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Originally published In Press as doi:10.1074/jbc.M705999200 on September 21, 2007

J. Biol. Chem., Vol. 282, Issue 47, 34194-34203, November 23, 2007
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Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages*Formula

Shigeru Tsuboi{ddagger}1 and Jennifer Meerloo§

From the {ddagger}Infectious and Inflammatory Disease Center and §Cell Imaging Facility, Burnham Institute for Medical Research, La Jolla, California 92037

Received for publication, July 23, 2007 , and in revised form, September 4, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP·WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Wiskott-Aldrich syndrome (WAS)2 is an X chromosome-linked immunodeficiency disorder. Patients with WAS suffer from severe bleeding, eczema, recurrent infections, auto-immune diseases, and an increased risk of lymphoreticular malignancy (1-3). The causative gene underlying WAS encodes Wikott-Aldrich syndrome protein (WASP) (4). WASP is a 62-kDa cytosolic protein comprising distinct domains that interact with other cellular factors to regulate, mediate, and target the many functions of WASP (Fig. 3A) (5, 6). WASP is expressed predominantly in hematopoietic cells and functions in the assembly of the actin cytoskeleton (7, 8), signal transduction (9-12), apoptosis (13, 14), and the regulation of gene expression (15, 16). Furthermore, WASP translocates to lipid rafts after T-cell receptor ligation, localizing to immune synapses, the junctions between T cells, and antigen-presenting cells (17, 18), and recent investigations of T-cell profiles from WASP-deficient mice have revealed a critical role for WASP in regulatory T-cell function (19-21).

The WASP C-terminal region (residues 178-502) regulates the organization of the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate binds to the Basic Region, whereas Cdc42 binds the GTPase binding domain. Binding of these cellular factors activates the WASP C-terminal VCA (verprolin/cofilin/acidic) domain, which in turn activates the actin-related protein complex (Arp2/3 complex), stimulating actin polymerization through the formation of F-actin branch junctions (8, 22, 23). Recently, the Toca-1 protein (transducer of Cdc42-dependent actin assembly) was identified as a mediator of actin polymerization induced by the Cdc42·N-WASP·Arp2/3 complex (24). The WASP C terminus is also required for myoblast fusion in Drosophila (25).

The WASP N-terminal region (residues 1-177) comprises an N-terminal segment and the WH1/EVH1 domain. Two classes of interacting proteins have been identified; (i) the mammalian verprolins bind to the WH1/EVH1 domain, a linkage that is critical for T-cell function (17, 26) and monocyte chemotaxis (27); (ii) calcium- and integrin-binding protein provides a functional bridge between WASP and integrins that plays an important role in cell adhesion by platelets (28).

Verprolin was originally identified as a yeast protein implicated in cell growth, cytoskeletal organization, and endocytosis (29). There are three mammalian homologues of verprolin that were identified through their ability to bind to the WASP N-terminal region; these are WIP (30), WICH/WIRE (WIP and CR16 homologous protein/WIP related protein) (31, 32), and CR16 (33). The verprolins have a common domain organization consisting of an actin binding verprolin homologous region, a glycine-rich region, a proline-rich region, and a WASP binding (WB) domain (34). WIP is a widely expressed protein and is the best characterized of the mammalian verprolins. WIP plays a key role in regulating WASP, with which it appears to form a constitutive complex that functions as a unit in many cellular processes (26, 27, 35, 36). For example, WIP is required for targeting WASP to the immune synapse after T-cell receptor ligation (6, 17). WIP expression is required for the functional expression of WASP in hematopoietic cells (37, 38), and in WIP-deficient mice, T cells fail to proliferate or polarize in response to T-cell-receptor ligation and form smaller T cell-antigen-presenting cells conjugate interfaces (26). WIP also binds directly to F-actin and inhibits Ccd42-mediated actin polymerization; it synergizes with N-WASP to induce filopodia when overexpressed in fibroblasts (39). A general role in filopodia formation is consistent with the requirement of WIP in pathogenic settings, notably the intracellular motility of vaccinia virus and Shigella, which involves the formation of an actin comet-tail mediated by the N-WASP·WIP complex (40), and the transendothelial migration of macrophages, which involves the formation of the podosomes mediated by the WASP·WIP complex (35, 41).

Mutation or deletion of WASP causes various functional abnormalities in hematopoietic cells. In macrophages from WAS patients, WASP deficiency causes abnormal morphology (42), adhesion defects (43-46), chemotactic defects (47, 48), and phagocytic defects (49-52).

Phagocytosis involves the ingestion and digestion of foreign materials (such as microorganisms, insoluble particles, damaged or dead host cells, cell debris, and activated clotting factors (53-55)) by specialized cells called phagocytes, which include macrophages and neutrophils. Phagocytosis is, therefore, essential for host defense against infection, neoplastic proliferation, wound healing, and tissue remodeling.

The phagocytic cup is an actin-based membrane structure formed at the plasma membrane of a phagocyte upon stimulation with foreign materials such as bacteria. Efficient phagocytosis requires opsonins, which are host molecules (e.g. IgG antibodies or complement proteins such as C3b) that bind to the surface of foreign materials. The opsonins are then recognized by phagocytic receptors (e.g. the Fc receptor for antibodies or CR3 for complement proteins), which induce a signal that results in polymerization of actin at the site of ingestion and the transient formation of the "phagocytic cup." Formation of the phagocytic cup is an essential first step in phagocytosis leading to digestion of foreign materials (56-58). Although it has been reported that WASP plays an important role in the formation of the phagocytic cup (49), the molecular details remain unknown.

