To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcαRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcαRI tail is important for FcαRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcαRI activation. PP2A binds to the intracellular tail of FcαRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcαRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcαRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcαRI ligand binding regulation, a key step in initiating an immune response.

Antibodies play a fundamental role in immune defense, providing protection against invading microorganisms. IgA represents the most abundantly produced Ab isotype in the body and makes essential contributions to immune protection both systemically and at mucosal sites (1, 2). The myeloid receptor for IgA, FcαRI (CD89), is a low affinity receptor for monomeric IgA (Ka ∼ 106 M−1). Polymeric IgA and IgA immune complexes, however, bind with greater avidity. FcαRI expression, restricted to cells of the myeloid lineage (3, 4), is constitutive and ligand independent since receptor expression is not altered in patients deficient in IgA (5). IgA-immune complexes can trigger numerous cellular responses via FcαRI, including phagocytosis, Ag presentation, cytokine release, and Ab-dependent cellular cytotoxicity (3, 4).

The FcαRI ligand binding subunit is composed of two extracellular Ig-like domains, a transmembrane region and a short cytoplasmic tail of 41 amino acids devoid of known signaling motifs. Given the strong cellular responses that can be initiated by FcαRI triggering, regulation of receptor activity is likely to be controlled (6, 7, 8, 9). For FcαRI, it was demonstrated in cells of allergic patients that FcαRI is “primed” and can be continuously activated by immune complexes, indicating the importance of a proper regulation of receptor activity (10). As described for FcγRI, FcγRIIa, and FcαRI, ligand binding can be rapidly modulated in response to intracellular signals without effects on receptor expression levels (11, 12, 13). This process was termed inside-out regulation, similar to that observed in the regulation of the integrin family of cell surface adhesion receptors (14). This inside-out signaling regulating FcRs is a rapid mechanism that allows cells to respond quickly to their environment.

For FcαRI, the molecular components regulating this pathway are currently unknown. In the present study, we identify the ubiquitously expressed Serine/Threonine phosphatase, PP2A, to specifically interact with the FcαRI intracellular domain. Using novel phosphorylation specific Abs, we characterized the role of FcαRI intracellular Serine 263 phosphorylation in cytokine-induced inside-out signaling of FcαRI and involvement of PP2A in this process. Furthermore, we demonstrated that cytokine induced increase of FcαRI ligand binding capacity involves PP2A activation, and dephosphorylation of FcαRI. Finally, we showed the biological relevance of this regulatory mechanism as PP2A activity modulation critically influences FcαRI mediated phagocytosis of pathogens.

The following reagents were purchased as here cited: anti-Flag Ab, anti-PP2A A-subunit Ab from Sigma-Aldrich, anti-PP1 Ab from Upstate Biotechnology, mAb A59 PE, mAb CD14 from BD Pharmingen, mIgG1-RPE from Dako, anti-HA Ab from Covance, Goat (Fab′)2 anti-mIgG1-RPE from (Southern Technologies), goat (Fab′)2 anti-rIgG-FITC from (Jackson ImmunoResearch Laboratory), anti-GST-Ab from Amersham Biosciences, and the anti-FcαRI rabbit serum was a kind gift from C. van Kooten (Leiden University Medical Center, Leiden, The Netherlands) (15). The inhibitor Okadaic Acid (OA) was from Alexis Biochemicals, and catharidin (Cat), fostriecin (Fos), and ascomycin (Acs) were from Sigma-Aldrich. Prot A/G PLUS agarose was from Santa Cruz Biotechnology, hygromycin was from Invitrogen, and Calcein-AM was from Molecular Probes. Ficoll-Histopaque and Percoll were from Amersham Biosciences. IL-3, IL-5, and GM-CSF were provided by Dr. H. Honing and described in Ref. 11, 16 . V-gene matched IgA and IgG Abs directed against Porin A are described in Ref. 6 . Anti-FcαRI-pho-Serine 263 and anti-FcαR-Serine 263 peptides (LTFARTPphoSVCK and LTFARTPSVCK) and Abs were generated by Covance Research Products.

The constructs FcαRI (wild type (Wt)),3 human FcγRI, FcγRIIa, FcRγIIIa, FcεRI, and murine (m) Fcγ in the yeast two-hybrid vector pGBT9 were described in Ref. 12 . The human FcαRI intracellular domain Serine 263A (S263A) and Serine 263D (S263D) mutants were generated by site directed mutagenesis. The FcαRI-Wt-Gly6 construct, served as a positive control, was generated by introduction of six glycines between the sequence of the GAL4 binding domain (BD) and the sequence of FcαRI to expose the FcαRI tail sequence to putative interacting proteins. FLAG-tagged PP2A A-subunit was inserted in pCB7. PP2A C-subunit Wt, H59Q, and H118Q were described in Ref. 17 and pMT-GST-FcαRI-tail, GST-FcαRI-S263A mutant, and the empty vector in Ref. 16 .

Oligo(dT) primed human dendritic cell (DC) library cloned in pACT-2 was a gift from G. Adema (Department of Tumor Immunology, Nijmegen, The Netherlands) (18). Screening for FcαRI-CY interacting proteins and interaction of PP2A with other FcRs was described in Ref. 12 .

The human monocytic cell line U937 was used in coimmunoprecipitation experiments. BaF3 cells stably transfected with FcαRI Wt, FcαRI S263A, and FcαRI S263D cell line were described in Ref. 16 . Eosinophil isolation was described in Ref. 16 . Monocytes were isolated as follows: first PBMC were collected using Ficoll-Histopaque gradient centrifugation. Next, the monocytes were isolated from PBMC using three-layer Percoll-gradients (34, 47.5, and 60%). After centrifugation monocytes were collected from upper interphase, purity of monocytes was checked by CD14 staining.

For overexpression of FcαRI and PP2A A-subunit, 293T cells were transfected using FuGENE 6 reagent (Roche) according to the manufactures recommendations. Overexpression of PP2A constructs in BaF3-FcαRI cells were performed with AMAXA nucleofector kit V (AMAXA Biosystems) according to the manufacturer’s protocol.

Fusion proteins of FcαRI cytosolic tail (Wt) with GST or GST only were purified from Escherichia coli lysates with glutathione Sepharose 4B beads (Amersham Biosciences). U937 cells were lysed in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl2, 4 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM DTT, and 1 mM PMSF) and after centrifugation to clarify the lysate, the supernatant was incubated with the GST fusion proteins for 2.5 h at 4°C. Next, beads were collected by centrifugation, washed in lysis buffer, and resuspended in Laemmli sample buffer. Subsequently, GST fusion proteins were analyzed for associating proteins by SDS-PAGE and Western blotting as described below.

293T cells transfected with FcαRI and PP2A A-subunit, or U937 cells, were collected by centrifugation and lysed in RIPA lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% DOC, 0.1% SDS, and 10 mM EDTA) for 30 min at 4°C. The lysate was clarified by centrifugation at 15,000 rpm for 10 min at 4°C, and the supernatant was incubated with indicated Ab-absorbed beads for 2.5 h at 4°C by end-over-end rotation. Next, beads were isolated, washed twice in RIPA lysis buffer, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE and Western blotting.

