The CY Domain of the FcγRIa α-Chain (CD64) Alters γ-Chain Tyrosine-based Signaling and Phagocytosis
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m207835200
ISSN1083-351X
AutoresJeffrey C. Edberg, Hongwei Qin, Andrew Gibson, Arthur M.F. Yee, Patricia Redecha, Zena K. Indik, Alan D. Schreiber, Robert P. Kimberly,
Tópico(s)T-cell and B-cell Immunology
ResumoAlthough the cytoplasmic domain of the human FcγRIa α-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcγRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcγRIa, we developed a series of human FcγRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the α-chain of FcγRIa and suggest that the cytoplasmic domain of FcγRI regulates the different functional capacities of the FcγRIa-receptor complex. Although the cytoplasmic domain of the human FcγRIa α-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcγRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcγRIa, we developed a series of human FcγRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the α-chain of FcγRIa and suggest that the cytoplasmic domain of FcγRI regulates the different functional capacities of the FcγRIa-receptor complex. The γ-chain, initially described as a component of the FcεRI signaling complex, is able to form multichain complexes with the ligand-binding α-chain of several Fc receptors (1Kinet J.P. Annu. Rev. Immunol. 1999; 17: 931-972Google Scholar, 2Ravetch J.V. Curr. Opin. Immunol. 1997; 9: 121-125Google Scholar, 3McKenzie S.E. Schreiber A.D. Curr. Opin. Hematol. 1998; 5: 16-21Google Scholar). The FcγRIa (CD64), FcγRIIIa (CD16A), FcαRI (CD89), and FcεRI α-chains associate with the γ-chain as a common molecule in signal transduction. The stoichiometry of the assembly of the receptor complex is generally αγ2, except in mast cells, which may have FcεRI and FcγRIIIa complexes where there is, in addition, a β chain (αβγ2) (4Kurosaki T. Gander I. Wirthmueller U. Ravetch J.V. J. Exp. Med. 1992; 175: 447-451Google Scholar, 5Dombrowicz D. Flamand V. Miyajima I. Ravetch J.V. Galli S.J. Kinet J.P. J. Clin. Invest. 1997; 99: 915-925Google Scholar, 6Hiraoka S. Furumoto Y. Koseki H. Takagaki Y. Taniguchi M. Okumura K. Ra C. Int. Immunol. 1999; 11: 199-207Google Scholar). In all of these Fc receptor complexes, the γ-chain with its tyrosine activation motif (ITAM) is necessary for receptor signaling (7Lowry M.B. Duchemin A.M. Robinson J.M. Anderson C.L. J. Exp. Med. 1998; 187: 161-176Google Scholar, 8Indik Z.K. Hunter S. Huang M.M. Pan X.Q. Chien P. Kelly C. Levinson A.I. Kimberly R.P. Schreiber A.D. Exp. Hematol. 1994; 22: 599-606Google Scholar, 9Davis W. Harrison P.T. Hutchinson M.J. Allen J.M. EMBO J. 1995; 14: 432-441Google Scholar). In the case of FcεRI, the β-chain serves as an amplifier of receptor function (10Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Google Scholar, 11Donnadieu E. Jouvin M.H. Kinet J.P. Immunity. 2000; 12: 515-523Google Scholar). FcγRIa, a receptor with high affinity for IgG (109m−1) (12Miller K.L. Duchemin A.M. Anderson C.L. J. Exp. Med. 1996; 183: 2227-2233Google Scholar), has received attention over the past few years as a potential therapeutic target in malignancy. Targeting of tumors to FcγRI with bispecific mAbs 1The abbreviations used are: mAb, monoclonal antibody; Ab, antibody; OA, okadaic acid; huFcγRIa, human FcγRIa; IL, interleukin; WT, wild-type; GAM, goat anti-mouse IgG; S4→A4, S328A/S331A/ S339A/S340A. 1The abbreviations used are: mAb, monoclonal antibody; Ab, antibody; OA, okadaic acid; huFcγRIa, human FcγRIa; IL, interleukin; WT, wild-type; GAM, goat anti-mouse IgG; S4→A4, S328A/S331A/ S339A/S340A. can facilitate tumor killing via FcγRI-expressing macrophages, and therapeutic humanized bispecific reagents targeting human FcγRIa are currently in clinical trials (13Posey J.A. Raspet R. Verma U. Deo Y.M. Keller T. Marshall J.L. Hodgson J. Mazumder A. Hawkins M.J. J. Immunother. 1999; 22: 371-379Google Scholar, 14Repp R. Valerius T. Wieland G. Becker W. Steininger H. Deo Y. Helm G. Gramatzki M. Van de Winkel J.G. Lang N. et al.J. Hematother. 1995; 4: 415-421Google Scholar, 15Deo Y.M. Graziano R.F. Repp R. van de Winkel J.G. Immunol. Today. 1997; 18: 127-135Google Scholar, 16Graziano R.F. Goldstein J. Sundarapandiyan K. Somasundaram C. Keler T. Deo Y.M. Cancer Immunol. Immunother. 1997; 45: 124-127Google Scholar, 17Keler T. Graziano R.F. Mandal A. Wallace P.K. Fisher J. Guyre P.M. Fanger M.W. Deo Y.M. Cancer Res. 1997; 57: 4008-4014Google Scholar, 18Pullarkat V. Deo Y. Link J. Spears L. Marty V. Curnow R. Groshen S. Gee C. Weber J.S. Cancer Immunol. Immunother. 1999; 48: 9-21Google Scholar, 19Somasundaram C. Sundarapandiyan K. Keler T. Deo Y.M. Graziano R.F. Hum. Antib. 