The podosome is also an actin-based structure formed at the plasma membrane of monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (76). Podosomes are micron-scale, dynamic, actin-rich protrusions and play essential roles in chemotactic migration and extravasation of those monocyte-derived cells (77). In WASP-deficient WAS patients, the podosomes are completely absent, indicating that WASP is a critical component of the podosomes (43). The phagocytic cup is also absent in WAS patients (49). These observations led us to postulate that WASP plays critical roles in the formation of both phagocytic cup and podosomes. However, molecular mechanisms of the formation of these structures including WASP are unclear. The present study focuses on understanding of the molecular basis of phagocytosis. Here we investigate how WASP regulates the formation of the phagocytic cup.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Recombinant human macrophage-colony stimulating factor-1 was purchased from R&D Systems. Anti-WASP monoclonal antibody and anti-WIP polyclonal antibody were purchased form Santa Cruz Biotechnology. Anti-phosphotyrosine monoclonal antibody (4G10) was purchased from Upstate. Anti-green fluorescence protein (GFP) monoclonal antibody (JL-8) was obtained from BD Clontech. Phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, aprotinin, IGEPAL CA-630, sodium orthovanadate, anti-mouse IgG agarose, saponin, bovine serum albumin, 3-methyladenine (3-MA), latex beads (3 µm, diameter), and phorbol 12-myristate 13-acetate (PMA) were purchased form Sigma-Aldrich. RPMI1640, other tissue culture reagents, Alexa 568-labeled phalloidin, and Alexa 488-labeled secondary antibodies were obtained from Invitrogen.

Cells and Transfection—Human monocyte cell line THP-1 was obtained from American Type Culture Collection and cultured in RPMI1640 containing 10% fetal calf serum (FCS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. THP-1 cells (1-2 x 106 cells) were transfected with the WASP constructs (2 µg) using Cell Line Nucleofector Kit V and Amaxa Nucleofector (Amaxa Inc.) according to the manufacturer's instructions. After transfection, cells were cultured in 6-well plates for 2 days in RPMI1640 containing 10% FCS supplemented with 12.5 ng/ml PMA. For human primary monocyte isolation, 20-40 ml of peripheral blood was drawn from healthy volunteers after informed consent was obtained. Monocytes were isolated from blood samples using Monocyte Isolation Kit II (Miltenyi Biotech). Cells were cultured in RPMI1640 containing 10% FCS supplemented with 20 ng/ml of recombinant human macrophage-colony stimulating factor-1. Monocytes cultured for 4 days in this medium attained morphology characteristics of macrophages, and their differentiated state was confirmed by a flow cytometric analysis for CD14+ status. Monocytes cultured for 2 days were harvested and transfected with the WASP constructs using Human Monocyte Nucleofector kit and Amaxa Nucleofector (Amaxa Inc.) according to the manufacturer's instructions. After transfection, cells were cultured for 2 days additionally. Cells were co-transfected with GFP expressing plasmid, pmaxGFP (Amaxa Inc.), as a transfection marker. The efficiency of transfection measured using pmaxGFP was 40-60% for THP-1 cells or monocytes. Transfection of short interfering RNA (siRNA) was performed using DharmaFECT 2 (Dharmacon Inc.). The following sequences were chosen to generate siRNA for WASP, 5'-GCCGAGACCTCTAAACTTA-3' (sense) and 5'-CGGCCAGATCTCAATATCAT-3' (scrambled). The efficiency of siRNA transfection measured using fluorescein isothiocyanate (FITC)-conjugated control siRNA, BLOCK-IT (Invitrogen), was 40-60%. The Internal Review Board of Burnham Institute for Medical Research approved these experiments.

Immunofluorescence Microscopy—THP-1 cells seeded on coverslips were stimulated with 12.5 ng/ml PMA for 2 days. Cells were fixed with 4% (w/v) paraformaldehyde (Fluka) for 10 min at room temperature. Fixed cells were permeabilized and blocked with phosphate-buffered saline containing 0.1% (w/v) saponin and 1% (w/v) bovine serum albumin. Cells were stained with Alexa 568-labeled phalloidin (Invitrogen) and anti-WIP or anti-WASP (Santa Cruz Biotechnology) in the presence of Fc-BlockTM (BD Pharmingen). The secondary antibody, Alexa 488-labeled anti-rabbit or mouse IgG (Invitrogen), was used. Cell staining was examined under a fluorescence microscope (Zeiss Axioplan AR) or MRC 1024 SP Bio-Rad laser point scanning confocal microscope (Bio-Rad).

Immunoprecipitation—For immunoprecipitation of WASP, 0.5-1 x 107 cells were lysed in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630, 1 mM phenylmethylsulfonyl fluoride, 3 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin). Lysates were centrifuged at 10,000 x 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-Aldrich) at 4 °C for 1 h. The resin binding the immune complex was washed with 0.5 ml of buffer A 3 times, and the complex was eluted with 1x Laemmli SDS-PAGE sample buffer. Eluted proteins were subjected to SDS-PAGE and analyzed by immunoblotting using anti-WASP antibody, anti-WIP polyclonal antibody (Santa Cruz Biotechnology), or anti-phosphotyrosine monoclonal antibody (4G10) (Upstate).