In vitro phosphorylation of GST-FcαRI intracellular domain proteins was performed as described in Ref. 16 . After phosphorylation, samples were washed extensively in a phosphatase buffer (20 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, 1 mM EDTA, and 0.1 mM MnCl2) and subsequently incubated with or without recombinant PP1 or 2A in phosphatase buffer. After incubation for 30 min at 30°C, samples were washed and resuspended in Laemmli buffer, and analyzed by electrophoresis on 15% SDS-PAGE gels. To assess the contribution of endogenous PP2A on FcαRI phosphorylation, BaF3 cell lysates (not cytokine-starved) were prepared as described above; however, NaF as general Serine/Threonine phosphatase inhibitor was substituted for the PP2A inhibitor OA (10−6 M). Substrate phosphorylation was detected by autoradiograph, and, if indicated, the relative intensities of specific bands were determined with a PhosphorImager STORM280 and Image quant software (Molecular Dynamics). Phosphorylation of GST-FcαRI-tail was set at 100%.

The IgA or IgG assays with Dynal beads were used for eosinophils and monocytes and performed as described previously in Ref. 16 . The ligand binding assays via IgA-coated plates were performed with BaF3 FcαRI-transfected cells as was described in Ref. 19 . PP2A inhibitors OA (10−9-10−6 M), Cat (10−6 M), or Fos (10−6 M) were used to inhibit PP2A and the PP2B inhibitor Acs (10−7 M) (20, 21, 22) was used as a negative control. The inhibitors were added to cells, incubated at 37°C before the assay. Nontreated cells refer to cells treated with only the solvent of the indicated inhibitor.

PP2A activity was measured with the PP2A immunoprecipitation phosphatase assay kit (Upstate Biotechnologies). The assay was performed according to the manufacturer’s recommendations. In brief, BaF3-FcαRI-Wt transfected cells were starved overnight in low serum concentrations (0.5%) with or without cytokine, lysed in 0.2% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, and 1 mM EDTA, and tested for PP2A activity. Immunoprecipitation by mIgG1 and OA (10−6 M), added during the phosphatase assay, served as negative controls. Released phosphate in pmol/25 μl was measured by malachite green assay and plotted as fold induction compared with unstimulated cells.

Cell surface FcαRI expression levels were measured by incubating the cells with CD89 mAb A59-PE or with an isotype control. Cells were washed twice with PBS containing 1% BSA and 0.1% NaN3. For detection of intracellular FcαRI-Serine 263, BaF3-transfected cells were cultured overnight in 0.5% FCS with or without cytokines and freshly isolated eosinophils were, if indicated, stimulated with IL-5. Then, cells were fixed by 1.5% PFA and incubated at room temperature for 10 min, pelleted, and permeabilized by resuspending with vigorous vortexing in 500 μl ice-cold MeOH per 106 cells followed by minimal 10 min at 4°C or stored at −20°C. Subsequently, staining with 10 μg/ml Ab FcαRI-phospho/non-phospho Serine 263 and GαRIgG-FITC was performed and analyzed on a FACSCalibur (BD Biosciences). Each FACS staining was performed in triplicate. Values of p were determined on FcαRI-phospho/non-phospho stainings using a nonpaired, two-tailed Students t test; values of p < 0.05 are considered significant.

Samples were prepared in reducing Laemmli sample buffer and analyzed by SDS-PAGE. Proteins were transferred on polyvinylidene fluoride membranes (Immobilon-P; Millipore) and blocked with 5% low fat milk powder in PBS for 1 h at room temperature. For PP2A detection, the membranes were probed with anti-PP2A followed by incubation for 45 min with goat anti-rabbit IgG (H+L)-HRP (Pierce). After extensive washing in PBST (PBS and 0.05% Tween 20), bound Abs were detected by chemiluminescence (Amersham Biosciences).

Internalization of FcαRI was described in Ref. 19 . In brief, cells were loaded with mAb anti-human CD89 or mIgG1 isotype control for 60 min at 4°C. After washing, the cells were incubated with a second Ab goat F(ab′)2 anti-mouse IgG1. At this point, the samples were split in two. One sample was put at 4°C to measure total surface expression of FcαRI, the other sample was put at 37°C for indicated time points. After these incubation periods, the surface FcαRI expression was measured by staining the retained surface receptors with a third Ab (RαG-IgG (H+L) FITC-conjugated; Jackson ImmunoResearch Laboratories) for 30 min at 4°C. After washing, the samples were analyzed for FcαRI surface expression on a FACSCalibur. Internalization of FcαRI in the 37°C samples was calculated as a percentage of the total FcαRI expression measured in the 4°C samples.

Influence of PP2A inhibition on internalization was assessed by incubation of samples with OA for 15 min at 37°C prior to the cross-linking step and during incubation with cross-linking Ab. Internalization of the nontreated samples was set at 100%.

Human monocytes were isolated as described under “isolation of blood cells.” A total of 1 × 105 cells were, if indicated, treated with OA (10−6 M) and opsonized by incubation for 30 min at 4°C with 5 × 106 IgA1 or IgG1 FITC-labeled Neisseria meningitidis in 5% heat inactivated FCS (final volume of 100 μl). Nonattached bacteria were separated from monocytes by centrifugation at 300 × g for 5 min. Following washing, cells were resuspended in 400 μl RPMI 1640 containing 10% FCS. Monocytes were split into two aliquots and further incubated either at 4 or 37°C. After 30 min, phagocytosis was stopped by addition of ice-cold PBS. After washing, cell surface-bound bacteria were detected by incubation with either goat IgG F(ab′)2-anti-hIgA-RPE (Southern Biotechnology Associates) or GIgG F(ab′)2-anti-hIgG-RPE (Southern Biotechnology Associates) for 30 min at 4°C. Samples were analyzed on a FACSCalibur. As a control for aspecific binding, nonopsonized bacteria were used. Ab-mediated phagocytosis was expressed as the decrease of the number double positive monocytes (PE-fluorescent FITC) incubated at 37°C compared with the 4°C samples. Number of double positive monocytes at 4°C was set at 100% (23).