1999; 9: 47-54Google Scholar). Bispecific mAb-based antigen targeting to FcγRI can also enhance antigen presentation by dendritic cells with clear applications to enhanced immunization strategies (20Liu C. Goldstein J. Graziano R.F., He, J. O'Shea J.K. Deo Y. Guyre P.M. J. Clin. Invest. 1996; 98: 2001-2007Google Scholar). Expression of the FcγRIa α-chain in the presence or absence of the γ-chain has allowed an assessment of the functional capacity of each chain. For example, the γ-chain is necessary for FcγRIa-mediated phagocytosis and FcγRIa-induced activation of tyrosine kinase activity (7Lowry M.B. Duchemin A.M. Robinson J.M. Anderson C.L. J. Exp. Med. 1998; 187: 161-176Google Scholar, 8Indik Z.K. Hunter S. Huang M.M. Pan X.Q. Chien P. Kelly C. Levinson A.I. Kimberly R.P. Schreiber A.D. Exp. Hematol. 1994; 22: 599-606Google Scholar, 9Davis W. Harrison P.T. Hutchinson M.J. Allen J.M. EMBO J. 1995; 14: 432-441Google Scholar). However, the α-chain is sufficient for endocytosis (7Lowry M.B. Duchemin A.M. Robinson J.M. Anderson C.L. J. Exp. Med. 1998; 187: 161-176Google Scholar, 9Davis W. Harrison P.T. Hutchinson M.J. Allen J.M. EMBO J. 1995; 14: 432-441Google Scholar). Whereas expression of the FcγRIa α-chain without a CY domain can also induce pseudopod extension and endocytosis, recent data from our group and others have provided the first evidence that the α-chain of FcγRIa can alter receptor function downstream of IgG binding (21van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. van Dijk M.A. Leusen J.H. Geuze H.J. van de Winkel J.G. Blood. 1999; 94: 808-817Google Scholar, 22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 23Indik Z. Chien P. Levinson A.I. Schreiber A.D. Immunobiology. 1992; 185: 183-192Google Scholar). Expression of an FcγRIa α-chain lacking the CY domain in a murine macrophage cell line results in quantitative differences in the phagocytic and endocytic capacity of the αγ2 receptor complexes compared with wild-type FcγRI. Lack of the CY domain also changes the Ca2+ dependence of receptor-specific phagocytosis and abolishes the receptor-elicited IL-6 response. Expression of a similar receptor mutant in B cells results in alterations in the intracellular cycling of the internalized receptor (21van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. van Dijk M.A. Leusen J.H. Geuze H.J. van de Winkel J.G. Blood. 1999; 94: 808-817Google Scholar). These data suggest a role for the α-chain CY domain in the regulation of FcγRI function. We have begun to dissect the molecular basis for the α-chain involvement in FcγRI signaling and cell activation. We now show that the CY domain of FcγRIa is constitutively phosphorylated and that receptor engagement and cross-linking result in a time dependent dephosphorylation. Inhibition of the dephosphorylation with okadaic acid, an inhibitor of type 1 protein serine/threonine phosphatase and type 2A protein serine/threonine phosphatase, blocks wild-type receptor-mediated phagocytosis and reduces tyrosine phosphorylation of the γ-chain. In contrast, both truncation and mutation of CY serines to alanine abrogate the effect of OA on phagocytosis and γ-chain tyrosine phosphorylation. These data suggest that serine phosphorylation of the FcγRIa α-chain inhibits early receptor-initiated tyrosine phosphorylation events. Furthermore, the selective reduction in IL6 production with truncation, but not with serine to alanine mutation, indicates that cytokine production is likely influenced by other elements engaged by the FcγRIa α-chain. The murine macrophage cell line P388D1 (obtained from American Type Culture Collection, Manassas, VA) was stably transfected with a cDNA encoding human FcγRIa (WT) or a mutant form of FcγRIa containing a stop codon after the first amino acid of the cytoplasmic domain (Lys315→Stop 315) (CY−) as we have described previously (8Indik Z.K. Hunter S. Huang M.M. Pan X.Q. Chien P. Kelly C. Levinson A.I. Kimberly R.P. Schreiber A.D. Exp. Hematol. 1994; 22: 599-606Google Scholar, 22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar). FcγRIa constructs encoding serine to alanine mutations (S328A/S331A, S339A/S340A, and S328A/S331A/S339A/S340A (S4→A4)) were prepared by overlap PCR and stably transfected as described previously (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). In all cases, two independently prepared cell lines stably expressing each FcγRI construct were analyzed. P388D1 cells transfected with human FcγRIIa have been described previously (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar,24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). Cell lines were maintained as adherent cultures (Corning tissue culture dishes) in RPMI 1640 medium as described previously (24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). The