Assay for the Phagocytic Cup Formation—Latex beads (3 µm diameter) (Sigma-Aldrich) were opsonized with 0.5 mg/ml human IgG (Sigma-Aldrich) for 16 h at room temperature, washed with phosphate-buffered saline extensively, and suspended in RPMI1640 containing 1% FCS and 10 mM 3-methyladenine (3-MA) (Sigma-Aldrich). PMA-differentiated THP-1 cells or primary macrophages grown on coverslips in 6-well culture plates were incubated in prewarmed serum-free medium containing 10 mM 3-MA for 30 min at 37 °C. Cells on coverslips were changed to 0.5 ml of ice-cold opsonized latex bead suspension at a ratio of 10-50 beads per cell and incubated for at 4 °C for 10 min to allow beads to adhere to cells. The formation of the phagocytic cups was initiated by the addition of 1.5 ml of prewarmed RPMI1640 containing 1% FCS and 10 mM 3-MA, and cells were incubated at 37 °C for a further 15 min. The phagocytic cup formation was stopped by fixation in 4% (w/v) paraformaldehyde for 10 min, and cells were permeabilized and blocked by incubating for 5 min in phosphate-buffered saline containing 0.1% (w/v) saponin and 1% (w/v) bovine serum albumin. Phagocytic cups were visualized by F-actin staining with Alexa 568-phalloidin (Invitrogen) and examined under a fluorescence microscope (Zeiss Axioplan AR).

Assay for Phagocytosis—To assay phagocytosis, we measured the phagocytic uptake of IgG-opsonized fluorescence dye-conjugated latex beads (TransFluoSpheres; excitation at 488 nm/emission at 560 nm; Invitrogen) by THP-1 cells in the absence of 3-MA using a flow cytometer, FACSort (BD Biosciences). Cells were co-transfected with FITC-conjugated control siRNA and then incubated with IgG-opsonized fluorescence dye-conjugated latex beads. FITC-positive transfected cells were gated (FL1), and phagocytosis was expressed as the difference in the mean fluorescence intensity of ingested beads (FL2) measured at 37 and 4 °C.

RNA Isolation and Reverse Transcription-PCR—Total RNA was isolated from THP-1 cells using PureLink 96 Total RNA Purification kit (Invitrogen) according to the manufacturer's instructions. After reverse transcription of 2 µg of total RNA by oligo(dT) priming, the resulting single strand cDNA was amplified using Expand High Fidelity PCR system (Roche Applied Science). PCR primers used were beta-actin sense (5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'), beta-actin antisense (5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'), WASP sense (5'-TGGACCTAGCCCAGCTGATA-3'), and WASP antisense (5'-AGGGGTCTTGTTCAGCTGA-3'). PCR was done on 100 ng of single-stranded cDNA in the presence of 5 µM each oligonucleotide primer in an Applied Biosystems 2720 Thermal Cycler (40 cycles, denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min). Aliquots of 10 µl of the amplification products were separated by 1.0% agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by Alpha Imager analysis.

Statistical Analysis—The significance of differences between groups was calculated by the Student's t test. Confidence (95%) was set a priori as the desired level of statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Examination of the Phagocytic Cup Formation—When phagocytic receptors on macrophages are stimulated with opsonized foreign materials such as bacteria, phagocytic cups are transiently formed. After the macrophages completely ingest the materials, the phagocytic cups disappear (Fig. 1A). This rapid turnover of phagocytic cups makes it difficult to examine their formation. To circumvent this problem, we have developed a new experimental system. A phosphatidylinositol 3-phosphate kinase inhibitor, 3-MA, inhibits phagocytosis without affecting the formation of phagocytic cups, resulting in stabilization of the phagocytic cups (59) (supplemental Fig. S1A). We took advantage of this effect of 3-MA on phagocytosis. To test this system, we incubated macrophages with IgG-opsonized latex beads (3-µm diameter) for 30 min. Cells were fixed, permeabilized, and stained with Alexa 568-phalloidin to visualize the phagocytic cups. Cell staining was examined under a fluorescence microscope. To quantify phagocytosis and the phagocytic cup formation, we scored the percentage of the cells with completely ingested beads (supplemental Fig. S1B, left panel) and the cells with phagocytic cups (supplemental Fig. S1B, right panel), respectively. The cells completely ingested most beads, and few phagocytic cups were observed in the absence of 3-MA (supplemental Fig. S1B, open bars). By contrast, in the presence of 3-MA (10 mM), uptake of beads was inhibited, and many phagocytic cups were observed at the cell surface (supplemental Fig. S1B, closed bars). A representative cell from each experiment after a 30-min incubation is shown. Typical phagocytic cups were observed in the presence of 3-MA (supplemental Fig. S1C, indicated by arrows). This new system enables us to examine the phagocytic cup formation more precisely.

THP-1 cells were differentiated by stimulation with PMA to obtain macrophage-like phenotypes, which closely resemble human monocyte-derived macrophages, as previously reported (60, 61). Human primary monocytes were differentiated into macrophages by incubating with macrophage-colony stimulating factor-1 (35, 43). In both PMA-differentiated THP-1 cells and human primary macrophages, typical phagocytic cups were observed in the presence of 3-MA, when cells were incubated with IgG-opsonized latex beads (Fig. 1, B and C). Additional examples of typical phagocytic cups formed on the cell surface upon stimulation with IgG-opsonized latex beads were presented in supplemental Fig. S2 (supplemental Fig. S2, PMA-differentiated THP-1 cell (A) and human primary macrophage (B)).


