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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00853

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Rac2^D57N, a dominant inhibitory Rac2 mutant that inhibits p38 kinase signaling and prevents surface ruffling in bone-marrow-derived macrophages

Amy N. Abell^1,*,, Aimee M. DeCathelineau⁴, Scott A. Weed², Daniel R. Ambruso³, David W. Riches⁴ and Gary L. Johnson^1,*,

¹ Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262, USA
² Department of Craniofacial Biology, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262, USA
³ Bonfils Blood Center and the Department of Pediatrics, University of Colorado School of Medicine, Denver, CO 80230, USA
⁴ Program in Experimental Pathology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA

View larger version (29K): [in a new window]   Fig. 1. Effect of Rac2wt and Rac2D57N expression in bone-marrow-derived macrophages on the actin cytoskeleton and the microtubule networks. Macrophages plated on coverslips were transfected with expression constructs for Rac2wt in pIRES2-EGFP (A-D,G), Rac2D57N in pIRES2-EGFP (E,F), empty vector pIRES2-EGFP (H) or FLAG-Rac2D57N in pCDNA3.1 (I-L). Cells were incubated for 24 hours in macrophage medium and treated as described in Materials and Methods. F-actin was stained with rhodamine-phalloidin (A,C-E,J,L). Microtubules were immunostained with an antibody to -tubulin (F-H). Transfected cells were identified by EGFP expression (A-H) or by immunostaining for FLAG (I-L). Arrowheads indicate transfected cells. Arrows indicate colocalization of Flag-Rac2D57N and F-actin at the cell membrane. Asterisks indicate colocalization of Rac2D57N with aggregates of F-actin. Scale bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 2. Rac2D57N expression in RAW macrophages promotes an elongated morphology. RAW cells were transfected with indicated expression constructs. IRES-EGFP-vector-, Rac2wt- and Rac2D57N-expressing cells were identified by EGFP expression. FLAG-tagged Rac1wt, Rac1Q61L, Rac1D57N and Rac1T17N were identified by immunostaining for FLAG. F-actin was visualized with rhodamine phalloidin. Scale bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 3. Rac2D57N expression in bone-marrow-derived macrophages inhibits M-CSF-stimulated surface ruffling. (A) M-CSF stimulates Rac activation in primary macrophages. Macrophages deprived of MCSF for 3 hours were stimulated for the indicated times with 100 ng ml-1 of recombinant M-CSF. Cell lysates were incubated with GST-PAKcrib, and analysed with a monoclonal antibody to Rac. Rac activity was normalized to total Rac expression and data are expressed as the fold stimulation of Rac activation compared with basal activity. Representative blots from three experiments are shown. (B) F-Actin staining of M-CSF-starved and M-CSF-stimulated macrophages expressing Rac2wt. F-actin was visualized with rhodamine-phalloidin. Scale bar, 10 µm. (C-E). Macrophages were transfected with empty vector, Rac2wt or Rac2D57N in pIRES2-EGFP treated as described above. Transfected cells were identified by EGFP expression. F-Actin was visualized with rhodamine-phalloidin. Quantitation of transfected cells with actin structures is shown. Data are expressed as mean±range of the percentage of transfected cells with actin structures from two independent experiments. A minimum of 50 transfected cells was examined for each condition.

View larger version (26K): [in a new window]   Fig. 4. Rac2D57N expression in bone-marrow-derived macrophages inhibits M-CSF-stimulated macropinocytosis. (A) M-CSF stimulates macropinocytosis in untransfected macrophages. Cells plated on coverslips were deprived of M-CSF in the presence of 10% serum. Cells were subsequently unstimulated (basal) or stimulated for 5 minutes with 100 ng ml-1 recombinant M-CSF. Subsequently, both basal and stimulated cells were incubated for 5 minutes with lucifer yellow (LY). LY uptake was observed by immunostaining with an antibody to LY. (B) Macrophages were transfected as described in Materials and Methods, and treated as described above. (Top) MCSF-stimulated macrophages that were immunostained for LY to show macropinocytosis. (Bottom) EGFP-positive cells indicating transfected cells. Scale bar, 10 µm. (C) Quantification of macropinocytosis. Data shown are the percentage of transfected cells with five or more macropinosomes under basal or M-CSF-stimulated conditions. Data are expressed as the mean ± s.e.m. of a minimum of 60 cells examined from three independent experiments. *, M-CSF-stimulated macropinocytosis by Rac2D57N-expressing macrophages was statistically different from M-CSF-stimulated vector-expressing or wild-type cells (P<0.005).

