FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m805638200
ISSN1083-351X
AutoresShigeru Tsuboi, Hidetoshi Takada, Toshiro Hara, Naoki Mochizuki, Tomihisa Funyu, Hisao Saitoh, Yuriko Terayama, Kanemitsu Yamaya, Chikara Οhyama, Shigeaki Nonoyama, Hans D. Ochs,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoMacrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients. Macrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients. Podosomes (see Fig. 1A) are micron-scale, dynamic, actin-based protrusions observed in motile cells such as macrophages, dendritic cells, osteoclasts, certain transformed fibroblasts, and carcinoma cells (1Linder S. Aepfelbacher M. Trends Cell Biol. 2003; 13: 376-385Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Podosomes play an important role in macrophage chemotactic migration, which is critical for recruitment of leukocytes to inflamed tissues. Podosomes are both adhesion structures and the sites of extracellular matrix degradation (2Linder S. Trends Cell Biol. 2007; 17: 107-117Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Adhesion to and degradation of the extracellular matrix are essential processes for the successful migration of macrophages in tissues. Podosomes occur in most macrophages and can be observed by differentiating human primary monocytes into macrophages with macrophage-colony stimulating factor-1 (M-CSF-1) 2The abbreviations used are: M-CSF-1, macrophage-colony stimulating factor-1; FBP17, formin-binding protein 17; WAS, Wiskott-Aldrich syndrome; WASP, Wiskott-Aldrich syndrome protein; N-WASP, neuronal WASP; WIP, WASP interacting-protein; EFC domain, extended FER-CIP4 homology domain; F-BAR domain, FER-CIP4 homology and Bin-amphiphysin-Rvs domain; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescence protein; siRNA, short interfering RNA; FITC, fluorescein isothiocyanate; PDZ-GEF, PDZ-guanine nucleotide exchange factor; HEK293 cells, human embryonic kidney 293 cells; HA, hemagglutinin; SH3, src homology 3 domain; dSH3, SH3 domain deletion; GST, glutathione S-transferase;, PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; siFBP, siRNA for FBP17; siC, scrambled control siRNA. and staining the F-actin using phalloidin (3Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (357) Google Scholar, 4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar). Podosomes labeled in this way appear as F-actin-rich dots (see Fig. 1C). Podosome formation has recently been directly observed in vitro and in vivo in leukocyte migration through the endothelium, diapedesis (5Carman C.V. Sage P.T. Sciuto T.E. Fuente M.A.d.l. Geha R.S. Ochs H.D. Dvorak H.F. Dvorak A.M. Springer T.A. Immunity. 2007; 26: 784-797Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Phagocytosis of bacterial pathogens is one of the most important primary host defense mechanisms against infections. The phagocytic cup (see Fig. 1B) is an actin-based membrane structure formed at the plasma membrane of phagocytes, including macrophages, upon stimulation with foreign materials such as bacteria. The phagocytic cup captures and ingests foreign materials, and its formation is an essential first step in phagocytosis leading to the digestion of foreign materials (6Underhill D.M. Ozinsky A. Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (847) Google Scholar, 7Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). When macrophages are stimulated by foreign materials, podosomes disappear, and phagocytic cups, which are also rich in F-actin, are formed to ingest the foreign materials (see Fig. 1D). Wiskott-Aldrich syndrome (WAS) is an X chromosome-linked immunodeficiency disorder. Patients with WAS suffer from severe bleeding, eczema, recurrent infection, autoimmune diseases, and an increased risk of lymphoreticular malignancy (8Wiskott A. Monatsschr. Kinderheilkd. 1937; 68: 212-216Google Scholar, 9Aldrich R.A. Steinberg A.G. Campbell D.C. Pediatrics. 1954; 13: 133-139PubMed Google Scholar, 10Notarangelo L.D. Miao C.H. Ochs H.D. Curr. Opin. Hematol. 