Figure 1
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  		FIGURE 1.
Formation of a phagocytic cup. A, a schematic of the formation of a phagocytic cup during ingestion of foreign materials. Binding of opsonized foreign materials to phagocytic receptors in phagocytes results in formation of a cup-shaped F actin-rich structure called the phagocytic cup. B and C, phagocytic cups can be visualized with phalloidin because they are rich in F-actin. PMA-differentiated THP-1 cells (a human monocyte cell line) (B) and human primary macrophages (C) were incubated with IgG-opsonized latex beads and then stained with Alexa 568-phalloidin. Arrows indicate typical phagocytic cups. D and E, phagosomes are barely stained with Alexa 568-phalloidin. PMA-differentiated THP-1 cells were transfected with activated c-src to enhance phagocytosis. src-untransformed THP-1 cells (Baseline) (D) and src-transformed THP-1 cells (E) were incubated with IgG-opsonized latex beads in the absence of 3-MA and stained with Alexa 568-phalloidin. In B-E, left panels and right panels indicate the phase-contrast images and fluorescence images stained with Alexa 568-phalloidin, respectively. The bar is 10 µm.

 
Phagocytes form the phagocytic cups upon stimulation with foreign materials, and subsequently they ingest the materials into the phagosomes. To clarify the difference between the phagocytic cups and phagosomes, phagosomes were induced and examined. THP-1 cells were co-transfected with activated c-src (Y527F) and pmaxGFP as a transfection marker, and then cells were incubated with IgG-opsonized latex beads in the absence of 3-MA. Cells were stained with Alexa 568-phalloidin. Expression of activated c-src enhances phagocytosis (Fig. 1E) compared with src-untransformed cells (Baseline) (Fig. 1D), since activity of src tyrosine kinases is required for phagocytosis (78). In both src-untransformed cells (base line) and src-transformed cells, phagosomes were barely stained with phalloidin (Fig. 1, D and E), demonstrating that the phagocytic cups are evidently distinct from the phagosomes.

Requirement for WASP in the Phagocytic Cup Formation—We asked if WASP is required for the formation of the phagocytic cup, since it has been shown that the formation of the phagocytic cup is impaired in macrophages from WASP-deficient WAS patients (49). To address this question, we examined whether knockdown of WASP affects the phagocytic cup formation. To do this expression of WASP was knocked down in THP-1 cells by transfection of siRNA, and then the transfected THP-1 cells were examined for the phagocytic cup formation.

We transfected 8 x 106 cells with 1 nmol of siRNAs, prepared total lysates or total RNAs from total transfected cells, and analyzed the expression level of WASP by immunoblotting or reverse transcription-PCR. Transfection of siRNA for WASP decreased both the amount of WASP (Fig. 2A, lane 2) and the expression of WASP mRNA (Fig. 2A, lane 6) in PMA-differentiated THP-1 cells compared with transfection of the scrambled control siRNA (Fig. 2A, lanes 1 and 5). Transfection of the WASP siRNA and its scrambled control siRNA had no effect on the expression of beta-actin (Fig. 2A, lanes 3, 4, 7, and 8). Cells were co-transfected with siRNAs and FITC-conjugated control siRNA, and then FITC-positive cells were examined for the phagocytic cup formation. To quantify the phagocytic cup formation, we scored the percentage of cells with the phagocytic cups per siRNA-transfected cells (FITC-labeled control siRNA-positive cells). When expression of WASP was knocked down, the phagocytic cup formation of THP-1 cells was reduced (Fig. 2B, left panel). To verify the result obtained from the experiments using a cell line, THP-1, we performed the same experiments using human primary macrophages. The phagocytic cup formation of macrophages was also reduced by transfection of siRNA for WASP (Fig. 2B, right panel). N-WASP expression is not high enough to compensate for reduced WASP expression, since N-WASP expression in THP-1 cells is much lower than WASP (less than 5% of WASP) based on reverse transcription-PCR results (27). These results suggest that WASP is necessary for the formation of the phagocytic cup.

WASP siRNA transfection also decreased phagocytosis expressed as phagocytic uptake of IgG-opsonized latex beads (supplemental Fig. S3), as it had decreased phagocytic cup formation. This result confirms that there is a good correlation between phagocytic cup formation and phagocytosis.

To determine whether WASP is required for the formation of the phagocytic cup, we tested if reduced phagocytic cup formation in the WASP knockdown cells would be reversed by re-expression of WASP. To do this, PMA-differentiated THP-1 cells were co-transfected with human WASP siRNA and mouse WASP cDNA and then examined for the phagocytic cup formation in the transfected cells. This experiment was designed based on our previous study (35). A mouse WASP cDNA and the human WASP siRNA were used for co-transfection experiments. The mouse cDNA contains 4 mismatches with the 19 bases of the human WASP siRNA sequence. When human WASP expression was silenced by the human WASP siRNA, mouse WASP was successfully expressed, and the mouse WASP expression reversed reduced formation of podosomes, the actin-based structures for macrophage migration, in the human WASP knockdown cells (35). FLAG-tagged mouse WASP was expressed in the WASP knockdown THP-1 cells (Fig. 2C, lanes 3 and 6). As a negative control, FLAG-tagged PDZ-guanine nucleotide exchange factor (PDZ-GEF) C-terminal fragment (residues 1146-1429) (F-C) (62) was expressed in cells because this fragment is stable in cytosol and does not interact with any WASP-related proteins (35). The human WASP knockdown cells expressing FLAG-tagged mouse WASP showed a significant increase in the phagocytic cup formation compared with cells expressing a FLAG-tagged PDZ-GEF (F-C) (Fig. 2D). This result indicates that reduced formation of the phagocytic cups are reversed by re-expression of WASP in human WASP knockdown cells. These results taken together demonstrate that WASP is required for the formation of the phagocytic cup (Fig. 2).