View larger version (40K): [in a new window]   Fig. 5. Rac2D57N expression in serum-starved bone-marrow-derived macrophages inhibits filopodia/retraction fiber formation upon readdition of macrophage medium. (A) The Rac activity is shown of macrophages that were either unstarved (steady state) or starved (basal) for 2 hours in the absence of M-CSF and serum, and then stimulated with macrophage medium. Rac activity was normalized to total Rac expression, and data are expressed as fold stimulation of Rac activity relative to basal (starved) macrophages. (B) Data are expressed as the percentage of total transfected cells with filopodia/retraction fibers. The actin structures of between 50 and 150 transfected cells were examined for each condition. Data represent two independent experiments. (C) Macrophages plated on coverslips were transfected with pIRES2-EGFP expression constructs for empty vector, Rac2wt or Rac2D57N and starved for 2 hours in the absence of M-CSF and serum. Cells were either unstimulated or stimulated for 30 minutes in macrophage medium containing 10% serum and 20% L cell medium containing M-CSF. Actin was visualized with rhodamine-phalloidin. Scale bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 6. LPS stimulates Rac and p38 kinase activities in bone-marrow-derived macrophages and Rac2D57N expression inhibits LPS stimulation of p38 kinase. (A) LPS stimulates Rac activation. Macrophages were stimulated for the indicated times with 10 ng ml-1 LPS and cell lysates were incubated with GST-PAKcrib and analysed with a monoclonal antibody to Rac. Rac activity was normalized to the total Rac expression, and data are expressed as the fold stimulation of Rac activation compared with basal activity. A representative experiment of three such experiments is shown. (B) Measurements of the average nuclear phosphorylated p38 kinase intensity for either basal or LPS stimulation of macrophages is shown. Data are from seven independent experiments with duplicate coverslips. n indicates the number cells examined for each condition. (C) Immunoblot of lysates prepared from macrophages that were stimulated for 20 minutes with the indicated concentrations of LPS is shown. Blots were probed with an antibody that recognizes the phosphorylated form of p38 kinase. The figure shows a representative blot of two experiments. (D) Nuclear and phosphorylated p38 kinase immunostaining of untransfected macrophages unstimulated or stimulated for 20 minutes with 10 ng ml-1 LPS is shown. Coverslips were treated as described in Materials and Methods. Nuclei were visualized with DAPI and phosphorylated p38 kinase was immunostained with an antibody to phosphorylated p38 kinase. A single section for each example is shown. (E) Data are expressed as the average nuclear phosphorylated p38 kinase intensity per nucleus of 15-25 transfected cells per treatment. Cells are from five independent experiments with duplicate coverslips. P values were calculated using Student's t test analysis. *, p38 kinase activity in Rac2D57N-expressing cells stimulated with LPS was significantly different from both LPS-stimulated vector and Rac2wt (P<0.01). **, Rac2D57N basal expression was significantly different from both basal vector and Rac2wt (P<0.005).

View larger version (56K): [in a new window]   Fig. 7. Inhibition of JNK and p38 kinase signaling by Rac2D57N. (A) COS7 cells were co-transfected with HA-JNK2 and the indicated expression constructs. Cells were stimulated with EGF for 20 minutes or exposed to UV radiation. Data shown are a representative experiment of two independent experiments. (Top) Autoradiogram of an in vitro kinase assay by immunoprecipitated HA-JNK2 using GST-Jun as the substrate. A phosphoimager was used to measure levels of phosphorylated GST-Jun. Fold increases in phosphorylation of GST-Jun are relative to basal GST-Jun phosphorylation of COS7 cells co-transfected with HA-JNK2 and empty vector. (Middle, bottom) Immunoblots using an antibody to HA to detect the presence of HA-JNK2 and an antibody specific to Rac2 to measure Rac2 expression. (B) COS7 cells were co-transfected with FLAG-p38 kinase and the indicated expression constructs. Cells were stimulated with sorbitol for 30 minutes, or exposed to UV irradiation. Data shown are a representative blot from four independent experiments. (Top) A blot with an antibody to the phosphorylated form of p38 kinase. Fold increases in phosphorylation of p38 kinase are relative to basal phosphorylation of p38 kinase in COS7 cells co-transfected with FLAG-p38 kinase and empty vector. (Middle, bottom) Immunoblots using the FLAG antibody to detect FLAG-p38 kinase and an antibody specific to Rac2 to measure Rac2 expression.

View larger version (31K): [in a new window]   Fig. 8. Guanine-nucleotide binding to GST-Rac2wt and GSTRac2D57N, and association with upstream regulators of Rac. Guaninenucleotide binding to purified GST-Rac2wt and GST-Rac2D57N was performed as described in Materials and Methods. Each closed square represents GST-Rac2wt and each open square represents GSTRac2D57N. The data shown are a representative experiment of two such experiments. Each data point represents the mean±range of duplicate determinations. (A) Binding of [3H]GDP to GST-Rac2. GST-Rac2 was incubated for the indicated times with [3H]GDP. Data are expressed as picomoles of [3H]GDP bound per µg GST-Rac2. (B) Binding of [35S]GTPS to GST-Rac2. GST-Rac2 was incubated for the indicated times with [35S]GTPS. Data are expressed as picomoles of [35S]GTPS bound per µg of GST-Rac2. (C) Dissociation of [3H]GDP from GST-Rac2 in the presence of an excess of unlabeled GDP. Prebound [3H]GDP was displaced by the addition of an excess of unlabeled GDP. The remaining bound [3H]GDP was measured at the indicated times. Data are expressed as the percentage of total [3H]GDP bound before the addition of unlabeled GDP. (D) Dissociation of [3H]GDP from GST-Rac2 in the presence of an excess of unlabeled GTPS. Prebound [3H]GDP was displaced by the addition of an excess of unlabeled GTPS. The remaining bound [3H]GDP was measured at the indicated times. Data are expressed as the percentage of total [3H]GDP bound before the addition of unlabeled GTPS. (E,F) COS7 cells were co-transfected with either RhoGDI or Myc-Tiam1 and the indicated expression constructs. An antibody to RhoGDI or to Myc was used to immunoprecipitate RhoGDI or Myc-Tiam1, respectively. The ability of Rac to immunoprecipitate with RhoGDI or Myc-Tiam1 is shown. Data are representative of between two and four experiments. (G,H) COS7 cells were transfected with indicated expression constructs and GST-PAKcrib binding was assessed. Data are representative of two such experiments.

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