Since the FcαRI cytosolic tail bears no known intrinsic signaling motifs (Fig. 1,A) (scansite.mit.edu) and cytokine-induced inside-out signaling of FcαRI is dependent on the intracellular domain, we searched for proteins interacting with the tail of FcαRI (13, 16, 24). We identified the regulatory or scaffold subunit (A-subunit) of the PP2A enzyme (four independent clones) as an interacting protein in yeast two-hybrid screens performed in a human DC cDNA library. PP2A selectively interacted with the FcαRI tail (FcαRI-Wt and FcαRI-Wt-Gly6) but not with bait vector (data not shown and Fig. 1,B). Additionally, PP2A binding to FcαRI-S263A, FcαRI-S263D mutant receptors (representing the constitutive active and inactive receptor, respectively), and other FcRs was tested (Fig. 1, A and B). PP2A only interacts with FcαRI-Wt, FcαRI S263D mutant, and FcγRIIa but not with the other receptors tested. Of note, PP2A A-subunit association to FcγRIIa has not been described before. To confirm FcαRI-PP2A interaction in yeast, we performed GST pulldown experiments by using recombinant GST-fusion proteins of FcαRI-Wt cytosolic tail (aa 226–266) and recombinant GST protein only (as a control). PP2A was coprecipitated with the GST-Wt-FcαRI cytosolic tail (GST-FcαR-tail) and not with the GST-fusion protein alone (GST) (Fig. 1,C). Also the interaction between full-length proteins was shown by cotransfected full-length FcαRI and FLAG-tagged PP2A A-subunit in 293T cells (Fig. 1,D, the reverse experiment showed FcαRI to be coimmunoprecipitated with the PP2A A-subunit, data not shown, n = 2). Next, coimmunoprecipitations performed with the human monocytic cell line U937 (that endogenously expresses FcαRI, FcR γ-chain, and PP2A) verified an endogenous interaction between FcαRI and PP2A (Fig. 1 E). Taken together, these experiments demonstrated the PP2A regulatory subunit (A-subunit) to interact with FcαRI intracellular domain, independently of FcR γ-chain.

FIGURE 1.

Association of PP2A A-subunit with FcαRI. A, Protein sequences of the intracellular domains of indicated FcR. Underlined FcαRI Serine 263 mutated to an alanine (A) or aspartic acid (D). B, β-galactosidase activity of indicated FcR or empty vector cotransfected with PP2A A-subunit as described in Materials and Methods (n ≥ 2). C, Pulldown experiments of recombinant GST-Wt-FcαRI intracellular tail fusion proteins (GST-FcαRI-tail) and (GST) marks recombinant protein of GST only. Western blots were immunoblotted for PP2A A-subunit. Reprobing with an anti-GST Ab served as loading control (n = 3). Coimmunoprecipitation of 293T cells overexpressing full-length FcαRI and FLAG-tagged PP2A A-subunit (D, n = 2) or with the human monocytic cell line U937 (E, n = 3). Immunoprecipitated with anti-FcαRI beads absorbed beads is indicated (+) and isotype Ab absorbed beads (-). Western blots were immunoblotted for PP2A A-subunit and IgG H chain bands served as a loading control.

FIGURE 1.

Association of PP2A A-subunit with FcαRI. A, Protein sequences of the intracellular domains of indicated FcR. Underlined FcαRI Serine 263 mutated to an alanine (A) or aspartic acid (D). B, β-galactosidase activity of indicated FcR or empty vector cotransfected with PP2A A-subunit as described in Materials and Methods (n ≥ 2). C, Pulldown experiments of recombinant GST-Wt-FcαRI intracellular tail fusion proteins (GST-FcαRI-tail) and (GST) marks recombinant protein of GST only. Western blots were immunoblotted for PP2A A-subunit. Reprobing with an anti-GST Ab served as loading control (n = 3). Coimmunoprecipitation of 293T cells overexpressing full-length FcαRI and FLAG-tagged PP2A A-subunit (D, n = 2) or with the human monocytic cell line U937 (E, n = 3). Immunoprecipitated with anti-FcαRI beads absorbed beads is indicated (+) and isotype Ab absorbed beads (-). Western blots were immunoblotted for PP2A A-subunit and IgG H chain bands served as a loading control.

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To assess the effect of PP2A on FcαRI cytosolic tail phosphorylation, we studied recombinant GST-FcαRI-tail proteins in in vitro phosphatase experiments. The FcαRI tail was phosphorylated as described previously (16) and, subsequently, incubated in the absence or presence of recombinant PP2A. This revealed recombinant active PP2A to be capable of dephosphorylating the FcαR tail (Fig. 2,A). Selective dephosphorylation by PP2A activity and not other family members (such as PP1) is shown in Fig. 2, B and C. To further establish PP2A activity involvement we tested OA, which is a commonly used PP2A inhibitor (20, 25) in the in vitro phosphorylation assay. Fig. 2 D shows OA treatment to be linked with increased FcαRI phosphorylation. Together, these data suggest involvement of active PP2A in regulation of FcαRI tail phosphorylation.

FIGURE 2.

PP2A dephosphorylates FcαRI intracellular tail. A, Beads binding GST-Wt-FcαRI intracellular tail fusion proteins (GST-FcαRI-tail) or GST alone (GST) were used in an in vitro phosphatase assay with cytokine-starved BaF3 lysates. After phoshorylation of GST recombinant proteins, samples were incubated with (+) or without (−) recombinant active PP2A. Phosphorylation was detected by autoradiography (upper panel). Coomassie staining was used to verify equal loading of GST recombinant proteins (lower panel) (n = 5). B, As described under A, additionally recombinant PP1C was included to determine specificity of PP2A dephosphorylation of GST-Wt-FcαRI recombinant protein (n = 3). C, Quantification of phosphorylation levels, depicted as % relative density measured by the PhosphoImager STORM 280 and Image Quant software. Phosphorylation of nontreated samples was set at 100%. Bars indicate mean ± SEM. D, In vitro kinase assay as described in Material and Methods. BaF3 cells were treated with OA, as indicated. Phosphorylation was detected by autoradiography (upper panel). Coomassie staining served to verify equal loading of GST recombinant proteins (lower panel) (n = 4). E, Beads binding GST-FcαRI intracellular tail fusion proteins of FcαRI-Wt (GST-FcαRI-tail), FcαRI-S263A (GST-FcαRI-S263A), and GST alone (GST) were used in an in vitro kinase assay with cytokine-starved BaF3 cell lysates. Phosphorylation was detected by autoradiography. Coomassie staining was used to verify equal loading of GST recombinant proteins. F, Results of quantification of phosphorylation levels, depicted as % relative density measured by a PhosphoImager STORM 280 and Image Quant software (n = 4). GST-FcαRI-Wt represented by S263, GST-FcαRI-S263A by S263A, and GST only by a minus sign (−). Bars indicate mean ± SEM.

FIGURE 2.

PP2A dephosphorylates FcαRI intracellular tail. A, Beads binding GST-Wt-FcαRI intracellular tail fusion proteins (GST-FcαRI-tail) or GST alone (GST) were used in an in vitro phosphatase assay with cytokine-starved BaF3 lysates. After phoshorylation of GST recombinant proteins, samples were incubated with (+) or without (−) recombinant active PP2A. Phosphorylation was detected by autoradiography (upper panel). Coomassie staining was used to verify equal loading of GST recombinant proteins (lower panel) (n = 5). B, As described under A, additionally recombinant PP1C was included to determine specificity of PP2A dephosphorylation of GST-Wt-FcαRI recombinant protein (n = 3). C, Quantification of phosphorylation levels, depicted as % relative density measured by the PhosphoImager STORM 280 and Image Quant software. Phosphorylation of nontreated samples was set at 100%. Bars indicate mean ± SEM. D, In vitro kinase assay as described in Material and Methods. BaF3 cells were treated with OA, as indicated. Phosphorylation was detected by autoradiography (upper panel). Coomassie staining served to verify equal loading of GST recombinant proteins (lower panel) (n = 4). E, Beads binding GST-FcαRI intracellular tail fusion proteins of FcαRI-Wt (GST-FcαRI-tail), FcαRI-S263A (GST-FcαRI-S263A), and GST alone (GST) were used in an in vitro kinase assay with cytokine-starved BaF3 cell lysates. Phosphorylation was detected by autoradiography. Coomassie staining was used to verify equal loading of GST recombinant proteins. F, Results of quantification of phosphorylation levels, depicted as % relative density measured by a PhosphoImager STORM 280 and Image Quant software (n = 4). GST-FcαRI-Wt represented by S263, GST-FcαRI-S263A by S263A, and GST only by a minus sign (−). Bars indicate mean ± SEM.