Figure 2
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  		FIGURE 2.
The role of WASP in the phagocytic cup formation. A and B, expression of WASP was reduced by transfection of siRNA. THP-1 cells were transfected with siRNA for WASP (siWASP) (lanes, 2, 4, 6, and 8) or its scrambled control siRNA (siC) (lanes 1, 3, 5, and 7). Total lysates and total RNAs were prepared from total transfected cells, and the expression levels of WASP and beta-actin were analyzed by immunoblotting (left panels) or reverse transcription -PCR (right panels). B, effects of WASP siRNA on the phagocytic formation. Cells were co-transfected with WASP siRNA and FITC-conjugated control siRNA, and FITC-positive transfected cells were examined for the phagocytic cup formation. PMA-differentiated THP-1 cells (left panel) and human primary macrophages (right panel) were transfected with siRNAs. The percentage of cells with the phagocytic cups per transfected cells was scored. The results of cells transfected FITC-conjugated control siRNA only (open bars) and cells transfected with siRNAs (+siC and +siWASP) (closed bars) were shown. C, re-expression of WASP in WASP knockdown cells. PMA-differentiated THP-1 cells were co-transfected with siRNAs and cDNAs. Cells were transfected with the control siRNA (+siC, the scrambled control siRNA for WASP), siRNA for WASP (+siW), the control cDNA (+F-C, FLAG-tagged PDZ-GEF cDNA), or the mouse WASP cDNA (+F-mW/+F-mWASP, FLAG-tagged mouse WASP cDNA). Total lysates were prepared from transfected cells and analyzed by immunoblotting (IB) with anti-FLAG antibody (lanes 1-3) and anti-WASP antibody (lanes 4-6). Anti-human WASP monoclonal antibody recognizes with mouse WASP. D, effect of re-expression of WASP in WASP knockdown cells on the phagocytic cup formation. Cells were co-transfected with siRNAs, cDNAs, and FITC-conjugated control siRNA, and then FITC-positive cells were examined for the phagocytic cup formation. The phagocytic cup formation of cells transfected with control siRNA (+siC) (open bar) and cells transfected with WASP siRNA (+siW) and cDNAs (+F-C or +F-mW) (closed bars) was scored. Data represent the mean ± S.D. of triplicate measurements.

 
The Role of the VCA Domain in the Phagocytic Cup Formation—The WASP C-terminal VCA domain has the activity to stimulate actin polymerization by interacting with Arp2/3 complex (Fig. 3A) (7, 8, 22, 23). We then asked if this domain plays an important role in the formation of the phagocytic cup, since the phagocytic cup is an F-actin-rich structure. To evaluate the importance of the activity of the VCA domain in the phagocytic cup formation, we examined if a deletion of the VCA domain affects the formation of the phagocytic cup. PMA-differentiated THP-1 cells were co-transfected with the Myc-tagged WASP constructs and pmaxGFP as a transfection marker, and then GFP-positive cells were examined for the phagocytic cup formation. The expression of the Myc-tagged WASP (residues 1-502) and the VCA domain-deleted WASP mutant (dVCA) (residues 1-435) in transfected cells was detected by immunoblotting (Fig. 3, A and B). The phagocytic cups were extensively formed in the cells expressing the full-length WASP, whereas the phagocytic cup formation was barely induced in the cells expressing the VCA domain-deleted WASP mutant (dVCA) (Fig. 3C). This result indicates that the activity of the WASP C-terminal VCA domain plays a critical role in the phagocytic cup formation.


Figure 3
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  		FIGURE 3.
The role of the VCA domain in the phagocytic cup formation. A, domain organization of WASP. BR, Basic Region; GBD, GTPase binding domain; Pro-rich, proline-rich region. B, expression of the Myc-WASPs. PMA-differentiated THP-1 cells were transfected with the Myc-tagged full-length WASP (lane 1) or the Myc-tagged VCA domain-deleted WASP mutant (+dVCA) (lane 2). Total lysates prepared from the transfected cells were analyzed by immunoblotting with anti-Myc monoclonal antibody. C, cells were co-transfected with the WASP constructs (WASP, full-length WASP; dVCA, the VCA domain-deleted WASP mutant) and pmaxGFP, and GFP-positive transfected cells were examined for the phagocytic cup formation. Data represent the mean ± S.D. of triplicate measurements.