2008; 15: 30-36Crossref PubMed Scopus (163) Google Scholar). The causative gene underlying WAS encodes Wiskott-Aldrich syndrome protein (WASP) (11Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78 (Correction (1994) Cell 79, 922): 635-644Abstract Full Text PDF PubMed Scopus (839) Google Scholar). WASP deficiency due to the mutation or deletion causes defects in adhesion, chemotaxis, phagocytosis, and the development of hematopoietic cells in WAS patients (10Notarangelo L.D. Miao C.H. Ochs H.D. Curr. Opin. Hematol. 2008; 15: 30-36Crossref PubMed Scopus (163) Google Scholar). The formation of podosomes and phagocytic cups is severely affected in macrophages from WAS patients (3Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (357) Google Scholar, 12Lorenzi R. Brickell P.M. Katz D.R. Kinnon C. Thrasher A.J. Blood. 2000; 95: 2943-2946Crossref PubMed Google Scholar, 42Calle Y. Anton I.M. Thrasher A.J. Jones G.E. J. Microsc. (Oxf.). 2008; 231: 494-505Crossref PubMed Scopus (39) Google Scholar), suggesting that WASP is involved in the formation of these structures. However, the detailed molecular mechanisms of their formation remain unknown. WASP is complexed with a cellular WASP-interacting partner, WASP-interacting protein (WIP) (13Ramesh N. Anton I.M. Hartwig J.H. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14671-14676Crossref PubMed Scopus (309) Google Scholar, 14Anton I.M. de la Fuente M.A. Sims T.N. Freeman S. Ramesh N. Hartwig J.H. Dustin M.L. Geha R.S. Immunity. 2002; 16: 193-204Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Recently, two groups (including us) have demonstrated that WASP and WIP form a complex and that the WASP-WIP complex is required for the formation of podosomes (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 15Chou H.C. Anton I.M. Holt M.R. Curcio C. Lanzardo S. Worth A. Burns S. Thrasher A.J. Jones G.E. Calle Y. Curr. Biol. 2006; 16: 2337-2344Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and phagocytic cups (16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Here, we identified formin-binding protein 17 (FBP17) as a protein interacting with the WASP-WIP complex and examined the role of FBP17 in the formation of podosomes and phagocytic cups. Reagents and Antibodies-Recombinant human macrophage-colony stimulating factor-1 (M-CSF-1) was purchased from R&D Systems (Minneapolis, MN). Phenylmethylsulfonyl fluoride, leupeptin, pepstain A, aprotinin, IGEPAL CA-630, paraformaldehyde, saponin, bovine serum albumin, 3-methyladenine, latex beads (3 μm in diameter), phorbol 12-myristate 13-acetate (PMA), human IgG, glycerol, Triton X-100, anti-FLAG monoclonal antibody (M2), and anti-β-actin antibody were purchased from Sigma-Aldrich. The anti-WASP monoclonal antibody, anti-WIP polyclonal antibody, and anti-Myc monoclonal antibody (9E10) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-dynamin-2 antibody was purchased from BD Biosciences. The rat anti-hemagglutinin (HA) monoclonal antibody (3F10) was purchased from Boehringer Ingelheim (Ridgefield, CT). The Cy2-labeled anti-rat IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Yeast Two-hybrid Screening-We screened a human lymphocyte cDNA library (Origene Technology Inc., Rockville, MD) using a full-length WIP as bait. A cDNA encoding full-length WIP was cloned into pGilda (BD Biosciences Clontech). The EGY48 yeast strain was transformed with pGilda-WIP, the human lymphocyte cDNA library, and pSH18–34, a reporter plasmid for the β-galactosidase assay. Transformants were assayed for Leu prototrophy, and a filter assay was performed for β-galactosidase measurement (17Tsuboi S. Nonoyama S. Ochs H.D. EMBO Rep. 2006; 7: 506-511Crossref PubMed Scopus (38) Google Scholar). Cells and Transfection-THP-1 and human embryonic kidney (HEK) 293 cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 and Dulbecco's modified Eagle's high glucose medium (Invitrogen), respectively, both supplemented with 10% fetal bovine serum. For human primary monocyte isolation, 10–30 ml of peripheral blood was drawn from healthy volunteers and WAS patients after informed consent was obtained. Monocytes were prepared from peripheral blood samples (10–30 ml) using a monocyte isolation kit II (Miltenyi Biotech Inc., Auburn, CA). Transfection of THP-1 cells and monocytes was performed with a Nucleofector device using a cell line Nucleofector kit V and a human monocyte Nucleofector kit, respectively, according to the manufacturer's