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Next, we assessed whether cytokine-induced FcαRI activation was correlated with dephosphorylation of Serine 263. The contribution of Serine 263 phosphorylation to total phosphorylation levels was determined by comparing phosphorylation levels of the GST-FcαRI-tail and the GST-FcαRI-S263A mutant. The decreased phosphorylation levels in the FcαRI-S263A mutant (Fig. 2, E and F) indicated that Serine 263 contributes to the FcαRI-tail phosphorylation levels. To monitor the phosphorylation level of FcαRI S263 during cytokine-induced FcαRI activation we generated two FcαRI Serine 263 specific Abs recognizing phosphorylated (pS263) or nonphosphorylated (S263) Serine 263. We tested these Abs in the BaF3-FcαRI-transfected cell lines and in primary eosinophils, known for their cytokine-induced FcαRI activation upon IL-3 or IL-5 stimulation, respectively (13, 16). Specificity of both Abs was assessed by immunoprecipitation of FcαRI from the different cell lines. These data showed recognition of the FcαRI-Wt cell line and FcαRI-S263D cells with the FcαRI-pS263 Ab on Western blot, whereas the FcαRI-S263A cell line and untransfected cells were negative. The FcαRI-S263 Ab only recognized the FcαRI-Wt cell line (Fig. 3,A). FcαRI expression of all cell lines is shown in Fig. 3,B. Similar results were obtained by staining the different FcαRI cell lines with both Abs in an intracellular FACS staining (Fig. 3, C and D), indicating specificity of the FcαRI-pS263 Ab for pFcαRI Serine 263 and the FcαRI-S263 Ab recognizing FcαRI-Wt. Correlation between FcαRI S263 phosphorylation level and FcαRI functionality was shown by comparing cytokine stimulated or cytokine-starved cells with the FcαRI-pS263 Ab (Fig. 3,E, p = 0.0135), i.e., nonstimulated cells exhibited a modestly higher FcαRI-S263 phosphorylation. Also, freshly isolated eosinophils showed higher levels of S263 phosphorylation compared with IL-5-stimulated eosinophils stained with the FcαRI-pS263 Ab (Fig. 3,F, p = 0.0249). Involvement of PP2A activity in this process was supported by increased S263 phosphorylation of BaF3-FcαRI Wt cells upon OA treatment (Fig. 3,G, p = 0.0245). OA did not affect FcαRI expression levels (Fig. 3,H). Expression of FcαRI-S263 was not altered by cytokine stimulation as determined with α-FcαRI A59 Ab (data not shown and Refs. 10, 16, 24). Moreover, dephosphorylation of Serine 263 due to cytokine stimulation was shown by the FcαRI-S263 Ab recognizing FcαRI-Wt (Fig. 3 I, p = 0.0011). Lower staining levels with this Ab in nonstimulated cells maybe attributed to hindrance by phosphorylation of Serine 263. In conclusion, unstimulated cells expressing inactive FcαRI correlated with phosphorylated intracellular FcαRI Serine and cytokine-stimulated FcαRI capable of binding IgA-immune complexes showed reduced Serine 263 phosphorylation. Enhanced S263 phosphorylation by OA treatment implicated PP2A involvement.

FIGURE 3.

Phosphorylation of FcαRI-Serine 263 correlates with receptor activity. A–E, G, and I, Cell lines used: BaF3 FcαR-Wt (BaF3-FcαRI Wt), transfected with cDNA of full-length Wt FcαRI, FcαRI-S263A (FcαRI-S263A), and FcαRI-S263D or untransfected cells (untr). A, Immunoprecipitation via anti-FcαRI mAb of indicated BaF3 transfected cell lines, stained with anti-FcαRI pSerine 263 Ab (α-FcαRI-pS263, upper panel or with anti-FcαRI Serine 263 Ab (α-FcαRI-S263, middle panel), L chain served as loading control (lower panel). B, FcαRI expression levels of transfected BaF3 cells. C and D, Indicated BaF3-transfected cells, cytokine starved overnight, stained with anti-FcαRI-pS263 Ab (C) or nonstarved cells stained with anti-FcαRI-S263 Ab (D). E, BaF3 cells were cultured in low sera without (starv) or with mIL-3 (IL-3) and stained with α-FcαRI-pS263 (p = 0.0135). F, Eosinophils were stimulated with IL-5 for 15 min and stained with α-FcαRI-pS263 (p = 0.0249). G, S263 phosphorylation of BaF3-FcαRI Wt cells upon OA treatment determined with α-FcαRI-pS263 Ab in the presence of IL-3 (p = 0.0245). H, Surface expression of FcαRI with or without treatment with OA (10−6M). I, Nonstimulated or stimulated BaF3 were stained with the α-FcαRI-S263 Ab (p = 0.0011). All experiments were n ≥ 2. Each FACS staining was performed in triplicate. p values were determined using a nonpaired, two-tailed Students t test, p values <0.05 are considered significant.

FIGURE 3.

Phosphorylation of FcαRI-Serine 263 correlates with receptor activity. A–E, G, and I, Cell lines used: BaF3 FcαR-Wt (BaF3-FcαRI Wt), transfected with cDNA of full-length Wt FcαRI, FcαRI-S263A (FcαRI-S263A), and FcαRI-S263D or untransfected cells (untr). A, Immunoprecipitation via anti-FcαRI mAb of indicated BaF3 transfected cell lines, stained with anti-FcαRI pSerine 263 Ab (α-FcαRI-pS263, upper panel or with anti-FcαRI Serine 263 Ab (α-FcαRI-S263, middle panel), L chain served as loading control (lower panel). B, FcαRI expression levels of transfected BaF3 cells. C and D, Indicated BaF3-transfected cells, cytokine starved overnight, stained with anti-FcαRI-pS263 Ab (C) or nonstarved cells stained with anti-FcαRI-S263 Ab (D). E, BaF3 cells were cultured in low sera without (starv) or with mIL-3 (IL-3) and stained with α-FcαRI-pS263 (p = 0.0135). F, Eosinophils were stimulated with IL-5 for 15 min and stained with α-FcαRI-pS263 (p = 0.0249). G, S263 phosphorylation of BaF3-FcαRI Wt cells upon OA treatment determined with α-FcαRI-pS263 Ab in the presence of IL-3 (p = 0.0245). H, Surface expression of FcαRI with or without treatment with OA (10−6M). I, Nonstimulated or stimulated BaF3 were stained with the α-FcαRI-S263 Ab (p = 0.0011). All experiments were n ≥ 2. Each FACS staining was performed in triplicate. p values were determined using a nonpaired, two-tailed Students t test, p values <0.05 are considered significant.