 
Phosphorylation of WASP—The activity of the WASP C-terminal VCA domain is regulated by the Basic Region, the GTPase binding domain, and the proline-rich region (Fig. 3A) (22, 23, 63-65). Phosphorylation of WASP on tyrosine 291 (Tyr-291) within the GTPase binding domain stabilizes the active conformation of WASP, which increases the basal activity of the VCA domain toward actin polymerization (64, 65). In addition, it has been shown that some phosphorylated proteins accumulate at the phagocytic cup (49, 66). Based on these observations, we hypothesized that WASP is phosphorylated in macrophages and that the WASP phosphorylation plays an important role in the phagocytic cup formation. To test this hypothesis, WASP was immunoprecipitated from PMA-differentiated THP-1 cells and immunoblotted with anti-phosphotyrosine monoclonal antibody (4G10). Immunoprecipitated WASP was recognized with 4G10, indicating that WASP was tyrosine-phosphorylated in PMA-differentiated THP-1 cells (Fig. 4A, lane 6). When cells were transfected with the Myc-tagged WASP wild-type construct, Myc-WASP was phosphorylated (Fig. 4B, lane 5). By contrast, when cells were transfected with a non-phosphorylatable WASP mutant construct (Y291F) in which the tyrosine at residue 291 was replaced with phenylalanine, WASP phosphorylation was not detected (Fig. 4B, lane 6). These results indicate that WASP is phosphorylated on tyrosine 291 in macrophages.


Figure 4
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  		FIGURE 4.
Phosphorylation of WASP. A, THP-1 cells starved in serum-free medium for 6 h (-) (lanes 1, 3, and 5) and PMA-differentiated THP-1 cell (+) (lanes 2, 4, and 6) were lysed, and WASP was immunoprecipitated with anti-WASP monoclonal antibody from the cell lysates. Total lysates were immunoblotted for WASP (lanes 1 and 2), and the WASP immunoprecipitates (IP) were immunoblotted (IB) by anti-WASP antibody (lanes 3 and 4) or anti-phosphotyrosine monoclonal antibody (4G10) (lanes 5 and 6) for WASP phosphorylation. B, PMA-differentiated THP-1 cells were transfected with the Myc-tagged wild-type WASP (wild) (lanes 1, 3, and 5) or the Myc-tagged non-phosphorylatable WASP mutant (Y291F) (lanes 2, 4, and 6). Cells were lysed, and Myc-tagged WASPs were immunoprecipitated with anti-Myc monoclonal antibody from cell lysates. Total lysates were immunoblotted for Myc-tagged WASPs (lanes 1 and 2), and the Myc immunoprecipitates were immunoblotted by anti-Myc monoclonal antibody (lanes 3 and 4) or anti-phosphotyrosine monoclonal antibody (4G10) (lanes 5 and 6) for WASP phosphorylation.

 
Importance of WASP Phosphorylation in the Phagocytic Cup Formation—To evaluate the importance of the WASP phosphorylation in the formation of the phagocytic cup, we made a comparison of the activity to induce the phagocytic cup formation between a non-phosphorylatable WASP mutant (Y291F) and a phosphomimetic WASP mutant (Y291E). PMA-differentiated THP-1 cells were transfected with Y291F, Y291E, or the dVCA as a negative control. Expression of the Myc-tagged WASP mutants was detected by immunoblotting (Fig. 5A). Cells were co-transfected with pmaxGFP as a transfection marker, and then GFP-positive cells were examined for the phagocytic cup formation. The non-phosphorylatable WASP mutant (Y291F) displayed a significant activity to induce the phagocytic cup formation compared with dVCA (Fig. 5B, left panel). The phosphotyrosine-mimetic WASP mutant (Y291E) induced extensive phagocytic cup formation compared with Y291F (Fig. 5B, left panel). This result was verified by the experiments using primary macrophages. The phagocytic cup formation in the macrophages expressing Y291F was significantly higher than dVCA, and the phagocytic cup formation in the macrophages expressing Y291E was extensively enhanced compared with Y291F (Fig. 5B, right panel). These results suggest that the phosphorylation of WASP on tyrosine 291 enhances WASP-induced effects on actin polymerization, potentiating the phagocytic cup formation.


Figure 5
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  		FIGURE 5.
Effect of the WASP phosphorylation on the phagocytic cup formation. A, PMA-differentiated THP-1 cells were transfected with the Myc-tagged dVCA, Y291F (non-phosphorylatable WASP mutant), or Y291E (phospho-mimetic WASP mutant) and then lysed. Total lysates were immunoblotted for the expression of the Myc-tagged WASP mutants. B, cells were co-transfected with the WASP constructs (dVCA, Y291F, or Y291E) and pmaxGFP, and GFP-positive transfected cells were examined for the phagocytic cup formation. Data represent the mean ± S.D. of triplicate measurements.

 
Complex Formation of WASP with WIP in Macrophages—A complex of WASP with WIP functions in important cellular processes such as the formation of the immune synapse (17, 26), cell polarization (27), and podosome formation (35, 41). We, therefore, hypothesized that the WASP·WIP complex may play a role in the formation of the phagocytic cup. To determine whether WASP binds WIP during the phagocytic cup formation, PMA-differentiated THP-1 cells were stimulated with IgG-opsonized latex beads and then lysed in the buffer A for immunoprecipitation. WASP was immunoprecipitated from the cell lysates with anti-WASP monoclonal antibody followed by immunoblotting for WASP and WIP. WIP co-immunoprecipitated with WASP in the lysates from THP-1 cells before and after stimulation with IgG-opsonized latex beads (Fig. 6A, lanes 11 and 12), indicating that WASP and WIP form a complex during the phagocytic cup formation in macrophages.

To determine the subcellular localization of the WASP·WIP complex, PMA-differentiated THP-1 cells were double-stained with phalloidin and anti-WASP or anti-WIP antibody. F-actin staining with phalloidin visualized phagocytic cups (Fig. 6B, center panels). Double staining revealed the co-localization of F-actin and WASP (Fig. 6B, upper panels). Double staining also revealed the co-localization of F-actin and WIP (Fig. 6B, lower panels). These results taken together suggest that WASP and WIP form the complex at the phagocytic cup (Fig. 6).

