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Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency

2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês

10.1074/jbc.m010445200

1083-351X

Yi Gu, Baoqing Jia, Feng‐Chun Yang, Maria D’Souza, Chad E. Harris, C W Derrow, Yi Zheng, David A. Williams,

Pancreatic function and diabetes

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 O2− responses to formyl-methionyl-leucyl-phenylalanine, reflecting the same cellular phenotype in these differentiated cells as those described previously in patient cells. These data suggest that the phenotypic abnormalities associated with D57N Rac2 may involve not only neutrophil cellular functions, but also abnormal cell survival in other hematopoietic cells and that overexpression of Rac leads to increased proliferation of normal cells in vitro, whereas deficiency of Rac leads to increased apoptosis. 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 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 mdithiothreitol (DTT), 1 mm MgCl2, and 5% glycerol at −80 °C. [γ-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 (29Chuang T.H. Xu X. Quilliam L.A. Bokoch G.M. Biochem. J. 1994; 303: 761-767Crossref PubMed Scopus (38) Google Scholar,30Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar). 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 mmMgCl2) to the preloaded reaction mixture (26Xu X. Wang Y. Barry D.C. Chanock S.J. Bokoch G.M. Biochemistry. 1997; 36: 626-632Crossref PubMed Scopus (29) Google Scholar), and the remaining bound radioactivity was examined at different time points by filtration (Midwest Scientific, Valley Park, MO). 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. Complex formation of His-tagged WT Rac2 and D57N Rac2 with GST-TrioN was carried out as described for the Dbl-G-protein interactions (31Zhu K. Debreceni B. Li R. Zheng Y. J. Biol. Chem. 2000; 275: 25993-26001Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). 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. WT mouse Rac2 and mutant Rac2 (human D57N and Q61L) cDNAs were cloned into a modified murine stem cell virus-based bicistronic vector (MIEG3; (1Williams 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. Boxer L. Blood. 2000; 96: 1646-1654PubMed Google Scholar)) 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. (1Williams 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. Boxer L. Blood. 2000; 96: 1646-1654PubMed Google Scholar). 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. (48Felts K. Bauer J.C. Vaillancourt P. strategies. 1999; 12: 74-77Google Scholar). High titer E86 clones (>1 × 105/ml) were used to collect viral supernatant to infect mouse bone marrow cells. Whole bone marrow was collected from WT and Rac2−/− 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. Virus-mediated LDBM transduction was performed as described previously (32Hanenberg H. Hashino K. Konishi H. Hock R.A. Kato I. Williams D.A. Hum. Gene Ther. 1997; 8: 2193-2206Crossref PubMed Scopus (178) Google Scholar). 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). 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 mmMgCl2, 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 GTPγS or 1 mm GDP. The loading was stopped by the addition of MgCl2 to 30 mm (30Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar). 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. 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 (33Yee N.S. Paek I. Besmer P. J. Exp. Med. 1994; 179: 1777-1787Crossref PubMed Scopus (151) Google Scholar). 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 mmHEPES/NaOH, pH 7.4, 140 mm NaCl, 2.5 mmCaCl2) 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. 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. (22Roberts 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. Williams D.A. Immunity. 1999; 10: 183-196Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). 10−6 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 ra