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The FcαRI mutants and our FcαRI-pS263 Ab data suggested that FcαRI dephosphorylation was linked to an active receptor, i.e., capable of binding IgA-complexes. Next, we functionally assessed the role of PP2A on cytokine-induced FcαRI ligand binding capacity. First, upon IL-3 stimulation of BaF3-FcαRI-Wt, FcαRI ligand binding capacity was increased (Fig. 4,A) as described previously (16). Subsequently, we monitored PP2A activity during this process and showed increased PP2A phosphatase activity upon IL-3 stimulation in the BaF3 cells (Fig. 4,B), suggesting a cytokine-mediated regulatory mechanism for FcαRI dephosphorylation by PP2A. Of note, OA treatment of the cells served as a positive control for inhibition of PP2A activity. Furthermore,we determined the effect of PP2A on FcαRI ligand binding capacity and found reduced FcαRI ligand binding when cells were treated with PP2A inhibitors (OA, Cat, and Fos) but not with cells treated with a PP2B inhibitor (Acs) (Fig. 4, C and D). To test whether PP2A exclusively acted on inside-out signaling, we analyzed receptor internalization, as documented to also be an FcR γ-chain-independent process (26). For this, we cross-linked the FcαR using Abs binding outside the FcαRI ligand BD. Inhibition of PP2A by OA did not affect receptor internalization in BaF3-FcαRI-transfected cells (Fig. 4 E). These data implicate a role for PP2A in the regulation of ligand binding rather than receptor internalization.

FIGURE 4.

PP2A inhibition reduces FcαRI ligand binding but not receptor internalization. A, IgA-coated plates served as ligand for FcαRI-transfected BaF3 cells in ligand binding assays, described under Materials and Methods. Ligand binding capacity (y-axis) was plotted as retained fluorescence as a percentage of input. FcαRI-transfected cells ligand binding capacity was determined upon starvation from IL-3 (−), and subsequent incubation with recombinant lL-3 (+IL-3). B, PP2A activity was determined of BaF3 cells expressing FcαRI-Wt, stimulated (IL-3) or nonstimulated (−) and, if indicated, upon OA treatment using a PP2A-specific immunoprecipitation phosphatase assay. ISO represents immunprecipitation via an isotype controle Ab, which served as a negative control (n = 3). PP2A activity plotted as fold induction compared with nonstimulated cells. Bars indicate mean ± SEM. C, Ligand binding capacity of FcαRI-transfected cells was assessed as described for A, after incubation with different concentrations of the PP2A inhibitor OA (10−9, 10−8, 10−7, 10−6 M) in the presence of IL-3. D, Upon incubation with PP2A inhibitors OA (10−6 M), Cat (10−6 M), Fos (10−6 M), and the PP2B inhibitor Acs (10−7 M) (gray) compared with nontreated cells (black bar). Graph represents % ligand binding capacity compared with nontreated cells (set at 100%) (n = 3). Bars indicate mean ± SD. E, FcαRI internalization on BaF3-FcαRI-transfected cells was assessed upon FcαRI cross-linking. FcαRI internalization at 37°C (white bar) was calculated as percentage of total FcαRI expression measured at 4°C (set at 100%). The effect of OA treatment on internalization is depicted as the black bar. Bars indicate mean ± SEM. (n = 5). F, Ligand binding capacity of BaF3-FcαRI, transfected with PP2A C-subunit Wt (black bar), C-subunit H59Q mutant (dark gray bar), or C-subunit H118Q mutant (light gray bar). Bars indicate mean ± SD (n = 3).

FIGURE 4.

PP2A inhibition reduces FcαRI ligand binding but not receptor internalization. A, IgA-coated plates served as ligand for FcαRI-transfected BaF3 cells in ligand binding assays, described under Materials and Methods. Ligand binding capacity (y-axis) was plotted as retained fluorescence as a percentage of input. FcαRI-transfected cells ligand binding capacity was determined upon starvation from IL-3 (−), and subsequent incubation with recombinant lL-3 (+IL-3). B, PP2A activity was determined of BaF3 cells expressing FcαRI-Wt, stimulated (IL-3) or nonstimulated (−) and, if indicated, upon OA treatment using a PP2A-specific immunoprecipitation phosphatase assay. ISO represents immunprecipitation via an isotype controle Ab, which served as a negative control (n = 3). PP2A activity plotted as fold induction compared with nonstimulated cells. Bars indicate mean ± SEM. C, Ligand binding capacity of FcαRI-transfected cells was assessed as described for A, after incubation with different concentrations of the PP2A inhibitor OA (10−9, 10−8, 10−7, 10−6 M) in the presence of IL-3. D, Upon incubation with PP2A inhibitors OA (10−6 M), Cat (10−6 M), Fos (10−6 M), and the PP2B inhibitor Acs (10−7 M) (gray) compared with nontreated cells (black bar). Graph represents % ligand binding capacity compared with nontreated cells (set at 100%) (n = 3). Bars indicate mean ± SD. E, FcαRI internalization on BaF3-FcαRI-transfected cells was assessed upon FcαRI cross-linking. FcαRI internalization at 37°C (white bar) was calculated as percentage of total FcαRI expression measured at 4°C (set at 100%). The effect of OA treatment on internalization is depicted as the black bar. Bars indicate mean ± SEM. (n = 5). F, Ligand binding capacity of BaF3-FcαRI, transfected with PP2A C-subunit Wt (black bar), C-subunit H59Q mutant (dark gray bar), or C-subunit H118Q mutant (light gray bar). Bars indicate mean ± SD (n = 3).

Close modal

To further assess PP2A specificity in FcαRI ligand binding capacity, we determined the ligand binding capacity of FcαRI-transfected cells overexpressing HA-tagged PP2A catalytic inactive mutants, CH59Q and CH118Q (17). Both catalytic inactive PP2A C-subunit mutants inhibited binding of IgA complexes to FcαRI compared with overexpression of PP2A C-subunitWt (Fig. 4 F). Successful expression of the different PP2A constructs was determined by intracellular FACS staining (data not shown).

To study the effect of PP2A inhibition in primary cells, we isolated eosinophils, known for their inside-out regulation of FcαRI upon IL-5 stimulation, but also monocytes from human blood and incubated cells with OA prior to the stimulation with IL-5, or GM-CSF, respectively. As shown in Fig. 5, A and B, cytokine-induced FcαRI ligand binding was induced by cytokine stimulation on both cell types and interestingly this could be decreased by OA treatment in both cell types, supporting a role for PP2A. OA or GM-CSF treatment did not affect FcαRI expression levels (data not shown, n = 3). IgG binding capacity of monocytes was not affected in the presence of OA in agreement with Edberg et al. (27) (Fig. 5 E and data not shown).

FIGURE 5.