instructions (Amaxa Biosystems, Gaithersburg, MD). Transfection of HEK293 cells was performed using SuperFect transfection reagent (Qiagen, Valencia, CA). THP-1 cells and monocytes were co-transfected with the FBP17 constructs and a GFP-expressing plasmid, pmaxGFP (Amaxa Biosystems Inc.), as a transfection marker. The transfection efficiency measured using pmaxGFP was 40–50% for THP-1 cells and 10–20% for monocytes. RNA Interference-A short interfering RNA (siRNA) for FBP17 and its scrambled control siRNA was synthesized by Dharmacon (Lafayette, CO). The targeting sequence was 5′-CCCACTTCATATGTCGAAGTCTGTT-3′ (18Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172: 269-279Crossref PubMed Scopus (304) Google Scholar). THP-1 cells and monocytes were transfected with siRNA using a cell line Nucleofector kit V and a human monocyte Nucleofector kit, respectively, and a Nucleofector device. Cells were co-transfected with an fluorescein isothiocyanate (FITC)-conjugated control siRNA, BLOCK-IT (Invitrogen), as a transfection marker. The transfection efficiency measured using BLOCK-IT was 40–50% for THP-1 cells and 10–20% for monocytes. Immunoprecipitation-For immunoprecipitation of WASP from THP-1 cells, 2 × 107 cells were lysed in buffer A (50 mm Tris-HCl, pH 7.5, 75 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin). Lysates were centrifuged at 10,000 × g at 4 °C for 15 min. The supernatant was incubated with 2 μg/ml anti-WASP monoclonal antibody (Santa Cruz Biotechnology) at 4 °C for 2 h and then incubated with anti-mouse IgG agarose (Sigma). The resin binding the immune complex was washed three times with 0.5 ml of buffer B (50 mm Tris-HCl, pH 7.5, 10% glycerol, 0.1% Triton X-100), and the complex was eluted with 1× Laemmli's SDS-PAGE sample buffer. Eluted proteins were subjected to SDS-PAGE and analyzed by immunoblotting for WASP, WIP, and FBP17. GST Pull-down Assay-Glutathione S-transferase (GST) and a fusion protein of GST and the src homology 3 (SH3) domain of FBP17 (548–609 amino acids) (GST-FSH3) were purified from Escherichia coli (XL-1B) extracts using glutathione-Sepharose-4B. HEK293 cells were transfected with the cDNAs of Myc- or FLAG-tagged protein and lysed in buffer A. Lysates from the transfected cells were incubated with the affinity matrices of GST alone or GST-FSH3 at 4 °C for 1 h. After a 1-h incubation, the matrices were washed five times with buffer A, and pull-down samples were analyzed by immunoblotting using anti-Myc or anti-FLAG antibody. Immunofluorescence Microscopy-THP-1 cells and monocytes grown on coverslips were differentiated into macrophages by incubation with 12.5 ng/ml PMA (Sigma) and 20 ng/ml M-CSF-1 (R&D Systems), respectively, for 72 h. HEK293 cells were transfected with various cDNA constructs and then cultured on coverslips for 48 h. Cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.1% (w/v) saponin, and blocked with 1% (w/v) bovine serum albumin. Cells were stained with primary antibodies and Alexa Fluor 488- or Alexa Fluor 564-labeled secondary antibodies (Invitrogen). Cells were also stained with Alexa Fluor 568-labeled phalloidin (Invitrogen). Cell staining was examined under a fluorescence microscope (Zeiss Axioplan AR) or an MRC 1024 SP laser point scanning confocal microscope (Bio-Rad). Assays for the Formation of Podosomes and Phagocytic Cups-The formation of podosomes and phagocytic cups was assayed by visualizing these actin-based membrane structures by F-actin staining as described previously (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Briefly, podosomes in differentiated THP-1 cells or macrophages were visualized by F-actin staining with Alexa Fluor 568-phalloidin. To form phagocytic cups in differentiated THP-1 cells or macrophages, latex beads (3 μm, Sigma) were opsonized with 0.5 mg/ml human IgG (Sigma), and cells grown on coverslips were incubated with the IgG-opsonized latex beads at 37 °C for 10 min in the presence of 10 mm 3-methyladenine (Sigma) to stabilize the phagocytic cups (16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The phagocytic cups were then also visualized with Alexa Fluor 568-phalloidin. Cells were examined under a fluorescence microscope (Zeiss Axioplan AR). Assays for Macrophage