human myelomonocytic cell line U937 (American Type Culture Collection) was maintained as a suspension culture in RPMI 1640 medium. All tissue culture reagents were from Invitrogen. For 32P studies, cells were cultured for 24 h in phosphate-free RPMI 1640 medium in the presence of 5 mCi of 32Pi. The protein tyrosine kinase inhibitor genistein was obtained from Invitrogen. The type 1/2A protein serine/threonine phosphatase inhibitor okadaic acid was obtained from Calbiochem. An okadaic acid analog, 1-Nor-okadaone, that does not possess protein phosphatase inhibitory activity (25Nishiwaki S. Fujiki H. Suganuma M. Furuya-Suguri H. Matsushima R. Iida Y. Ojika M. Yamada K. Uemura D. Yasumoto T. et al.Carcinogenesis. 1990; 11: 1837-1841Google Scholar) was obtained from Alexis Biochemicals (San Diego, CA). F(ab′)2 fragments of the anti-FcγRIa mAb 22.2 and Fab fragments of the anti-FcγRIIa mAb IV.3 were obtained from Medarex (Annandale, NJ). Mouse F(ab′)2 fragments and F(ab′)2 goat anti-mouse IgG (GAM) were obtained from Jackson ImmunoResearch (West Grove, PA). Mouse IgG was obtained from Sigma. All other reagents were from Sigma. Quantitative huFcγRI expression was matched for cells expressing the WT and the cytoplasmic domain deletion mutant by fluorescence-activated cell sorting using anti-FcγRI mAb 22.2-fluorescein isothiocyanate (Medarex). Polyclonal anti-γ-chain Abs prepared in rabbits immunized with a C-terminal peptide sequence that is shared by both human and murine γ-chain were used for immunoprecipitations and blotting as we have described previously (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar). A polyclonal rabbit antiserum raised against the C-terminal 11 amino acids of the cytoplasmic domain of FcγRIa was prepared. Anti-phosphotyrosine mAb 4G10 was obtained from Upstate Biotechnology (Lake Placid, NY). A rabbit polyclonal anti-phosphoserine Ab, soluble phosphoserine, and epidermal growth factor-stimulated A431 cell lysate (used as a positive control) were obtained from Zymed Laboratories Inc. FcγRI was immunoprecipitated from the transfected cell lines or U937 cells using either mAb 22.2 or mAb 197 (kindly provided by Dr. Paul Guyre, Dartmouth University Medical School) (26Guyre P.M. Graziano R.F. Vance B.A. Morganelli P.M. Fanger M.W. J. Immunol. 1989; 143: 1650-1655Google Scholar) prebound to protein G-agarose (Pharmacia Corp.). γ-Chain from transfected cells was immunoprecipitated by polyclonal rabbit anti-γ-chain Abs bound to protein G-agarose. Cells (10–20 × 106 cells/ml) were lysed in phosphate-buffered saline containing 1% Nonidet P-40 (Sigma) and protease inhibitors (EDTA/pepstatin/aprotinin/sodium orthovanadate/pefabloc). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. For immunoblotting analysis, protein immunoprecipitates were separated by SDS-PAGE and blotted onto nitrocellulose membranes (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 27Edberg J.C. Kimberly R.P. J. Immunol. 1994; 152: 5826-5835Google Scholar). Membranes were blocked with 10% nonfat milk or 3% bovine serum albumin followed by incubation with the blotting Ab/mAb. Blots were washed three times with phosphate-buffered saline-0.1% Tween 20 and probed with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Biosciences or Jackson ImmunoResearch). After three more washes, bound horseradish peroxidase-conjugated Ab was detected using ECL (Amersham Biosciences) according to the manufacturer's directions. Membranes were stripped by incubation with Tris-HCl, pH 2.3, for 30 min at room temperature and then reprobed as described above. Phagocytosis by transfected P388D1 cells was determined in an adherent assay system (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). Biotinylated mAb 22.2 F(ab′)2, mAb IV.3 Fab, and biotinylated bovine erythrocytes were prepared as described previously (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). Biotinylated bovine erythrocytes were saturated with streptavidin and washed. The resulting bovine erythrocytes were coated with biotinylated mAb, and the level of mAb binding was verified by flow cytometry. P388D1 cells, adhered to round glass coverslips at 5 × 105 cells/ml, were incubated with anti-FcγRIa mAb 22.2 F(ab′)2-coated bovine erythrocytes (E-22.2) or anti-FcγRIIa mAb IV.3 Fab-coated bovine erythrocytes (E-IV.3) in RPMI 1640 medium/20% fetal calf serum (50 μl at 5 × 107bovine erythrocytes/ml) for 1 h at 37 °C. Noninternalized bovine erythrocytes were lysed by brief immersion of the coverslip in distilled H2O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as the phagocytic index (number of bovine erythrocytes internalized per 100 P388D1 cells). Treatment of cells with genistein (100 nm) to block protein