PP2A inhibition induces decreased FcαRI ligand binding and affects IgA-mediated phagocytosis of N. meningitidis. Eosinophils (A) and monocytes (B) were isolated from human blood and ligand binding was assessed by the use of Ig-coated beads as described in Materials and Methods. More than two beads binding to a cell were scored positive. A, IgA ligand binding. White bar represents unstimulated eosinophils, and black bar represents eosinophils stimulated with IL-5. Eosinophils were incubated with various concentrations of OA (gray bars) before IL-5 stimulation. The experiment was repeated three times yielding similar results. B, As described for A but with monocytes and GM-CSF as stimulus. Experiment was repeated thrice yielding essential identical results. C–F, GM-CSF-stimulated human monocytes were incubated with 10−6 M OA (gray bars) before incubation with FITC-labeled N. meningitidis (μg/ml) sensitized with different concentrations of IgA1 or IgG1 (black bars). Monocytes were split into two aliquots and incubated either at 4 (C and E) or 37°C (D and F). Upon incubation at 37°C, surface bound bacteria were detected. Phagocytosis was calculated as decrease of double-positive monocytes (in % of total) incubated at 37°C compared with 4°C (n = 4).

FIGURE 5.

PP2A inhibition induces decreased FcαRI ligand binding and affects IgA-mediated phagocytosis of N. meningitidis. Eosinophils (A) and monocytes (B) were isolated from human blood and ligand binding was assessed by the use of Ig-coated beads as described in Materials and Methods. More than two beads binding to a cell were scored positive. A, IgA ligand binding. White bar represents unstimulated eosinophils, and black bar represents eosinophils stimulated with IL-5. Eosinophils were incubated with various concentrations of OA (gray bars) before IL-5 stimulation. The experiment was repeated three times yielding similar results. B, As described for A but with monocytes and GM-CSF as stimulus. Experiment was repeated thrice yielding essential identical results. C–F, GM-CSF-stimulated human monocytes were incubated with 10−6 M OA (gray bars) before incubation with FITC-labeled N. meningitidis (μg/ml) sensitized with different concentrations of IgA1 or IgG1 (black bars). Monocytes were split into two aliquots and incubated either at 4 (C and E) or 37°C (D and F). Upon incubation at 37°C, surface bound bacteria were detected. Phagocytosis was calculated as decrease of double-positive monocytes (in % of total) incubated at 37°C compared with 4°C (n = 4).

Close modal

Inside-out signaling represents a mechanism of cells to rapidly respond in a controlled manner to environmental bacterial infection. To study the importance of PP2A in inside-out regulation of FcαRI in the innate immune response, we assessed phagocytosis of IgA1-opsonized N. meningitidis by GM-CSF-stimulated human monocytes (23) in the presence of OA. Monocytes incubated with OA (gray bars) were less capable of IgA1 binding (Fig. 5,C) and IgA1-mediated phagocytosis of N. meningitidis (Fig. 5,D) compared with nontreated monocytes (black bars). IgG1-mediated binding (Fig. 5,E) and phagocytosis (Fig. 5 F) were barely changed upon OA treatment. PP2A can thus have an impact on immunity to bacterial infections by regulation of FcαRI ligand binding.

Signal transduction by multi-subunit FcR is considered to be dominated by ITAM-containing subunits (28). The first step in FcR activation, however, is the capacity of FcR α-chain to bind ligand mediated by inside-out signals (13). In this study, we document that cytokine-induced inside-out activation of FcαRI is accompanied by dephosphorylation of the FcαRI intracellular Serine 263 with two novel generated (phosphorylation- and nonphosphorylation-specific) FcαRI-Serine 263 Abs in IL-3-dependent mBaF3-FcαRI cells and primary human eosinophils. In addition, we found the Serine/Threonine PP2A to specifically interact with the FcαRI α-chain intracellular domain, and to play a critical role in regulating FcαRI ligand binding and FcαRI-mediated immunity. Of note, in yeast, PP2A also binds to the intracellular tail of FcγRIIa receptor. Interestingly, on eosinophils, this receptor is also known for its cytokine-induced inside-out activation (11).

On a molecular level, we found that PP2A can dephosphorylate the FcαRI intracellular tail, as shown in an in vitro phosphatase assay, whereas an other ubiquitously expressed family member (PP1) could not. Finally, we document increased phosphatase activity of PP2A upon IL-3 stimulation.

Functionally, cytokine-induced inside-out activation of FcαRI has been described for human eosinophils (11) and hinted at for human neutrophils (29). In this study, we report that inhibition of PP2A activity resulted in decreased capability of FcαRI to bind IgA complexes in IL-3-stimulated BaF3-FcαRI cells, in IL-5-stimulated eosinophils, and in GM-CSF-stimulated monocytes. FcαRI internalization in the presence of OA was unaffected when crosslinked outside the ligand BD. This suggested a dominant role of PP2A activity in ligand binding rather than other FcR γ-chain-independent processes. Notably, PP2A inhibition on monocytes resulted in even less binding of IgA-immune complexes compared with in vitro unstimulated monocytes. These findings may indicate isolated monocytes to be already activated, an observation supported by other studies (30, 31, 32, 33, 34), and may also explain the rather low additional induction of ligand binding after GM-CSF treatment.

The importance of the role of PP2A in innate immunity was shown in an in vitro phagocytosis assay. Treatment of monocytes with OA abrogated phagocytosis of IgA1 opsonized N. meningitidis as a direct consequence of decreased binding of the IgA1-opsonized bacteria. Importantly, these results appeared specific for FcαRI-mediated ligand binding by monocytes, since phagocytosis of IgG1-opsonized bacteria was not hampered by OA treatment. Collectively, this suggests PP2A to be a novel mediator in cytokine-induced FcαRI inside-out signaling.

Little is known regarding the intracellular signals that mediate the communication between activated cytokine receptors and inside-out control of FcαRI. Though, PI3K was demonstrated to be responsible for a switch to a “high functionality” state of FcαRI, and PKC was shown to be a part of this activation pathway downstream of PI3K (24). In contrast to FcRs, inside-out signaling is a well-known regulatory process for integrins (14). Although limited sequence homology exists between integrins and FcαRI inside-out regulation, there are important similarities: 1) the importance of the intracellular domain for modulation of receptor affinity, 2) mutation of a single Serine within the C-terminal domain disrupts inside-out signaling, 3) correlation between phosphorylation of the intracellular domain and receptor activity, 4) the involvement of intracellular proteins such as PI3K, and 5) the involvement of the cytoskeleton (24, 35, 36, 37, 38, 39); finally, involvement of PP2A has been described for integrins (40, 41). In this study, we document a similar role of PP2A in FcαRI (de)phosphorylation and receptor functioning.