Migration and Phagocytosis-For the macrophage migration assay, human macrophages (2 × 105 cells) were plated onto chemotaxis membranes with 5-μm pores (Corning, Acton, MA) coated with 0.15% gelatin/phosphate-buffered saline placed within Boyden chamber inserts. M-CSF-1 was used as a chemoattractant and diluted in serum-containing RPMI 1640 medium in lower chambers. After a 4-h incubation, non-migrating cells were removed by gently wiping the upper surface of the filter. The filter was removed from the inserts using a razor blade and mounted onto glass plates, and the number of migrating cells was counted under a fluorescence microscope. For the phagocytosis assay, human macrophages (1 × 106 cells) were seeded on coverslips and incubated with 0.5 ml of RPMI 1640 medium containing IgG-opsonized latex beads (3 μm) at 4 °C for 10 min, allowing the beads to attach to cells. Phagocytosis was initiated by adding 1.5 ml of preheated RPMI 1640 medium, and the cells were incubated with the beads at 37 °C for 30 min. Control plates were incubated at 4 °C to estimate nonspecific binding of latex beads to the cells. After incubation, the cells were vigorously washed with phosphate-buffered saline, and the number of intracellular latex beads was determined by counting beads within cells under a fluorescence microscope. The percentage of phagocytosis was calculated as the total number of cells with at least one bead as a percentage of the total number of cells counted. At least 100 cells were examined. Cell Fractionation-To prepare the cytoplasmic and membrane fractions, macrophages (1 × 106 cells) were washed with ice-cold phosphate-buffered saline and suspended in 50 mm Tris-HCl buffer, pH 7.5, containing 1 mm EDTA and proteinase inhibitors as described above. The cell suspensions were sonicated four times on ice for 5 s each using a bath-type sonicator followed by ultracentrifugation at 265,000 × g at 4 °C for 2 h. The supernatant was used as the cytosolic fraction, and the pellet was resuspended in 50 mm Tris-HCl, pH 7.5, containing 1 mm EDTA and used as the membrane fraction. Anti-Caspase-3 (Santa Cruz Biotechnology) and anti-sodium potassium ATPase antibodies (AbCam, Inc., Cambridge, MA) were used to determine the purity of the cytosolic and membrane fractions, respectively. Statistics-Statistically significant differences were determined using the Student's t test. Differences were considered significant if p < 0.05. FBP17 Binds to the WASP-WIP Complex and Dynamin-2 in Macrophages-To explore the detailed molecular mechanisms of the formation of podosomes and phagocytic cups, we searched for a protein interacting with the WASP-WIP complex. We identified FBP17 as a WIP-binding protein in a yeast two-hybrid screen using the full-length WIP as bait. FBP17 was originally identified as a protein binding to formin, a protein that regulates the actin cytoskeleton (19Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Crossref PubMed Scopus (194) Google Scholar). FBP17 is a member of the Schizosaccharomyces pombe Cdc15 homology (PCH) protein family (20Ho H.Y. Rohatgi R. Lebensohn A.M. Le M. Li J. Gygi S.P. Kirschner M.W. Cell. 2004; 118: 203-216Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar) and contains an N-terminal extended FER-CIP4 homology (EFC) domain (also known as the FER-CIP4 homology and Bin-amphiphysin-Rvs (F-BAR) domain), protein kinase C-related kinase homology region 1 (HR1), and an SH3 domain (Fig. 1E). The EFC/F-BAR domain has membrane binding and deformation activities, and FBP17 is involved in endocytosis in transfected COS-7 cells (18Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172: 269-279Crossref PubMed Scopus (304) Google Scholar, 21Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Shimada A. Niwa H. Tsujita K. Suetsugu S. Nitta K. Hanawa-Suetsugu K. Akasaka R. Nishino Y. Toyama M. Chen L. Liu Z.J. Wang B.C. Yamamoto M. Terada T. Miyazawa A. Tanaka A. Sugano S. Shirouzu M. Nagayama K. Takenawa T. Yokoyama S. Cell. 2007; 129: 761-772Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). To confirm that FBP17 directly interacts with WIP or WASP, we performed GST pull-down assays using a fusion protein of GST and the SH3 domain of FBP17 (GST-FBPSH3). Purified GST and the