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The Protein-tyrosine Phosphatase SHP-1 Associates with the Phosphorylated Immunoreceptor Tyrosine-based Activation Motif of FcγRIIa to Modulate Signaling Events in Myeloid Cells

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m305078200

1083-351X

Latha P. Ganesan, Huiqing Fang, Clay B. Marsh, Susheela Tridandapani,

Glycosylation and Glycoproteins Research

FcγRIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by FcγRIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with FcγRIIa has been well studied, it is not clear how SHP-1 is activated by FcγRIIa to mediate its regulatory effect. Here we report that FcγRIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of FcγRIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of FcγRIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with FcγRIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, FcγRIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NFκB-dependent gene transcription in THP-1 cells activated by clustering FcγRIIa. FcγRIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by FcγRIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with FcγRIIa has been well studied, it is not clear how SHP-1 is activated by FcγRIIa to mediate its regulatory effect. Here we report that FcγRIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of FcγRIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of FcγRIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with FcγRIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, FcγRIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NFκB-dependent gene transcription in THP-1 cells activated by clustering FcγRIIa. IgG receptors (FcγR) on monocytes and macrophages mediate immune complex clearance by a process termed phagocytosis (1Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2029) Google Scholar). At least four classes of FcγR are expressed on monocytes and macrophages (2Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1028) Google Scholar); FcγRI, FcγRIIa, and FcγRIIIa are all activating receptors that are associated with immunoreceptor tyrosine-based activation motif (ITAM). 1The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; PtdIns, phosphatidylinositol; EGFP, enhanced green fluorescent protein; SH2, Src homology domain 2.1The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; PtdIns, phosphatidylinositol; EGFP, enhanced green fluorescent protein; SH2, Src homology domain 2. In contrast, FcγRIIb is an inhibitory receptor that is associated with an immunoreceptor tyrosine-based inhibition motif (ITIM). Of these receptors FcγRIIa is uniquely expressed in human cells and is the only ITAM-associated receptor that bears the ITAM within its cytoplasmic tail (3Van Den Herik-Oudijk I.E. Westerdaal N.A.C. Henriquez N.V. Capel P.J.A. van de Winkel J.G.J. J. Immunol. 1994; 152: 574-584PubMed Google Scholar, 4Van Den Herik-Oudijk I.E. Capel P.J. Van der Bruggen T. van de Winkel J.G. Blood. 1995; 85: 2202-2211Crossref PubMed Google Scholar). Of the ITAMs identified to date, the ITAM of FcγRIIa has the longest spacer region between the two YXXL motifs that together make the ITAM. The functional significance of this extended spacer is not fully understood. In addition, FcγRIIa is the most widely expressed FcγR in the human hematopoetic system. Clustering of FcγR by immune complexes initiates a cascade of signaling events, the first of which is the activation of the Src family of tyrosine kinases that phosphorylate the ITAMs of FcγR (5Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar, 6Cooney D.S. Phee H. Jacob A. Coggeshall K.M. J. Immunol. 2001; 167: 844-854Crossref PubMed Scopus (50) Google Scholar). The phosphorylated ITAMs serve as docking sites for SH2 domain-containing cytosolic enzymes and enzyme-adapter complexes including the tyrosine kinase Syk and the p85 adapter subunit of PtdIns 3-kinase (7Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Bio. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Association of Syk with the phosphorylated ITAM activates the enzyme resulting in autophosphorylation of Syk and tyrosine phosphorylation of multiple cytosolic proteins (8Ghazizadeh S. Bolen J.B. Fleit H.B. Biochem. J. 1995; 305: 669-674Crossref PubMed Scopus (59) Google Scholar, 9Kiener P.A. Rankin B.M. Burkhardt A.L. Schieven G.L. Gilliland L.K. Rowley R.B. Bolen J.B. Ledbetter J.A. J. Biol. Chem. 1993; 268: 24442-24448Abstract Full Text PDF PubMed Google Scholar). Likewise, association of p85 with the ITAM delivers PtdIns 3-kinase to the proximity of its lipid substrates in the membrane, resulting in the generation of 3′-phosphorylated inositol lipids that activate PH domain-containing enzymes to promote cytoskeletal changes required for the phagocytic process (10Krugmann S. Welch H. Curr. Biol. 1998; 8: R828Abstract Full Text Full Text PDF PubMed Google Scholar). Inactivation of either Syk or PtdIns 3-kinase has been shown to completely abrogate FcγR-mediated phagocytosis (11Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybuleewicz V.L.J. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (402) Google Scholar, 12Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 14Lowry M.B. Duchemin A.-M. Coggeshall K.M. Robinson J.M. Anderson C.L. J. Biol. Chem. 1998; 273: 24513-24520Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Phagocytosis is a complex process that is accompanied by the generation of reactive oxygen radicals and the production of inflammatory cytokines, which results in tissue damage. Therefore the phagocytic process is subject to a tight regulation. In this regard, several mechanisms have been proposed including the expression and function of the inhibitory receptor FcγRIIb (15Clynes R. Maizes J.S. Guinamard R. Ono M. Takai T. Ravetch J.V. J. Exp. Med. 1999; 189: 179-185Crossref PubMed Scopus (337) Google Scholar, 16Tridandapani S. Siefker K. Teillaud J.-L. Carter J.O. Wewers M.D. Anderson C.L. J. Biol. Chem. 2002; 277: 5082-5089Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 17Pricop L. Redecha P. Teillaud J.-L. Frey J. Fridman W.H. Fridman C.S. Salmon J.E. J. Immunol. 2001; 166: 531-537Crossref PubMed Scopus (199) Google Scholar), the function of intracellular inhibitory phosphatases such as the inositol phosphatases SHIP-1 (18Cox D. Dale B.M. Kishiwada M. Helgason C.D. Greenberg S. J. Exp. Med. 2001; 193: 61-71Crossref PubMed Scopus (130) Google Scholar, 19Tridandapani S. Wang Y. Marsh C.B. Anderson C.L. J. Immunol. 2000; 169: 4370-4378Crossref Scopus (50) Google Scholar, 20Nakamura K. Malykhin A. Coggeshall K.M. Blood. 2002; 100: 3374-3382Crossref PubMed Scopus (79) Google Scholar) and SHIP-2 (21Pengal R.A. Ganesan L.P. Fang H. Marsh C.B. Anderson C.L. Tridandapani S. J. Biol. Chem. 2003; 278: 22657-22663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and the protein-tyrosine phosphatase SHP-1 (22Kant A.M. De P. Peng X. Yi T. Rawlings D.J. Kim J.S. Durden D.L. Blood. 2002; 100: 1852-1859Crossref PubMed Google Scholar). Recent studies have revealed that the inositol phosphatases SHIP-1 and SHIP-2 not only work through the ITIM of FcγRIIb, but are also capable of associating with the ITAMs of FcγR to modulate activation events, thus providing an additional level of complexity to the regulation of phagocytosis (19Tridandapani S. Wang Y. Marsh C.B. Anderson C.L. J. Immunol. 2000; 169: 4370-4378Crossref Scopus (50) Google Scholar, 20Nakamura K. Malykhin A. Coggeshall K.M. Blood. 2002; 100: 3374-3382Crossref PubMed Scopus (79) Google Scholar, 23Galandrini R. Tassi I. Morrone S. Lanfrancone L. Pelicci P. Piccoli M. Frati L. Santoni A. Eur. J. Immunol. 2001; 31: 2016-2025Crossref PubMed Scopus (26) Google Scholar). Whereas the molecular details of ITAM-mediated activation of the SHIP proteins is well studied, it is not known how SHP-1 is activated by ITAM-bearing receptors. SHP-1 is a cytosolic tyrosine phosphatase that negatively regulates immune receptor signaling and growth factor signaling (24Zhang J. Somani A.K. Siminovitch K.A. Immunology. 