PP2A has traditionally been described as an enzyme that is constitutively active and terminates signals by removal of phosphate groups from phosphorylated proteins. However, PP2A can be turned “on” and “off” by subunit phosphorylation and carboxyl methylation in addition to binding of the various regulatory subunits as described for Src-related kinase p56Lck, insulin receptor, and the EGF receptor (42, 43, 44). Furthermore, IL-3 modulation of PP2A activity has been described for Jak2 (45), anti-apoptotic protein Bcl-2 (46), and Raf1 kinase (47). Since PP2A does not bind the FcαRI-S263A mutant in yeast, representing the active receptor, and PP2A activity increased upon IL-3 stimulation, we suggest PP2A to become more active upon cytokine stimulation, resulting in FcαRI dephosphorylation and dissociation of PP2A from the receptor. In this way, PP2A may regulate cytokine-induced FcαRI activation (Fig. 6).

FIGURE 6.

Schematic representation of the role of PP2A in FcαRI inside-out signaling. The intracellular domain of inactive FcαRI is phosphorylated (P) by kinases. PP2A associates with inactive FcαRI and, upon cytokine stimulation, PP2A becomes activated, dephosphorylates the intracellular tail, and dissociates leading to an active FcαR capable of binding IgA-immune complexes (but only one IgA is drawn for simplicity). IgA is presented by the typical T-shape, binding in a 1:2 stochiometry to FcαRI (based on crystallization studies by Herr et al. in Ref. 48 ). For PP2A, A represents the regulatory-, C the catalytic-, and B the variable-subunit of PP2A, IgA is represented and represents an immune complex.

FIGURE 6.

Schematic representation of the role of PP2A in FcαRI inside-out signaling. The intracellular domain of inactive FcαRI is phosphorylated (P) by kinases. PP2A associates with inactive FcαRI and, upon cytokine stimulation, PP2A becomes activated, dephosphorylates the intracellular tail, and dissociates leading to an active FcαR capable of binding IgA-immune complexes (but only one IgA is drawn for simplicity). IgA is presented by the typical T-shape, binding in a 1:2 stochiometry to FcαRI (based on crystallization studies by Herr et al. in Ref. 48 ). For PP2A, A represents the regulatory-, C the catalytic-, and B the variable-subunit of PP2A, IgA is represented and represents an immune complex.

Close modal

In summary, here we identify the activity of PP2A as a molecular switch for FcαRI regulation. PP2A is the first identified interacting protein to the intracellular tail of FcαRI and is critical for FcαRI-mediated pathogen phagocytosis.

We thank Prof. G. Adema (Department of Tumor Immunology, Nijmegen, The Netherlands) for providing the human DC cDNA library, Prof. D. C. Pallas (Department of Biochemistry and Winship Cancer Institute, Emory University School of Medicine, GA) for providing the PP2A catalytic inactive mutants and Wt constructs, the volunteers of the Sanquin Bloedbank Midden Nederland, Ing. J. A. van der Linden, Ing. M. Blokland for technical support, Dr. J. Beekman for help with Fig. 1B, and Prof. P. C. Coffer for critically reading this manuscript and helpful discussions (all: Department of Immunology, The Netherlands).

The authors have no financial conflict of interest.

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.

1

This work was supported by the Dutch Cancer Foundation (KWF/NKB) Grant UU 2002 2706 and by the Association for International Cancer Research Grant 03-119.

3

Abbreviations used in this paper: Wt, wild type; m, murine; BD, binding domain; DC, dendritic cell; Cat, catharidin; Fos, fostriecin; Acs, ascomycin; p, phosphorylated.