GST-FSH3 fusion protein were subjected to SDS-PAGE (Fig. 1F, lanes 1 and 2). The HEK293 transfected cells express the Myc- and FLAG-tagged proteins (Fig. 1F, lanes 3–6). The results from the GST pull-down assays were shown (Fig. 1F, lanes 7–14). Both WASP and WIP were pulled down by GST-FSH3 (Fig. 1, lanes 10 and 14), indicating that the SH3 domain of FBP17 directly interacts with both proteins. It has previously been shown that FBP17 binds to N-WASP and dynamin in transfected cells (18Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172: 269-279Crossref PubMed Scopus (304) Google Scholar, 21Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We examined whether FBP17 binds to WASP, WIP, and dynamin-2 in macrophages. THP-1 (human monocyte cell line) cells closely resemble monocyte-derived macrophages when differentiated by stimulation with PMA (23Auwerx J. Experientia (Basel). 1991; 47: 22-31Crossref PubMed Scopus (666) Google Scholar) and form podosomes and phagocytic cups that are morphologically and functionally indistinguishable from those in primary macrophages (supplemental Fig. 1) (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Auwerx J. Experientia (Basel). 1991; 47: 22-31Crossref PubMed Scopus (666) Google Scholar). WASP was immunoprecipitated from the lysates of PMA-differentiated THP-1 cells with an anti-WASP monoclonal antibody (Fig. 1G, lanes 2, 5, and 8) followed by immunoblotting using antibodies to FBP17 (21Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), WASP, and WIP. Both WIP and FBP17 co-immunoprecipitated with WASP (Fig. 1G, lanes 5 and 8). FBP17 also co-immunoprecipitated with dynamin-2 (Fig. 1G, lanes 14). These results, taken together with the results in Fig. 1F, suggest that FBP17 binds to the WASP-WIP complex and dynamin-2 in macrophages. We next used immunofluorescence to examine whether FBP17 localizes at podosomes and phagocytic cups because the WASP-WIP complex is an essential component of podosomes (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 15Chou H.C. Anton I.M. Holt M.R. Curcio C. Lanzardo S. Worth A. Burns S. Thrasher A.J. Jones G.E. Calle Y. Curr. Biol. 2006; 16: 2337-2344Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and phagocytic cups (16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). THP-1 cells transfected with FLAG-tagged FBP17 (FLAG-FBP17) and differentiated by stimulation with PMA were stained with an anti-FLAG monoclonal antibody to visualize FBP17 and with phalloidin to visualize the F-actin in podosomes and phagocytic cups (Fig. 1, H and I, left and middle panels). Merged images revealed that both F-actin and FBP17 are present in podosomes and phagocytic cups (Fig. 1, H and I, right panels), indicating that FBP17 localizes at podosomes and phagocytic cups. Importance of FBP17 in the Formation of Podosomes and Phagocytic Cups-To determine the importance of FBP17 in the formation of podosomes and phagocytic cups, we knocked down FBP17 in THP-1 cells with siRNAs. To confirm that the expression of FBP17 was knocked down in cells, we transfected THP-1 cells with siRNAs, prepared lysates from the total siRNAs-transfected cells, and analyzed the expression level of FBP17 by immunoblotting. THP-1 cells transfected with the siRNA for FBP17 expressed ∼40% less FBP17 than cells transfected with a scrambled control siRNA based on the immunoblots (Fig. 2A, lanes 1 and 2) but expressed the same level of β-actin (Fig. 2A, lanes 3 and 4). The transfection efficiency of THP-1 cells was estimated to be 40–50% from the expression of green fluorescent protein (GFP) used as a transfection control. Therefore, the decrease in FBP17 expression indicates that FBP17 was efficiently knocked down in most transfected cells. Human primary monocytes were co-transfected with the FBP17 siRNAs and a FITC-conjugated control siRNA as a transfection marker. After differentiation of the monocytes into macrophages with M-CSF-1, FITC-positive cells were examined for the formation of podosomes and phagocytic cups. To quantify their formation, we scored the percentage of cells with podosomes or phagocytic cups among FITC-positive cells. When the expression of FBP17 was knocked down, the formation of both podosomes and phagocytic cups in