2000; 12: 361-378Google Scholar, 25Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar). SHP-1 is predominantly expressed in hematopoetic cells and contains two NH2-terminal located SH2 domains, a central phosphatase domain and two tyrosine phosphorylation sites in the COOH-terminal region. The enzyme is regulated by intramolecular interactions such that the NH2-terminal SH2 domain folds over the catalytic domain to inactivate the enzyme (26Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 27Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (127) Google Scholar, 28Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (185) Google Scholar). Deletion of the NH2-terminal SH2 domain, or engagement of the SH2 domains with cognate phosphopeptides has been shown to activate the phosphatase. Enzyme activity of SHP-1 is further enhanced by phosphorylation of tyrosines (Tyr536 and Tyr564) in the COOH-terminal region (29Zhang Z. Shen k. Lu W. Cole P.A. J. Biol. Chem. 2002; 278: 4668-4674Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The significance of the regulatory role of SHP-1 in the hematopoetic system is best exemplified in mice homozygous for motheaten (me/me) or motheaten viable (mev/mev) mutations (30Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (685) Google Scholar, 31Shultz L.D. Green M.C. J. Immunol. 1976; 116: 936-943PubMed Google Scholar, 32Green M.C. Shultz L.D. J. Hered. 1975; 66: 250-258Crossref PubMed Scopus (149) Google Scholar). The me/me mice do not express any SHP-1 protein, whereas the mev/mev mice express inactive splice variants of SHP-1. Both of these mutations result in multiple hematopoetic defects including elevated levels of autoantibodies and chronic inflammation resulting in early mortality. In a recent study, Durden and colleagues (22Kant A.M. De P. Peng X. Yi T. Rawlings D.J. Kim J.S. Durden D.L. Blood. 2002; 100: 1852-1859Crossref PubMed Google Scholar) have demonstrated that SHP-1 down-regulates FcγR-mediated phagocytosis in the J774A.1 mouse macrophage cell line. In this study we have analyzed the molecular details of SHP-1 activation by the human FcγRIIa and the functional consequence of this activation. We report that SHP-1 phosphatase activity is induced upon FcγRIIa clustering in THP-1 human monocytic cells. FcγRIIa clustering results in membrane translocation of SHP-1 and association of SHP-1 with the phosphorylated ITAM of FcγRIIa. Co-precipitation experiments in cells transfected with ITAM tyrosine mutants of FcγRIIa revealed that SHP-1 associates with the NH2-terminal ITAM tyrosine, whereas Syk associates with the COOH-terminal ITAM tyrosine. Previous studies using substrate-trapping mutants of SHP-1 have demonstrated that SHP-1 associates with and dephosphorylates Syk, p85, and p62dok in other cell systems (33Dustin L.B. Plas D.R. Wong J. Hu Y.T. Soto C. Chan A.C. Thomas M.L. J. Immunol. 1999; 162: 2717-2724PubMed Google Scholar, 34Cuevas B. Lu Y. Watt S. Kumar R. Zhang J. Siminovitch K.A. Mills G.B. J. Biol. Chem. 1999; 274: 27583-27589Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 35Berg K.L. Siminovitch K.A. Stanley E.R. J. Biol. Chem. 1999; 274: 35855-35865Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Likewise, our current studies demonstrate association of SHP-1 with the above molecules upon FcγRIIa clustering suggesting that SHP-1 may dephosphorylate these molecules to down-regulate related signaling pathways. Consistent with this notion, analysis of functional consequence of SHP-1 phosphatase activity during FcγRIIa signaling demonstrated that overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 down-regulates NFκB-dependent gene transcription following FcγRIIa signaling. Taken together these results suggest that signaling events initiated by the ITAM of FcγRIIa are a composite of both positive and negative regulatory enzyme activation. Cells, Antibodies, and Reagents—THP-1 cells were obtained from ATCC and cultured in RPMI supplemented with 10% fetal bovine serum. P388D1 transfectants expressing human FcγRIIa were a generous gift from Dr. J. C. Edberg (University of Alabama) (36Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Anti-FcγRIIa antibody IV.3 was obtained from Medarex (Annandale, NJ). Rabbit polyclonal SHP-1, p85, Syk antibodies, and mouse monoclonal anti-phosphotyrosine antibody and phosphatase assay kits were purchased from Upstate Biotechnology (Charlottesville, VA). Immunoprecipitation and Western Blotting—THP-1 cells and transfected P388D1 cells were activated by clustering FcγRIIa with F(ab′)2 fragments of monoclonal antibody IV.3 and goat F(ab′)2 anti-mouse Ig secondary antibody. Resting and activated cells were lysed in TN1 buffer (50 mm Tris, pH 8.0, 10 mm EDTA, 10 mm Na4P2O7, 10 mm NaF, 1% Triton X-100, 125 mm NaCl, 10 mm Na3VO4, 10 μg/ml each aprotinin and leupeptin), and postnuclear lysates were incubated overnight with the antibody of interest and protein G-agarose beads (Invitrogen) or goat anti-mouse Ig covalently linked to Sepharose, depending on the antibody. Immunoprecipitations with control antibodies were performed in lysates of cells stimulated for 3 min. Immune complexes bound to beads were washed in TN1 and boiled in SDS sample buffer (60 mm Tris, pH 6.8. 2.3% SDS, 10% glycerol, 0.01% bromphenol blue, and 1% 2-mercaptoethanol) for 5 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the antibody of interest, and developed by enhanced chemiluminescence. Analysis of FcγRIIa Expression by Flow Cytometry—P388D1 transfectants were tested for expression of FcγRIIa by incubating with Fab fragments of anti-FcγRIIa monoclonal antibody IV.3, at a concentration of 10 μg/ml for 30 min at 4 °C. The cells were washed and incubated with fluorescein isothiocyanate-labeled goat F(ab′)2 anti-mouse Ig secondary antibody for 30 min at 4 °C. Cells were subsequently washed, fixed in 1% paraformaldehyde, and analyzed by flow cytometry on an Elite EPICS fluorescence-activated cell sorter (Coulter, Hialeah, FL). Data from 10,000 cells per condition were recorded to yield the percentage of cells expressing receptors. Phosphatase Assays—Phosphatase assays were performed as described previously (37Kon-Kozlowski M. Pani G. Pawson T. Siminovitch K.A. J. Biol. Chem. 1996; 271: 3856-3862Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), with slight modifications. To measure phosphatase activity associated with SHP-1, FcγRIIa, Syk, p85, p62dok, and Erk, these proteins were immunoprecipitated from resting and activated (FcγRIIa clustering) THP-1 cells. Immunoprecipitations with control antibodies were done in lysates of cells stimulated for 7 min. The immunoprecipitates were washed six times in wash buffer (10 mm Tris, pH 7.4), and subsequently incubated with tyrosine phosphopeptide substrate (RRLIEDAEpYAARG) (Upstate Biotechnology) in 10 mm Tris, pH 7.4, for 30 min. Reaction was stopped with 100 μl of malachite green solution, incubated for a further 15 min, and the absorbance was measured at 630 nm. All assays were performed at least three times and the values obtained were plotted as mean ± S.D. Transfection of THP-1 Cells and Luciferase Assays—For analysis of SHP-1 influence on NFκB transcriptional activity, THP-1 cells were transfected by electroporation (310 V, 950 μF; Bio-Rad Gene Pulser II) with 5 μg of wild-type SHP-1 or catalytically deficient (D419A) SHP-1 (a kind gift from Dr. R. Siraganian) (38Xie Z.H. Zhang J. Siraganian R.P. J. Immunol. 2000; 164: 1521-1528Crossref PubMed Scopus (55) Google Scholar), 1 μg of NFκB-luc plasmid, and 0.5 μg of pEGFP to normalize for transfection efficiency. Transfectants were harvested 24 h later, activated by clustering FcγRIIa by methods described above for 6 h at 37 °C. The cells were lysed in 100 μl of cell culture lysis reagent (Promega). Luciferase activity was measured using the Promega luciferase assay reagent. Data are represented as graphs indicating the % increase in NFκB activity in cells activated by clustering FcγRIIa over those that were not activated. Data points are expressed as mean ± S.D. of three independent experiments. Statistical analysis was performed by Student's t test. Transfection of COS-7 Cells—COS-7 cells were transfected as previously described (39Tridandapani S. Lyden T.W. Smith J.L. Carter J.E. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 2000; 275: 20480-20487Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, cells were grown on culture dishes until they were 60–70% confluent. Plasmids encoding wild-type SHP-1 and D419A SHP-1 were mixed with LipofectAMINE 2000 reagent (Invitrogen). The DNA mixture was added to cells in serum-free Dulbecco's modified Eagle's medium and incubated for 3 h at 37 °C in a CO2 incubator. The media was then replaced by Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were harvested 24 h later and analyzed for expression of the transfected cDNAs by Western blotting whole cell lysates and SHP-1 immunoprecipitates from the transfectants were assessed for phosphatase activity as described above. GFP-SHP-1 Construct—Wild-type SHP-1 cDNA in pSVL vector was obtained from Dr. R. Siraganian, and subcloned into pEGFP vector (Clontech) using the Xho and XbaI sites. Expression of GFP-SHP-1 was first confirmed by transfecting COS-7 fibroblasts with either empty EGFP vector or GFP-SHP-1 constructs, and subsequent Western blotting with anti-SHP-1 antibody. Transfection of P388D1 Cells and Confocal Microscopy—P388D1 cells stably expressing human FcγRIIa were transfected with GFP-SHP-1 plasmids using LipofectAMINE, as described above for COS-7 cells. Cells were harvested 24 h post-transfection, serum starved, and stimulated by clustering FcγRIIa for 5 min. Resting and activated cells were fixed in 1% paraformaldehyde, cytospun onto glass slides, and stained with Hoechst nuclear stain. Slides were then mounted using mounting media (Molecular Probes) and analyzed by confocal microscopy using a Zeiss LSM510 multiphoton confocal microscope. SHP-1 Is Activated by FcγRIIa Clustering—To assess whether SHP-1 is activated by FcγRIIa, THP-1 cells were stimulated by clustering FcγRIIa with Fab fragments of the receptor-specific monoclonal antibody IV.3, followed by secondary cross-linking with goat F(ab′)2 fragments of anti-mouse Ig antibody. SHP-1 was immunoprecipitated from resting and activated cells and analyzed first, for phosphatase activity (Fig. 1A) and second, for tyrosine phosphorylation by Western blotting (Fig. 1B). The use of Fab/F(ab′)2 fragments of the clustering antibodies precludes the engagement of other FcγR present on the THP-1 cells by IgG ligand interaction ensuring that the resultant signals are emanating from FcγRIIa alone. In Fig. 1A, SHP-1 phosphatase activity was measured in THP-1 cells activated for the various time points indicated in the figure. Results indicate that SHP-1 phosphatase activity is induced by FcγRIIa clustering and the activity peaks around 7 min post-stimulation. Previous studies have indicated that the enzyme activity of SHP-1 is enhanced upon tyrosine phosphorylation of SHP-1 (29Zhang Z. Shen k. Lu W. Cole P.A. J. Biol. Chem. 2002; 278: 4668-4674Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The results shown in Fig. 1B demonstrate that SHP-1 is tyrosine-phosphorylated upon FcγRIIa clustering. A reprobe of the same membrane with anti-SHP-1 antibody in the lower panel indicates equal loading of SHP-1 in all lanes. The last lane marked "C" is a control immunoprecipitate with normal rabbit IgG. To further confirm that the clustering antibodies used do not engage other FcγR expressed on THP-1 cells, specifically FcγRIIb, which bears a high level homology with FcγRIIa in the extracellular domain, binding of Fab fragments of monoclonal antibody IV.3 to FcγRIIb was analyzed. For this THP-1 cells were subjected to immunoprecipitation with Fab fragments of IV.3, intact IV.3 IgG, and two pan-FcγRII antibodies KB61 and AT10. The immunoprecipitates were probed with rabbit polyclonal antibodies specific for either FcγRIIb (Fig. 1C, upper panel) or FcγRIIa (Fig. 1C, lower panel). Results indicate that whereas all four antibodies used are able to immunoprecipitate FcγRIIa, Fab fragments of IV.3 are unable to bind FcγRIIb. The pan-FcγRII antibodies KB61 and AT10 bound FcγRIIb as expected. Intact IV.3 IgG was also able to precipitate some FcγRIIb presumably by ligand interaction, as we have previously reported. Taken together these results