1
Mestecky, J., M. W. Russell, C. O. Elson.
1999
. Intestinal IgA: novel views on its function in the defense of the largest mucosal surface.
Gut
44
:
2
-5.
2
Woof, J. M., J. Mestecky.
2005
. Mucosal immunoglobulins.
Immunol. Rev.
206
:
64
-82.
3
van Egmond, M., C. A. Damen, A. B. van Spriel, G. Vidarsson, E. van Garderen, J. G. van de Winkel.
2001
. IgA and the IgA Fc receptor.
Trends Immunol.
22
:
205
-211.
4
Monteiro, R. C., J. G. van de Winkel.
2003
. IgA Fc receptors.
Annu. Rev. Immunol.
21
:
177
-204.
5
Chevailler, A., R. C. Monteiro, H. Kubagawa, M. D. Cooper.
1989
. Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue.
J. Immunol.
142
:
2244
-2249.
6
Vidarsson, G., W. L. van Der Pol, J. M. van Den Elsen, H. Vile, M. Jansen, J. Duijs, H. C. Morton, E. Boel, M. R. Daha, B. Corthesy, J. G. van De Winkel.
2001
. Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B.
J. Immunol.
166
:
6250
-6256.
7
Dechant, M., G. Vidarsson, B. Stockmeyer, R. Repp, M. J. Glennie, M. Gramatzki, J. G. van De Winkel, T. Valerius.
2002
. Chimeric IgA antibodies against HLA class II effectively trigger lymphoma cell killing.
Blood
100
:
4574
-4580.
8
Zhang, W., J. Voice, P. J. Lachmann.
1995
. A systematic study of neutrophil degranulation and respiratory burst in vitro by defined immune complexes.
Clin. Exp. Immunol.
101
:
507
-514.
9
Otten, M. A., E. Rudolph, M. Dechant, C. W. Tuk, R. M. Reijmers, R. H. Beelen, J. G. van de Winkel, M. van Egmond.
2005
. Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors.
J. Immunol.
174
:
5472
-5480.
10
Bracke, M., E. van de Graaf, J. W. Lammers, P. J. Coffer, L. Koenderman.
2000
. In vivo priming of FcαR functioning on eosinophils of allergic asthmatics.
J. Leukocyte Biol.
68
:
655
-661.
11
Koenderman, L., S. W. Hermans, P. J. Capel, J. G. van de Winkel.
1993
. Granulocyte-macrophage colony-stimulating factor induces sequential activation and deactivation of binding via a low-affinity IgG Fc receptor, hFc γ RII, on human eosinophils.
Blood
81
:
2413
-2419.
12
Beekman, J. M., J. E. Bakema, J. G. van de Winkel, J. H. Leusen.
2004
. Direct interaction between FcγRI (CD64) and periplakin controls receptor endocytosis and ligand binding capacity.
Proc. Natl. Acad. Sci. USA
101
:
10392
-10397.
13
Bracke, M., G. R. Dubois, K. Bolt, P. L. Bruijnzeel, J. P. Vaerman, J. W. Lammers, L. Koenderman.
1997
. Differential effects of the T helper cell type 2-derived cytokines IL-4 and IL-5 on ligand binding to IgG and IgA receptors expressed by human eosinophils.
J. Immunol.
159
:
1459
-1465.
14
Williams, M. J., P. E. Hughes, T. E. O'Toole, M. H. Ginsberg.
1994
. The inner world of cell adhesion: integrin cytoplasmic domains.
Trends Cell Biol.
4
:
109
-112.
15
Van Zandbergen, G., R. Westerhuis, N. K. Mohamad, J. G. van De Winkel, M. R. Daha, C. van Kooten.
1999
. Crosslinking of the human Fc receptor for IgA (FcαRI/CD89) triggers FcR γ-chain-dependent shedding of soluble CD89.
J. Immunol.
163
:
5806
-5812.
16
Bracke, M., J. W. Lammers, P. J. Coffer, L. Koenderman.
2001
. Cytokine-induced inside-out activation of FcαR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor.
Blood
97
:
3478
-3483.
17
Ogris, E., X. Du, K. C. Nelson, E. K. Mak, X. X. Yu, W. S. Lane, D. C. Pallas.
1999
. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.
J. Biol. Chem.
274
:
14382
-14391.
18
Triantis, V., D. E. Trancikova, M. W. Looman, F. C. Hartgers, R. A. Janssen, G. J. Adema.
2006
. Identification and characterization of DC-SCRIPT, a novel dendritic cell-expressed member of the zinc finger family of transcriptional regulators.
J. Immunol.
176
:
1081
-1089.
19
Bakema, J. E., S. de Haij, C. F. den Hartog-Jager, J. Bakker, G. Vidarsson, M. van Egmond, J. G. van de Winkel, J. H. Leusen.
2006
. Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor γ-chain interaction.
J. Immunol.
176
:
3603
-3610.
20
Walsh, A. H., A. Cheng, R. E. Honkanen.
1997
. Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A.
FEBS Lett.
416
:
230
-234.
21
Honkanen, R. E..
1993
. Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A.
FEBS Lett.
330
:
283
-286.
22
Zhan, Q., Q. Ge, T. Ohira, T. Van Dyke, J. A. Badwey.
2003
. p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases.
J. Immunol.
171
:
3785
-3793.
23
Rodriguez, M. E., W. L. Van der Pol, J. G. Van de Winkel.
2001
. Flow cytometry-based phagocytosis assay for sensitive detection of opsonic activity of pneumococcal capsular polysaccharide antibodies in human sera.
J. Immunol. Methods
252
:
33
-44.
24
Bracke, M., E. Nijhuis, J. W. Lammers, P. J. Coffer, L. Koenderman.
2000
. A critical role for PI 3-kinase in cytokine-induced Fcα-receptor activation.
Blood
95
:
2037
-2043.
25
Schonthal, A. H..
1998
. Role of PP2A in intracellular signal transduction pathways.
Front Biosci.
3
:
D1262
-D1273.
26
Launay, P., C. Patry, A. Lehuen, B. Pasquier, U. Blank, R. C. Monteiro.
1999
. Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRγ association and protects against degradation of bound ligand.
J. Biol. Chem.
274
:
7216
-7225.
27
Edberg, J. C., H. Qin, A. W. Gibson, A. M. Yee, P. B. Redecha, Z. K. Indik, A. D. Schreiber, R. P. Kimberly.
2002
. The CY domain of the Fcγ RIa α-chain (CD64) alters γ-chain tyrosine-based signaling and phagocytosis.
J. Biol. Chem.
277
:
41287
-41293.
28
Daeron, M..
1997
. Fc receptor biology.
Annu. Rev. Immunol.
15
:
203
-234.
29
Weisbart, R. H., A. Kacena, A. Schuh, D. W. Golde.
1988
. GM-CSF induces human neutrophil IgA-mediated phagocytosis by an IgA Fc receptor activation mechanism.
Nature
332
:
647
-648.
30
Repo, H., P. Vuopio, M. Leirisalo, S. E. Jansson, T. U. Kosunen.
1979
. Impaired neutrophil chemotaxis in Pelger-Huet anomaly.
Clin. Exp. Immunol.
36
:
326
-333.
31
Treves, A. J., D. Yagoda, A. Haimovitz, N. Ramu, D. Rachmilewitz, Z. Fuks.
1980
. The isolation and purification of human peripheral blood monocytes in cell suspension.
J. Immunol. Methods
39
:
71
-80.
32
Stibenz, D., C. Buhrer.
1994
. Down-regulation of L-selectin surface expression by various leukocyte isolation procedures.
Scand. J. Immunol.
39
:
59
-63.
33
Lundahl, J., G. Hallden, M. Hallgren, C. M. Skold, J. Hed.
1995
. Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures.
J. Immunol. Methods
180
:
93
-100.
34
Macey, M. G., D. A. McCarthy, S. Vordermeier, A. C. Newland, K. A. Brown.
1995
. Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.
J. Immunol. Methods
181
:
211
-219.
35
Perez, O. D., D. Mitchell, G. C. Jager, S. South, C. Murriel, J. McBride, L. A. Herzenberg, S. Kinoshita, G. P. Nolan.
2003
. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1.
Nat. Immunol.
4
:
1083
-1092.
36
Otey, C. A., K. Burridge.
1990
. Patterning of the membrane cytoskeleton by the extracellular matrix.
Semin. Cell Biol.
1
:
391
-399.
37
Dedhar, S., G. E. Hannigan.
1996
. Integrin cytoplasmic interactions and bidirectional transmembrane signaling.
Curr. Opin. Cell Biol.
8
:
657
-669.
38
Shimizu, Y., S. W. Hunt, III.
1996
. Regulating integrin-mediated adhesion: one more function for PI 3-kinase?.
Immunol. Today
17
:
565
-573.
39
Jones, S. L., J. Wang, C. W. Turck, E. J. Brown.
1998
. A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function.
Proc. Natl. Acad. Sci. USA
95
:
9331
-9336.
40
Brockdorff, J., M. Nielsen, A. Svejgaard, P. Dobson, C. Ropke, C. Geisler, N. Odum.
1997
. Protein phosphatase 2A plays a critical role in interleukin-2-induced β 2-integrin dependent homotypic adhesion in human CD4+ T cell lines.
Cytokine
9
:
333
-339.
41
Takahashi, K., K. Suzuki.
2006
. Regulation of protein phosphatase 2A-mediated recruitment of IQGAP1 to β1 integrin by EGF through activation of Ca2+/calmodulin-dependent protein kinase II.
J. Cell Physiol.
208
:
213
-219.
42
Chen, J., B. L. Martin, D. L. Brautigan.
1992
. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation.
Science
257
:
1261
-1264.
43
Chen, J., S. Parsons, D. L. Brautigan.
1994
. Tyrosine phosphorylation of protein phosphatase 2A in response to growth stimulation and v-src transformation of fibroblasts.
J. Biol. Chem.
269
:
7957
-7962.
44
Sontag, E..
2001
. Protein phosphatase 2A: the Trojan Horse of cellular signaling.
Cell Signal.
13
:
7
-16.
45
Yokoyama, N., N. C. Reich, W. T. Miller.
2001
. Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.
J. Interferon Cytokine Res.
21
:
369
-378.
46
Deng, X., T. Ito, B. Carr, M. Mumby, W. S. May, Jr.
1998
. Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A.
J. Biol. Chem.
273
:
34157
-34163.
47
Adams, D. G., R. L. Coffee, Jr, H. Zhang, S. Pelech, S. Strack, B. E. Wadzinski.
2005
. Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.
J. Biol. Chem.
280
:
42644
-42654.
48
Herr, A. B., E. R. Ballister, P. J. Bjorkman.
2003
. Insights into IgA-mediated immune responses from the crystal structures of human FcαRI and its complex with IgA1-Fc.
Nature
423
:
614
-620.