tyrosine kinase or with okadaic acid (1 μm) to block protein type 1 and 2A protein serine/threonine phosphatase activity was performed by preincubating the coverslip-adherent cells for 30 min or 10 min, respectively, at 37 °C followed by addition of E-22.2 or E-IV.3 in RPMI 1640 medium/20% fetal calf serum as described above. Controls included loading cells with 0.1–1% Me2SO (depending on the concentration of inhibitor) for the same period of time. Cells were stimulated in 96-well tissue culture plates (Corning) with either phorbol 12-myristate 13-acetate, surface-absorbed rabbit IgG, or surface-adsorbed F(ab′)2GAM + mAb 22.2 F(ab′)2. Wells were coated with adsorbed protein (20 μg/ml rabbit IgG or F(ab′)2 GAM) for 2 h at 37 °C. For anti-FcγRI stimulation, mAb 22.2 F(ab′)2at 20 μg/ml was added to a suspension of cells for 30 min at 4 °C followed by two washes to remove unbound mAb. Cells (1–2.5 × 105 cells/ml) were added to the wells and cultured for varying periods of time. The level of murine IL-1β or IL-6 in diluted culture supernatants was quantitated by enzyme-linked immunosorbent assay. For IL-1β determination, recombinant standard, capture Ab (polyclonal rabbit Ab) and biotinylated detection and neutralization mAb (clone 1400.24.17) were obtained from Endogen (Woburn, MA). For IL-6 determination, recombinant standard, capture mAb (clone MP5-2-F3) and biotinylated detection mAb (clone MP5-32C11) were obtained from Pharmingen. Horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch) and then 3,3′,5,5′-tetramethylbenzidine substrate were added, and the A 450 was determined. Aliquots of cells at 5 × 106 cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4 °C followed by two washes. For indirect immunofluorescence, the cells were then incubated with saturating concentrations of fluorescein isothiocyanate-conjugated goat anti-mouse IgG F(ab′)2 at 4 °C for another 30 min. After washing, the cells were analyzed immediately for immunofluorescence using a FACScan (BD Biosciences). Analysis of flow cytometry listmode data was performed using CellQuest (BD Biosciences). Statistical comparisons were performed with the paired t test. A probability of 0.05 was used to reject the null hypothesis that there is no difference between the samples. The cytoplasmic domain of the ligand binding α-chain of the human FcγRIa receptor complex (αγ2) exerts a quantitative influence on receptor-specific phagocytosis (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar). The quantitative phagocytic capacity of a cytoplasmic domain-lacking mutant of huFcγRIa is lower than that of wild-type huFcγRIa expressed in the murine macrophage cell line P388D1. Likewise, the kinetics of phagocytosis are slower in the tail minus mutant form of huFcγRIa compared with WT huFcγRIa in these cells. To explore possible mechanisms for this effect, we quantitated huFcγRIa-specific phagocytosis in these cell lines in the presence of several kinase and phosphatase inhibitors. Previous work has demonstrated that FcγR-mediated phagocytosis is dependent on tyrosine kinase activation. For FcγRIa, phagocytosis is dependent on tyrosine phosphorylation of the γ-chain. In P388D1 cell lines stably expressing either WT huFcγRIa or the cytoplasmic tail minus mutant form of huFcγRIa (CY−), phagocytosis was completely inhibited by pretreatment of the cells with the tyrosine kinase inhibitor genistein (Fig. 1 A). Cell viability was unaffected by genistein during the time course of these studies, establishing a clear role for tyrosine phosphorylation in FcγRI phagocytosis. Receptor-specific phagocytosis was reestablished to normal levels in the WT cells during the 60-min time course of the phagocytosis assay if genistein was removed after the initial incubation period (Fig. 1 A). Interestingly, only 70% of the phagocytic capacity of the tail minus mutant was restored during the same time period. This observation was highly reproducible (p < 0.017, n = 9 pairs). Although the CY domain of huFcγRIa lacks tyrosine residues, it does contain four serine residues. We hypothesized that alteration in the phosphorylation status of the CY domain of huFcγRI might be important in regulation of receptor complex (αγ2) function. The type 1 and 2A protein serine/threonine phosphatase inhibitor OA significantly blocked WT huFcγRIa-specific phagocytosis (treatedversus untreated cells, p < 0.001,n = 15 pairs). In contrast, OA had no effect on phagocytosis mediated by mutant huFcγRIa lacking the CY domain (treated versus untreated cells, p > 0.05,n = 15 pairs) (Fig. 1 A). The addition of OA to the cells did not alter cell viability of either cell line during the 1-h phagocytic assay. Furthermore, pretreatment of the WT FcγRIa-expressing cell line with the OA analog 1-Nor-okadaone, which is structurally similar to OA but does not inhibit type 1 and 2A protein serine/threonine phosphatase (25Nishiwaki S. Fujiki H. Suganuma M. Furuya-Suguri H. Matsushima R. Iida Y. Ojika M. Yamada K. Uemura D. Yasumoto T. et al.Carcinogenesis. 