac2 protein function may be determined by localization of this protein in specific intracellular microenvironments and by interactions with other proteins at the right place.
We are indebted to Merv Yoder for his invaluable advice and helpful comments on the manuscript and to Gillian B. Bradford for her excellent advice and help on flow cytometry. We are also grateful to Philip Blomgren for his help with composing the SEM images and to Susan Rice and Jeffrey Lay for their technical assistance on the FACStar sorting. We thank Sharon Smoot, Eva Meunier, and Kerry Holleran for administrative assistance.
Submitted October 30, 2001; accepted April 17, 2002.
Supported by National Institutes of Health grant ROI DK59955.
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: David A. Williams, Division of Experimental Hematology, Children's Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail: david.williams{at}chmcc.org.
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© 2002 by The American Society of Hematology.
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  L. A. Samayawardhena, R. Kapur, and A. W. B. Craig Involvement of Fyn kinase in Kit and integrin-mediated Rac activation, cytoskeletal reorganization, and chemotaxis of mast cells Blood, May 1, 2007; 109(9): 3679 - 3686. [Abstract] [Full Text] [PDF]
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 Y. Tian and M. V. Autieri Cytokine expression and AIF-1-mediated activation of Rac2 in vascular smooth muscle cells: a role for Rac2 in VSMC activation Am J Physiol Cell Physiol, February 1, 2007; 292(2): C841 - C849. [Abstract] [Full Text] [PDF]
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 A. Yamauchi, C. C. Marchal, J. Molitoris, N. Pech, U. Knaus, J. Towe, S. J. Atkinson, and M. C. Dinauer Rac GTPase Isoform-specific Regulation of NADPH Oxidase and Chemotaxis in Murine Neutrophils in Vivo: ROLE OF THE C-TERMINAL POLYBASIC DOMAIN J. Biol. Chem., January 14, 2005; 280(2): 953 - 964. [Abstract] [Full Text] [PDF]
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R. Nijhara, P. B. van Hennik, M. L Gignac, M. J. Kruhlak, P. L. Hordijk, J. Delon, and S. Shaw Rac1 Mediates Collapse of Microvilli on Chemokine-Activated T Lymphocytes J. Immunol., October 15, 2004; 173(8): 4985 - 4993. [Abstract] [Full Text] [PDF]
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M. R. Saadatzadeh, K. Bijangi-Vishehsaraei, P. Hong, H. Bergmann, and L. S. Haneline Oxidant Hypersensitivity of Fanconi Anemia Type C-deficient Cells Is Dependent on a Redox-regulated Apoptotic Pathway J. Biol. Chem., April 16, 2004; 279(16): 16805 - 16812. [Abstract] [Full Text] [PDF]
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R. van Bruggen, E. Anthony, M. Fernandez-Borja, and D. Roos Continuous Translocation of Rac2 and the NADPH Oxidase Component p67phox during Phagocytosis J. Biol. Chem., March 5, 2004; 279(10): 9097 - 9102. [Abstract] [Full Text] [PDF]
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E. Werner GTPases and reactive oxygen species: switches for killing and signaling J. Cell Sci., January 15, 2004; 117(2): 143 - 153. [Abstract] [Full Text] [PDF]
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D. Pradip, X. Peng, and D. L. Durden Rac2 Specificity in Macrophage Integrin Signaling: POTENTIAL ROLE FOR Syk KINASE J. Biol. Chem., October 24, 2003; 278(43): 41661 - 41669. [Abstract] [Full Text] [PDF]
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P. B. van Hennik, J. P. t. Klooster, J. R. Halstead, C. Voermans, E. C. Anthony, N. Divecha, and P. L. Hordijk The C-terminal Domain of Rac1 Contains Two Motifs That Control Targeting and Signaling Specificity J. Biol. Chem., October 3, 2003; 278(40): 39166 - 39175. [Abstract] [Full Text] [PDF]
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M. Glogauer, C. C. Marchal, F. Zhu, A. Worku, B. E. Clausen, I. Foerster, P. Marks, G. P. Downey, M. Dinauer, and D. J. Kwiatkowski Rac1 Deletion in Mouse Neutrophils Has Selective Effects on Neutrophil Functions J. Immunol., June 1, 2003; 170(11): 5652 - 5657. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-MEM containing 20% FBS (HyClone),
4mM L-glutamine, P/S (GIBCO BRL Life
Technologies), recombinant rat stem cell factor (rSCF, 100 ng/mL;
Amgen, Thousands Oaks, CA), recombinant murine interleukin-3 (mIL-3,
100 U/mL), and recombinant human interleukin-6 (hIL-6, 100 U/mL; both
Pepro Tech, Rocky Hill, NJ). To assay neutrophil differentiation, the
cultured BM cells were cytocentrifuged onto a slide, stained with
Wright-Giemsa, and examined and photographed using a light microscope.
The percentage of abnormal neutrophils was determined based on
microscopic enumeration of more than 200 cells in at least 5 independent fields per sample.