To determine the requirement for the WASP·WIP complex formation in the phagocytic cup formation, we blocked WASP binding to WIP in cells using a EGFP-tagged WIP C-terminal fragment containing the WASP-binding domain (EGFP-WB; residues 321-503) as a competitor as previously described (27, 35), and then examined transfected cells for the phagocytic cup formation. Expression of the EGFP constructs in cells was detected by immunoblotting (Fig. 7A, lanes 5 and 6). WIP co-immunoprecipitated with WASP in the lysates from cells expressing EGFP alone (Fig. 7A, lane 9). The amount of the co-immunoprecipitated WIP with WASP decreased in the lysates from the cells expressing EGFP-WB detected with the cells expressing EGFP (Fig. 7A, lanes 9 and 10). Considering the 40-60% of the transfection efficiency based on the EGFP expression, the decrease in the amount of the co-immunoprecipitated WIP (Fig. 7A) indicated that the complex formation of WASP with WIP was efficiently blocked in most transfected cells. Formation of the phagocytic cup was reduced in the cells expressing EGFP-WB (Fig. 7B, left panel). This result was verified by the experiment using primary macrophages. Formation of the phagocytic cup was also reduced in the macrophages expressing EGFP-WB compared with EGFP (Fig. 7B, right panel). These results indicate that the phagocytic cup formation was impaired when WASP binding to WIP was blocked in cells, suggesting that the complex formation of WASP with WIP is necessary for the phagocytic cup formation (Fig. 7).


Figure 6
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  		FIGURE 6.
WASP binds WIP to form a complex at the phagocytic cup. A, PMA-differentiated THP-1 cells were stimulated with IgG-opsonized latex beads and then lysed. Total lysates were analyzed by immunoblotting (IB) for the expression of WASP (lanes 1 and 2) and WIP (lanes 3 and 4). The lysates were incubated with a control (cntl.) mouse IgG (lanes 5, 6, 9, and 10) or anti-WASP monoclonal antibody (lanes 7, 8, 11, and 12). The immunoprecipitates (IP) were analyzed by immunoblotting for WASP (lanes 5-8) and WIP (lanes 9-12). B, confocal laser scanning micrographs of PMA-differentiated THP-1 cells. Cells were incubated with IgG-opsonized latex beads and double-stained with anti-WASP or anti-WIP antibodies (left panels) and phalloidin (center panels). Yellow indicates co-localization of green (WASP or WIP) and red (F-actin) (right panels). The inset depicts additional magnification of a phagocytic cup. The bar is 10 µm.

 


Figure 7
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  		FIGURE 7.
The role of the WASP·WIP complex in the phagocytic cup formation. A, binding of WASP to WIP was blocked by the EGFP-tagged WASP binding fragment of WIP (EGFP-WB). PMA-differentiated THP-1 cells were transfected with EGFP alone (EGFP) as a negative control (lanes 1, 3, 5, 7, and 9) or EGFP-WB (lanes 2, 4, 6, 8, and 10). Cells were lysed and immunoblotted for WASP (lanes 1 and 2), WIP (lanes 3 and 4), and EGFP (lanes 5 and 6). WASP was immunoprecipitated (IP) from the lysates and analyzed by immunoblotting for WASP (lanes 7 and 8) and WIP (lanes 9 and 10). B, EGFP-positive transfected cells were examined for the phagocytic cup formation. PMA-differentiated THP-1 cells (left panel) or human primary macrophages (right panel) were transfected with EGFP alone as a negative control (open bars) or EGFP-WB (closed bars). Data represent the mean ± S.D. of triplicate measurements.

 
We next asked which is critical for the phagocytic cup formation, the WASP phosphorylation or the formation of the WASP·WIP complex. To address this question, we blocked WASP binding to WIP by expression of the WB fragment (EGFP-WB) in the cells expressing the WASP phosphorylation mutants and then examined cells for the phagocytic cup formation. Expression of the WASP mutants and the EGFP-tagged proteins was detected by immunoblotting (Fig. 8A). The nonphosphorylatable WASP mutant (Y291F) significantly induced the phagocytic cup formation, and the phosphotyrosine-mimetic WASP mutant (Y291E) greatly enhanced the phagocytic cup formation in the cells expressing EGFP (Fig. 8B), which is consistent with the results in Fig. 5. However, when WASP binding to WIP was blocked by the expression of EGFP-WB, the activities to induce the phagocytic cup formation in the cells expressing both WASP mutants, Y291F and Y291E, reduced to the same level as the VCA domain-deleted WASP mutant (Fig. 8B). This result suggests that the WASP·WIP complex formation is critical for the phagocytic cup formation rather than the WASP phosphorylation.


Figure 8
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  		FIGURE 8.
Complex formation of WASP with WIP is critical for the phagocytic cup formation. A, PMA-differentiated THP-1 cells were co-transfected with the Myc-tagged WASP constructs (dVCA as a negative control, Y291F, or Y291E) and the EGFP constructs (E, EGFP alone as a negative control; E-W, EGFP-WB). Total lysates were analyzed by immunoblotting for the WASP mutants (lanes 1-5) and EGFP (lanes 6-10). B, EGFP-positive transfected cells were examined for the phagocytic cup formation. Data represent the mean ± S.D. of triplicate measurements.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have demonstrated that the tyrosine-phosphorylated WASP and the WASP·WIP complex play essential roles in the efficient formation of the phagocytic cup in macrophages. Thus, WASP plays a critical role in phagocytosis.