demonstrate that SHP-1 is activated by the ITAM-bearing FcγRIIa in THP-1 cells, without the involvement of FcγRIIb. SHP-1 Translocates to the Membrane upon FcγRIIa Clustering—To test whether FcγRIIa clustering resulted in membrane translocation of SHP-1, GFP-SHP-1 constructs were generated and transiently transfected into P388D1 mouse macrophage cells stably expressing human FcγRIIa. Cells were stimulated for 5 min by clustering FcγRIIa and analyzed by confocal microscopy. Results indicated that SHP-1 is distributed in the cytoplasm in resting cells and translocates to the membrane in cells activated by clustering FcγRIIa (Fig. 1D). In parallel samples transfected with EGFP alone, no movement of GFP was observed in activated cells compared with resting cells (data not shown). SHP-1 Co-immunoprecipitates with FcγRIIa—We next assessed whether SHP-1 associates with FcγRIIa to become activated. Here, THP-1 cells were activated by clustering FcγRIIa by the methods described above. SHP-1 was immunoprecipitated from resting and activated cells, and analyzed for association with FcγRIIa by Western blotting with the FcγRIIa-specific antibody 260. Results indicated that SHP-1 associates with FcγRIIa upon activation (Fig. 2A, upper panel). No association was detectable in resting cells. The same membrane was reprobed with anti-SHP-1 antibody to ensure equivalent loading of SHP-1 in all lanes (lower panel). As a second approach to confirm association of SHP-1 with FcγRIIa, the receptors were immunoprecipitated from resting and activated THP-1 cells and subjected to a phosphatase assay with a phosphopeptide substrate. The amount of free phosphate released was detected by the addition of malachite green. Results are expressed as picomole of phosphate released by immunoprecipitates from activated cells after subtracting the values obtained from immunoprecipitates from resting cells (Fig. 2B). Control immunoprecipitates consistently showed values equal to or lower than resting cell immunoprecipitates. Together these experiments demonstrate that SHP-1 associates with FcγRIIa following receptor clustering. NH2-terminal ITAM Tyrosine of FcγRIIa Is Necessary for Association with SHP-1—To examine which of the two ITAM tyrosines of FcγRIIa were involved in the association with SHP-1, we used two experimental models. First, synthetic biotinylated peptides derived from the ITAM of FcγRIIa, which were either non-phosphorylated (P1), or singly phosphorylated on either the NH2-terminal ITAM tyrosine (P2) or the COOH-terminal ITAM tyrosine (P3), were applied to THP-1 lysates and the peptide-bound material was analyzed for the presence of SHP-1 by Western blotting. The results shown in Fig. 3A, upper panel, indicate that the phosphorylated NH2-terminal ITAM tyrosine, but not the COOH-terminal tyrosine, efficiently bound SHP-1. SHP-1 did not associate with the non-phosphorylated peptide (lane 1). In contrast, parallel experiments analyzing the binding properties of the peptides demonstrated that the peptide phosphorylated on the COOH-terminal ITAM tyrosine is functional and is able to associate with Syk (Fig. 3A, middle panel) and p85 (Fig. 3A, lower panel). These latter findings are consistent with earlier reports demonstrating that the COOH-terminal ITAM tyrosine of FcγRIIa is sufficient for association with Syk (40Kim M.K. Pan X.Q. Huang Z.Y. Hunter S. Hwang P.H. Indik Z.K. Schreiber A.D. Clin. Immunol. 2001; 98: 125-132Crossref PubMed Scopus (24) Google Scholar), and that p85 associates with both NH2- and COOH-terminal ITAM tyrosines of FcγRIIa (6Cooney D.S. Phee H. Jacob A. Coggeshall K.M. J. Immunol. 