1990; 11: 1837-1841Google Scholar), did not alter the phagocytic response. As an additional control for the OA treatment, we incubated P388D1 cells expressing human FcγRIIa with OA, and no inhibition of FcγRIIa-specific phagocytosis was observed (Fig. 1 A). These results strongly suggest that OA does not have nonspecific effects on the cells over the time course of these experiments and that serine dephosphorylation is important in regulation of huFcγRIa-specific phagocytosis in the presence of an intact FcγRIa cytoplasmic domain. We considered the possibility that engagement of the ligand-binding site of FcγRIa, which can augment receptor function (28Pfefferkorn L.C. van de Winkel J.G. Swink S.L. J. Biol. Chem. 1995; 270: 8164-8171Google Scholar), might alter the sensitivity of phagocytic to OA. However, saturation of the transfected P388D1 with murine IgG2a (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar) did not change the sensitivity of WT huFcγRIa to OA, nor did it change the lack of inhibition of CY− huFcγRIa phagocytosis (Fig. 1 B). To directly assess serine phosphorylation of the CY domain of the α-chain of FcγRIa, we examined anti-phosphoserine mAb binding to huFcγRIa immunoprecipitates from transfected P388D1 cells. Constitutive serine phosphorylation of huFcγRIa was clearly apparent in the murine P388D1 cells (Fig.2 A). The specificity of the anti-phosphoserine Ab was confirmed by the ability of soluble phosphoserine to completely block the reactivity of the Ab with FcγRIa (data not shown). As additional controls for the specificity of the anti-phosphoserine Ab, human FcγRIa in which all four cytoplasmic serine residues were mutated to alanine (stably transfected into P388D1 cells, see below) and the mutant FcγRIa lacking a cytoplasmic domain were immunoprecipitated and were nonreactive with this blotting Ab. Reprobing of the membrane with a blotting anti-human FcγRIa Ab confirmed loading of the serine-mutated form of the receptor (Fig. 2 A). Upon receptor-specific cross-linking, the level of phosphoserine decreased over a 5-min time period (Fig. 2 B). Reprobing of the membranes with a polyclonal rabbit anti-huFcγRI Ab confirmed equivalent levels of immunoprecipitated FcγRIa at each time point (Fig. 2 B). To verify the results of the anti-phosphoserine mAb, we also preloaded U937 cells with 32Pi and examined the level of phosphorylation of immunoprecipitated huFcγRI. Again, constitutive levels of 32P-labeled FcγRIa were detectable in resting cells, and, in agreement with the immunoblotting studies, cross-linking of the receptor resulted in a decrease in the level of 32P-labeled FcγRI immunoprecipitate (Fig.2 C). Treatment of the cells with OA before receptor cross-linking resulted in the preservation of FcγRI phosphorylation, demonstrating that the OA is acting, at least in part, at the level of the FcγRI α-chain. To directly demonstrate that dephosphorylation of the CY domain of FcγRIa is important in receptor function, we prepared three different constructs of huFcγRIa in which we mutated 1) the two membrane proximal serines to alanine (S328A/S331A), 2) the two membrane distal serines to alanine (S339A/S340A), and 3) all four cytoplasmic domain serines to alanine (S4→A4). These three variant forms of huFcγRI were stably expressed in P388D1 cells (Fig. 3 A), and all three receptors were functional, as demonstrated by their ability to mediate receptor-specific phagocytosis (Table I). For comparison, expression of the WT and CY− mutant FcγRIa is shown in Fig. 3 B. To determine whether dephosphorylation of any of the specific serine residues (or of some combination of serine residues) is required for receptor function, we examined the OA sensitivity of phagocytosis by each of these variant receptors. Like the CY domain truncation construct, receptor-specific phagocytosis mediated by each of the three serine mutant receptor forms was resistant to OA treatment (Table I). As expected, pretreatment of all lines with genistein resulted in complete inhibition of receptor-specific phagocytosis. These results, taken together, demonstrate a role for dephosphorylation of serine residues in the CY domain in FcγRIa-mediated phagocytosis.Table IFcγRIa-specific phagocytic index in P388D1 cellsBaseline phagocytic indexPhagocytic index (% control)aResults are reported as the percentage of control of number of internalized E-22 per 100 transfected P388D1 cells in the genistein (n = 3)- or okadaic acid (n = 8–10)-treated cells relative to control Me2SO-treated cells (n = 3 pairs for genistein, n = 8–10 pairs for OA).