macrophages was significantly reduced (p < 0.01; Fig. 2, B and C). These results suggest that FBP17 is necessary for the formation of podosomes and phagocytic cups. A representative cell from each experiment is shown in Fig. 2, F and G, for podosomes and in Fig. 2, H and I, for phagocytic cups. We then assayed macrophage migration as a podosome function and phagocytosis as a phagocytic cup function. When expression of FBP17 was knocked down, macrophage migration through a gelatin filter toward a chemoattractant was significantly reduced in cells transfected with FBP17 siRNA (p < 0.02; Fig. 2D). Phagocytosis of IgG-opsonized latex beads was also reduced (Fig. 2E). These results suggest that FBP17 is essential for chemotaxis and phagocytosis because of its role in forming podosomes and phagocytic cups, respectively. FBP17 Recruits the WASP-WIP Complex to the Plasma Membrane-Recent biochemical analyses revealed that FBP17 binds to a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), through its EFC/F-BAR domain and to N-WASP and dynamin via its SH3 domain (18Tsujita K. Suetsugu S. Sasaki N. Furutani M. Oikawa T. Takenawa T. J. Cell Biol. 2006; 172: 269-279Crossref PubMed Scopus (304) Google Scholar, 21Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Kakimoto T. Katoh H. Negishi M. J. Biol. Chem. 2006; 281: 29042-29053Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We have shown that although WASP and WIP are cytosolic proteins, the WASP-WIP complex localizes at podosomes and phagocytic cups (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We then examined whether FBP17 recruits the WASP-WIP complex to the plasma membrane in macrophages. We focused on the roles of the EFC and SH3 domains of FBP17 and constructed three FBP17 mutants for the recruitment experiments: a Lys-33 to Glu (K33E) substitution, a Lys-166 to Ala (K166A) substitution, and an SH3 domain deletion (dSH3). Both substitution mutations in the EFC domain (K33E and K166A) significantly reduce membrane binding and deformation (22Shimada A. Niwa H. Tsujita K. Suetsugu S. Nitta K. Hanawa-Suetsugu K. Akasaka R. Nishino Y. Toyama M. Chen L. Liu Z.J. Wang B.C. Yamamoto M. Terada T. Miyazawa A. Tanaka A. Sugano S. Shirouzu M. Nagayama K. Takenawa T. Yokoyama S. Cell. 2007; 129: 761-772Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), and the dSH3 mutant does not bind to WASP and WIP because the SH3 domain is the binding site of WASP and WIP (Fig. 1F). We co-transfected HEK293 cells with the FLAG-tagged FBP17 constructs, WASP, and WIP. A C-terminal fragment (1146–1429 amino acids) of PDZ-GDP exchange factor (PDZ-GEF) was used as a negative control for FBP17 because this fragment is stable in the cytosol and does not interact with any WASP-related proteins (4Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (48) Google Scholar, 16Tsuboi S. Meerloo J. J. Biol. Chem. 2007; 282: 34194-34203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 25Rebhun J.F. Castro A.F. Quilliam L.A. J. Biol. Chem. 2000; 275: 34901-34908Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). We confirmed the expression of FBP17 and its mutants in cells by immunoblotting (supplemental Fig. 2) and immunoprecipitated FLAG-tagged proteins from lysates of the transfected cells with anti-FLAG antibody (Fig. 3A, lanes 1–5). WASP and WIP were detected in the immunoprecipitates from cells expressing the FLAG-tagged FBP17, K33E, and K166A constructs (Fig. 3A, lanes 7–9 and 12–14) but not the FLAG-tagged PDZ-GEF and dSH3 constructs (Fig. 3A, lanes 6, 10, 11, and 15), indicating that FBP17 and its mutants K33E and K166A form a complex with WASP and WIP but that dSH3 not. Next, cells expressing the FLAG-tagged proteins, WASP, and WIP were examined under the immunofluorescence microscope for the localization of the FLAG-tagged proteins and WASP. WASP and WIP were localized in the cytosol in cells transfected with only the WASP cDNA and only the WIP cDNA, respectively, as well as in cells expressing both WASP and WIP (supplemental Fig. 3). In cells co-expressing FLAG-PDZ-GEF (control) with WASP and WIP, both FLAG-PDZ-GEF and WASP were cytosolic (Fig. 3B). In cells co-expressing FLAG-FBP17 with WASP an
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