It has been demonstrated that tyrosine phosphorylation of WASP (not N-WASP) is induced by several tyrosine kinases including Fyn (67), Lyn (68), Btk (69, 70), Hck (hematopoietic cell kinase) (71), and Pyk2 (72). On the other hand, WASP phosphorylation has been observed in several experimental systems including platelets (69, 73), B cells (74), and COS-7 cells (75). However, physiological roles of the WASP phosphorylation remain largely unknown.

Just recently, Chellaiah et al. (72) reported the importance of the WASP phosphorylation in a physiologically relevant system. They demonstrated that the WASP phosphorylation is critical in osteoclast bone resorption. Our results suggest that the WASP phosphorylation is essential for the efficient formation of the phagocytic cup (Figs. 4 and 5), providing a linkage between the WASP phosphorylation and a physiological function, phagocytosis.

The non-phosphorylatable WASP mutant (Y291F) significantly induced the formation of the phagocytic cup, and the phospho-mimetic WASP mutant (Y291E) was much more effective in the phagocytic cup formation than Y291F (Fig. 5B). This result is consistent with a previous study that the WASP mutant Y291E induced filopodia much more efficiently than Y291F (75). We suggest that the higher activity to induce the phagocytic cup formation by Y291E than Y291F is based on the fact that the phosphorylation on tyrosine 291 increases basal activity of WASP toward Arp2/3 complex (64). These results also suggest that the phosphorylation of WASP on tyrosine 291 is a regulatory mechanism essential for the efficient formation of the phagocytic cup.

It has been previously reported that the complex formation of WASP with WIP is critical for several physiologically important processes including the immune synapse formation in T cells (17, 26) and podosome formation in macrophages (35). We also demonstrated that the WASP·WIP complex is required for the phagocytic cup formation (Fig. 7). Because the immune synapse, podosome, and phagocytic cup are all actin-based membrane structures, these results suggest that the complex formation of WASP with WIP is a common molecular step in these specialized actin-based membrane structures.

WIP has two important functions toward WASP. One is to recruit WASP to an appropriate site where the WASP·WIP complex physiologically functions (17, 35, 40). The other one is to stabilize WASP in cells (37). We showed that blocking binding of the phospho-mimetic WASP mutant (Y291E) to WIP reduced the phagocytic cup formation to the same level as the VCA-deleted WASP mutant (Fig. 8B), although the WASP mutant (Y291E) has the high activities to stimulate actin polymerization and to induce the phagocytic cup formation (Fig. 5). This result suggests that the recruitment of WASP by WIP to the plasma membrane is critical for the phagocytic cup formation.

The podosome is another actin-rich structure formed at the plasma membrane of macrophages (76). Although the podosome is functionally and morphologically distinct from the phagocytic cup, the formation of the WASP·WIP complex is a common molecular step in the formation of both actin-based structures, phagocytic cup and podosomes (Figs. 6 and 7) (35). What makes the functional and morphological distinctions between these structures? We speculate that one is the difference of where the WASP·WIP complex localizes in macrophages. The WASP·WIP complex is recruited to a leading edge for the formation of the podosomes (76), and the complex is recruited to a site of ingestion of the foreign materials for the formation of phagocytic cup (Fig. 6) by interacting with a specific and unidentified component of each structure. Another one is the difference of signal transduction pathways mediating the formation of these structures. The WASP·WIP complex functions downstream of chemokine receptors for the formation of the podosomes (76), and the complex functions downstream of opsonin receptors for the formation of phagocytic cup (53-55).

In conclusion, we showed that the WASP phosphorylation and the complex formation of WASP with WIP are the essential molecular steps for the formation of the phagocytic cup. The absence of WASP impairs these molecular steps, resulting in defective formation of the phagocytic cup. Defective phagocytic cup formation accounts for phagocytic defects, contributing to recurrent infections in WAS patients. Thus, our results indicate that WASP is a key regulator of the phagocytic cup formation in macrophages, suggesting a possible disease mechanism underlying recurrent infections in WAS patients.


   FOOTNOTES
 
* This work was supported by National Institute of Health Grant R01HD042752 (to S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed: Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-795-5258; Fax: 858-795-5225; E-mail: stsuboi{at}burnham.org.

2 The abbreviations used are: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; F-actin, filamentous actin; VCA domain, verprolin/cofilin/acidic domain; dVCA, VCA domain-deleted; Arp2/3, actin-related protein; WH1/EVH1, WASP homology1/Ena/VASP (vasodilator-stimulated phosphoprotein) homology1; WIP, WASP-interacting protein; 3-MA, 3-methyladenine; PMA, phorbol 12-myristate 13-acetate; FCS, fetal calf serum; GFP, green fluorescence protein; EGFP, enhanced GFP; siRNA, short interfering RNA; FITC, fluorescein isothiocyanate; PDZ-GEF, PDZ-guanine nucleotide exchange factor; WB domain, WASP binding domain. Back


   ACKNOWLEDGMENTS
 
We greatly thank Dr. Robert C. Liddington (Burnham Institute for Medical Research) for critical reading of the manuscript. We appreciate the gift of the activated c-src construct from Dr. Sara Courtneidge (Burnham Institute for Medical Research).



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