+Genistein (100 nm)+Okadaic acid (1 μm)WT FcγRIa168.6 ± 18.28.5 ± 2.1bTreatment versuscontrol: p < 0.005.20.9 ± 14.9bTreatment versuscontrol: p < 0.005.S328A/S331A-FcγRIa94.2 ± 16.47.6 ± 5.2bTreatment versuscontrol: p < 0.005.98.1 ± 11.5S339A/S340A-FcγRIa62.2 ± 13.19.1 ± 4.2bTreatment versuscontrol: p < 0.005.87.5 ± 25.8S4→A4-FcγRIa75.0 ± 9.86.0 ± 3.5bTreatment versuscontrol: p < 0.005.94.0 ± 24.1a Results are reported as the percentage of control of number of internalized E-22 per 100 transfected P388D1 cells in the genistein (n = 3)- or okadaic acid (n = 8–10)-treated cells relative to control Me2SO-treated cells (n = 3 pairs for genistein, n = 8–10 pairs for OA).b Treatment versuscontrol: p < 0.005. Open table in a new tab We demonstrated previously that the CY domain of FcγRIa is required for early FcγRIa-induced IL-6 secretion (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar). In contrast to the CY domain truncation form of FcγRIa, which did not elicit IL-6 secretion, receptor-specific cross-linking of the S328A/S331A, S339A/S340A, and S4→A4 mutants resulted in both IL-1β and IL-6 secretion 8 h after stimulation (TableII). We attempted to determine the sensitivity of FcγRIa-induced IL-1β and IL-6 secretion to OA, but the incubation of cells with OA for the time periods necessary for cytokine production was toxic to the cells and thereby precluded measurement of cytokine release. These results are consistent with a model of FcγRIa function that requires dephosphorylation of the FcγRIa CY domain for receptor-mediated cell activation.Table IIFcγRIa-specific IL-1β and IL-6 production by P388D1 cellsFcγRIa-induced secretion ofaResults are reported as ng/ml (average ± S.D., n = 3 independent experimental determinations) of cytokine above mock-stimulated cells (GAM-coated wells).IL-1βIL-6ng/mlng/mlWT FcγRIa546.7 ± 118.7301.3 ± 49.3CY− FcγRIa537.0 ± 112.622.7 ± 9.0bCY− FcγRIaversus WT FcγRIa: p < 0.001.S328A/S331A-FcγRIa519.0 ± 148.1326.7 ± 55.1S339A/S340A-FcγRIa577.0 ± 117.5287.0 ± 29.5S4→A4-FcγRIa554.3 ± 115.0332.7 ± 21.8a Results are reported as ng/ml (average ± S.D., n = 3 independent experimental determinations) of cytokine above mock-stimulated cells (GAM-coated wells).b CY− FcγRIaversus WT FcγRIa: p < 0.001. Open table in a new tab Among the earliest signaling events that occur after cross-linking of FcγRIa are the activation of Src family tyrosine kinases such as Hck and tyrosine phosphorylation of the γ-chain (29Cambier J.C. J. Immunol. 1995; 155: 3281-3285Google Scholar, 30Wang A.V. Scholl P.R. Geha R.S. J. Exp. Med. 1994; 180: 1165-1170Google Scholar, 31Durden D.L. Kim H.M. Calore B. Liu Y. J. Immunol. 1995; 154: 4039-4047Google Scholar, 32Suzuki T. Kono H. Hirose N. Okada M. Yamamoto T. Yamamoto K. Honda Z. J. Immunol. 2000; 165: 473-482Google Scholar). Given the importance of serine dephosphorylation for FcγRIa phagocytosis and the dependence of phagocytosis on the tyrosine phosphorylation of the γ-chain, we reasoned that OA pretreatment might alter the tyrosine phosphorylation of the γ-chain. Indeed, pretreatment of WT FcγRI-expressing cells with OA resulted in a dramatic decrease in the level of tyrosine phosphorylation of the γ-chain (Fig. 4 A). Comparable loading of γ-chain in all lanes was confirmed by sequential analysis of the same blots with the anti-γ-chain Ab. In contrast to the WT FcγRI-expressing cells, incubation of the tail minus mutant FcγRIa or the S4→A4 mutant FcγRIa expressing P388D1 cells with OA showed no demonstrable effect on receptor-specific activation-dependent tyrosine phosphorylation of the γ-chain (Fig. 4 B). These results support the model that OA maintains serine phosphorylation of the CY domain of FcγRIa which in turn reduces WT FcγRIa mediated tyrosine phosphorylation of the γ-chain. The cytoplasmic domain of the FcγRIa α-chain can modulate the kinetics of both receptor-mediated endocytosis and phagocytosis (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar) and make receptor-specific phagocytosis insensitive to changes in [Ca2+]i (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar, 24Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Google Scholar). These observations suggest that the FcγRIa α-chain interacts with intracellular molecules that can modulate receptor signaling elements and function. Previous work has shown that murine FcγRIa is constitutively phosphorylated on serine (33Quilliam A.L. Osman N. McKenzie I.F. Hogarth P.M. Immunology. 