2001; 167: 844-854Crossref PubMed Scopus (50) Google Scholar, 41Gibbins J.M. Briddon S. Shutes A. Van Vugt M.J. De Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Because the above experiments were performed with synthetic peptides, we next asked whether the native FcγRIIa receptor would likewise demonstrate the differential ITAM tyrosine requirement for association with SHP-1 and Syk. For these experiments we used P388D1 mouse macrophage transfectants stably expressing single ITAM tyrosine mutants of human FcγRIIa. The P388D1 transfectants were activated by clustering FcγRIIa, the receptors were immunoprecipitated from resting and activated cells and analyzed by Western blotting for co-precipitating SHP-1 (Fig. 3B, upper panel) or Syk (Fig. 3B, lower panel). Results indicated that SHP-1 failed to associate with FcγRIIa when the NH2-terminal ITAM tyrosine was mutated to phenylalanine (Y252F). However, the Y252F receptor displayed efficient binding to Syk. These results are consistent with the above peptide binding experiments. To assess the signaling outcome of the ITAM tyrosine mutations, we compared the ability of these mutated receptors versus the wild-type receptor to induce signaling. For this we first ensured that the transfected receptors were expressed to comparable levels by flow cytometry (Fig. 4A). The transfected cells were stimulated by clustering FcγRIIa. FcγRIIa was immunoprecipitated from resting and activated cells and analyzed for tyrosine phosphorylation. Results indicated that all three receptors are capable of being tyrosine phosphorylated (Fig. 4B, upper panel). As might be expected the single ITAM tyrosine mutants displayed lower phosphorylation levels than the wild-type receptor. A reprobe of the membrane demonstrated equivalent receptor expression in the transfectants (Fig. 4B, lower panel). The reduced signal seen with anti-FcγRIIa antibody in the activated lane is because of the fact that the anti-FcγRIIa blotting antibody often displays lower efficiency of detection of the phosphorylated FcγRIIa in a reprobe. We, and others, have previously reported this property of the anti-FcγRIIa blotting antibody (19Tridandapani S. Wang Y. Marsh C.B. Anderson C.L. J. Immunol. 2000; 169: 4370-4378Crossref Scopus (50) Google Scholar, 42Vely F. Gruel N. Moncuit J. Cochet O. Rouard H. Dare S. Galon J. Sautes C. Fridman W.H. Teillaud J.-L. Hybridoma. 1997; 16: 519-528Crossref PubMed Scopus (36) Google Scholar). We next analyzed total cellular tyrosine phosphorylation in the transfectants stimulated by FcγRIIa clustering (Fig. 4C). Results indicated that, clustering of the NH2-terminal ITAM tyrosine mutant leads to enhanced overall cellular tyrosine phosphorylation in comparison to clustering of the wild-type receptor (lane 4 versus lane 1). In contrast, mutation of the COOH-terminal ITAM tyrosine completely abrogated overall cellular phosphorylation. These observations are consistent with the notion that SHP-1 associates with the NH2-terminal ITAM tyrosine to down-modulate tyrosine phosphorylation events, and that Syk associates with the COOH-terminal ITAM tyrosine to become activated and lead to the phosphorylation of signaling proteins in the cell. SHP-1 Associates with p85, Syk, and p62dok during FcγRIIa Signaling—The activation of SHP-1 during FcγRIIa signaling suggests that SHP-1 causes dephosphorylation of tyrosine-phosphorylated proteins. Numerous previous studies have identified the association of SHP-1 with tyrosine-phosphorylated signaling molecules, the subsequent dephosphorylation of these molecules, and down-regulation of the related signaling pathways (43Veillette A. Latour S. Davidson D. Annu. Rev. Immunol. 2002; 20: 669-707Crossref PubMed Scopus (210) Google Scholar). Drawing from these previous studies, we next analyzed whether SHP-1 associated with the tyrosine kinase Syk, the p85 adapter molecule of PtdIns 3-kinase, and the Ras GAP-binding protein p62dok during FcγRIIa signaling. Thus, THP-1 cells were activated by clustering FcγRIIa for various time points. SHP-1 was immunoprecipitated from resting and activated THP-1 cells and analyzed by Western blotting for the presence of co-precipitating Syk (Fig. 5A), p85 (Fig. 5B), and p62dok (Fig. 5C). As seen in the figure, SHP-1 associated with the above molecules in an activation-dependent manner. The membranes were reprobed with anti-SHP-1 antibody to ensure equal loading in all lanes. To further analyze whether active SHP-1 is associated with Syk, p85, and p62dok, phosphatase assays were performed on the respective immunoprecipitates