1993; 78: 358-363Google Scholar), and we now show that the serine residues in the CY domain of human FcγRIa are actively phosphorylated and dephosphorylated in relation to receptor cross-linking and that this phosphorylation is important in regulation of FcγRIa function. The constitutive phosphorylation of the huFcγRIa α-chain was observed both in transfected murine macrophages and in the human myelomonocytic cell line U937. Upon activation, the receptor is transiently dephosphorylated. Inhibition of serine dephosphorylation with the type 1 and 2A protein serine/threonine phosphatase inhibitor, okadaic acid, results in a marked decrease in receptor function, including both phagocytosis and tyrosine phosphorylation of the γ-chain. Mutagenesis of the four cytoplasmic domain serine residues suggests that at least several of these serines are involved in this modulation of FcγRIa α-chain function. Okadaic acid likely alters the phosphorylation of multiple intracellular targets, and we considered the possibility that OA might mediate its effects on phagocytosis by altering serine and threonine phosphorylation of the γ-chain. Indeed, the γ-chain is constitutively phosphorylated on threonine and upon receptor activation becomes phosphorylated on serine and tyrosine (34Durden D.L. Rosen H. Cooper J.A. Biochem. J. 1994; 299: 569-577Google Scholar, 35Paolini R. Jouvin M.H. Kinet J.P. Nature. 1991; 353: 855-858Google Scholar). It is unlikely, however, that the inhibitory effect of OA on receptor-specific phagocytosis is due to alterations in γ-chain phosphorylation. OA does not alter phagocytosis in either the tail minus mutant form of FcγRIa or the FcγRIa serine to alanine mutants. Furthermore, OA does not alter FcγRIIa-specific phagocytosis. Taken together with the observation that OA alters the phosphorylation state of the FcγRIa α-chain, these data indicate a direct effect of OA on the phosphorylation state of the FcγRIa α-chain and suggest that this effect is responsible for OA altering FcγRIa-specific phagocytosis. The kinase(s) responsible for phosphorylation of the CY domain is currently unknown. Although protein kinase C activity is required for FcγR phagocytosis (36Karimi K. Gemmill T.R. Lennartz M.R. J. Leukocyte Biol. 1999; 65: 854-862Google Scholar, 37Larsen E.C. DiGennaro J.A. Saito N. Mehta S. Loegering D.J. Mazurkiewicz J.E. Lennartz M.R. J. Immunol. 2000; 165: 2809-2817Google Scholar), it is unlikely that protein kinase C isoforms are directly involved in modulating the phosphorylation of the CY domain of FcγRIa during receptor activation. Notably, the receptor is dephosphorylated upon cross-linking, and analysis of the sequence of the CY domain using ProfileScan of the Prosite data base (www.isrec.isb-sib.ch/software/PFSCAN_form.html) does not reveal any potential protein kinase C consensus sites. Of course, it is possible that there are protein kinase C sites not identified by such analysis, but the observations that the classical protein kinase C α isoform is important in the FcγR-induced respiratory burst and that the novel isoforms protein kinase C δ and/or protein kinase C ε are involved in FcγR phagocytosis (37Larsen E.C. DiGennaro J.A. Saito N. Mehta S. Loegering D.J. Mazurkiewicz J.E. Lennartz M.R. J. Immunol. 2000; 165: 2809-2817Google Scholar) suggest that protein kinase C family members play a role downstream of the receptor per se. Interestingly, motif analysis does indicate two consensus sites for casein kinase II, a kinase implicated in CD5 signaling (38Raman C. Kuo A. Deshane J. Litchfield D.W. Kimberly R.P. J. Biol. Chem. 1998; 273: 19183-19189Google Scholar). However, analysis of the FcγRIa CY domain and casein kinase II constructs using the yeast two-hybrid system has not shown any evidence of interaction between FcγRIa and either the α- or β-subunits of casein kinase II. Future studies will be required to determine the nature of the kinase responsible for constitutive phosphorylation of FcγRIa. The identity of the phosphatase(s) responsible for activation-induced dephosphorylation is also unknown. Our data with the protein phosphatase 1/2A inhibitor, okadaic acid, strongly implicate these phosphatases. Screens of two human leukocyte cDNA libraries for binding partners to the FcγRIa CY domain have not identified candidate phosphatases, but these serine/threonine phosphatases may be targeted to a signaling complex rather than the phosphorylated target itself (39Barford D. Das A.K. Egloff M.P. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 133-164Google Scholar, 40Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Google Scholar). Perhaps the known interaction between FcγRIa and non-muscle filamin-280 (actin-binding protein ABP-280) regulates the localization of FcγRIa in the membrane (41Ohta Y. Stossel T.P. Hartwig J.H. Cell. 1991; 67: 275-282Google Scholar). The dissociation of this protein upon receptor engagement may change the relationship of FcγRIa with the actin cytoskeleton. As with other receptor systems, associations with cytoskeletal elements may be important in allowing localization of the receptor with a phosphatase(s) and other signaling elements activated by FcγR (42Tridandapani S. Lyden T.W. Smith J.L. Carter J.E. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 2000; 275: 20480-20487Google Scholar, 43Lowry M.B. Duchemin A.M. Coggeshall K.M. Robinson J.M. Anderson C.L. J. Biol. Chem. 1998; 273: 24513-24520Google Scholar, 44Melendez A.J. Gillooly D.J. Harnett M.M. Allen J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2169-2174Google Scholar, 45Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Google Scholar, 46Tilton B. Andjelkovic M. Didichenko S.A. Hemmings B.A. Thelen M. J. Biol. Chem. 1997; 272: 28096-28101Google Scholar, 47Wang J. Brown E.J. J. Biol. Chem. 1999; 274: 24349-24356Google Scholar, 48Mansfield P.J. Shayman J.A. Boxer L.A. Blood. 2000; 95: 2407-2412Google Scholar). Although the specific kinase(s) and phosphatase(s) targeting the FcγRIa α-chain remain unclear, our data indicate that the previous model suggesting that the γ-chain is both necessary and sufficient for FcγRIa function requires revision. We propose a model in which constitutive serine phosphorylation of the CY domain of FcγRIa regulates the ability of the receptor to initiate the tyrosine kinase-based signaling cascade necessary for receptor function. Upon serine dephosphorylation, the tyrosine-based signaling cascade is fully engaged, and receptor-induced cell activation proceeds normally. In the FcγRIa CY truncation mutant, the inhibitory influence of the phosphorylated tail is removed, allowing receptor function to proceed. However, the functional differences between this mutant form of the receptor and the wild-type receptor also suggest that the CY domain of FcγRIa facilitates signaling and is required for full receptor function, including the induction of IL-6 secretion (22Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Google Scholar) and sorting of internalized receptor to endosomes (21van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. van Dijk M.A. Leusen J.H. Geuze H.J. van de Winkel J.G. Blood. 1999; 94: 808-817Google Scholar). Thus, in addition to facilitating association with cytoskeletal components, the FcγRIa CY domain may serve as a scaffold for the binding of signaling elements critical for full receptor function. Our data also emphasize the potential for genetic variants of the CY domain to influence receptor function. Polymorphic variants of the extracellular domains of FcγRIIA, FcγRIIIA, and FcγRIIIB alter ligand binding and impact upon autoimmune disease susceptibility and severity (49Wu J. Edberg J.C. Redecha P.B. Bansal V. Guyre P.M. Coleman K. Salmon J.E. Kimberly R.P. J. Clin. Invest. 1997; 100: 1059-1070Google Scholar, 50Salmon J.E. Millard S. Schachter L.A. Arnett F.C. Ginzler E.M. Gourley M.F. Ramsey-Goldman R. Peterson M.G. Kimberly R.P. J. Clin. Invest. 1996; 97: 1348-1354Google Scholar, 51Salmon J.E. Millard S.S. Brogle N.L. Kimberly R.P. J. Clin. Invest. 1995; 95: 2877-2885Google Scholar, 52van der Pol W. van de Winkel J.G. Immunogenetics. 1998; 48: 222-232Google Scholar, 53Kimberly R.P. Salmon J.E. Edberg J.C. Arthritis Rheum. 1995; 38: 306-314Google Scholar). In contrast, the CY domain of FcγRIIA, which contains a tyrosine activation motif, is invariate (54Edberg J.C. Wainstein E., Wu, J. Csernok E. Sneller M.C. Hoffman G.S. Keystone E.C. Gross W.L. Kimberly R.P. Exp. Clin. Immunogenet. 1997; 14: 183-195Google Scholar), and little attention has been focused on the CY domain of the γ-chain-associated receptors. We have recently reported two single-nucleotide polymorphisms in the CY domain of FcγRIa (55Gibson A.W., Wu, J. Edberg J.C. Kimberly R.P. Kammer G.M. Tsokos G.C. Lupus: Molecular and Cellular Pathogenesis. Humana Press, Totowa, NJ1999: 557-573Google Scholar). By their proximity to the serines at 339 and 340, we can now speculate that these single-nucleotide polymorphisms may alter quantitative phosphorylation and resultant receptor function. An understanding of the biology of these single-nucleotide polymorphisms will no doubt provide insights into the molecular mechanisms of FcγRIa signaling and also into genetic susceptibility factors for altered immune function. Nonetheless, they provide evidence for the potential clinical significance of our current observations on the role of the FcγRIa cytoplasmic domain and its serine residues on receptor function. We thank Ka Chen, Jessica T. Leonard, Dana Lau, Paul Palavin, and James J. Moon for technical assistance and Andrew J. Beavis for flow cytometric analysis and cell sorting.
Referência(s)