from resting and activated THP-1 cells. Consistent with association of SHP-1 protein, results indicated that phosphatase activity was present in Syk, p85, and p62dok immunoprecipitates (Fig. 5D). In control experiments, no association of SHP-1 phosphatase activity was observed in Erk immunoprecipitates from activated THP-1 cells (data not shown). Taken together these data suggest that SHP-1 may dephosphorylate the above molecules to down-regulate activation events induced by FcγRIIa clustering. SHP-1 Down-regulates FcγRIIa-mediated Function—In recent reports we, and others, have demonstrated that FcγRIIa clustering results in the activation of NFκB-dependent gene transcription (19Tridandapani S. Wang Y. Marsh C.B. Anderson C.L. J. Immunol. 2000; 169: 4370-4378Crossref Scopus (50) Google Scholar, 21Pengal R.A. Ganesan L.P. Fang H. Marsh C.B. Anderson C.L. Tridandapani S. J. Biol. Chem. 2003; 278: 22657-22663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 44Sánchez-Mejorada G. Rosales C. J. Leukocyte Biol. 1998; 63: 521-533Crossref PubMed Scopus (161) Google Scholar). These activation events are subject to regulation by the inositol phosphatases SHIP-1 and SHIP-2, presumably as a result of the consumption of the lipid products of PtdIns 3-kinase and the downstream signaling thereof. Our present studies demonstrate that SHP-1 associates with the p85 subunit of PtdIns 3-kinase, suggesting that SHP-1 may modulate PtdIns 3-kinase activity. Therefore, we next asked whether SHP-1 also played a role in modulating NFκB-dependent gene transcription initiated by FcγRIIa clustering. In these experiments we used wild-type and catalytically inactive (D419A) SHP-1 constructs, which we first expressed in COS-7 fibroblasts by transient transfection and analyzed for SHP-1 protein expression and enzyme activity. The results shown in Fig. 6B demonstrate that both wild-type and D419A SHP-1 are expressed efficiently from these plasmids. COS-7 fibroblasts do not express any endogenous SHP-1 as is seen from the absence of SHP-1 in the mock-transfected cells (lane 1). Shown in Fig. 6B, lower panel, is the phosphatase activity of these two SHP-1 proteins expressed in COS-7 cells. Having ensured that we could achieve appropriate protein expression from these constructs, we then transiently transfected THP-1 cells with plasmids encoding the NFκB binding element coupled to a luciferase gene (NFκB-luc) either alone or with an excess of wild-type SHP-1 or D419A SHP-1. The cells were harvested 24 h post-transfection, activated by clustering FcγRIIa, and NFκB-dependent luciferase expression was assessed in a luciferase enzyme assay. Results from three independent experiments are shown in Fig. 6A. Overexpression of wild-type SHP-1 completely abrogated NFκB-dependent luciferase induction. In contrast, overexpression of the catalytically inactive D419A SHP-1 resulted in enhanced luciferase induction. These data demonstrate that SHP-1 negatively regulates FcγRIIa-mediated biological outcomes in human myeloid cells. The human-specific FcγRIIa is a low affinity IgG receptor that has several unique features to it. In addition to being the most widely expressed IgG receptor, it also contains an unusually lengthy ITAM in its cytoplasmic domain. Mutational analyses of the cytoplasmic domain of FcγRIIa have identified specific amino acid motifs that are important for the phagocytic process. For example, mutation of either of the two tyrosine residues within the ITAM of FcγRIIa have been reported to severely abrogate intracellular calcium mobilization and phagocytosis (36Edberg J.C. Lin C.T. Lau D. Unkeless J.C. Kimberly R.P. J. Biol. Chem. 1995; 270: 22301-22307Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 45Mitchell M.A. Huang M.M. Chien P. Indik Z.K. Pan X.Q. Schreiber A.D. Blood. 1994; 84: 1753-1759Crossref PubMed Google Scholar). An additional tyrosine residue located NH2-terminal to the ITAM also becomes phosphorylated upon receptor clustering and plays a role in FcγRIIa-mediated activation (45Mitchell M.A. Huang M.M. Chien P. Indik Z.K. Pan X.Q. Schreiber A.D. Blood. 1994; 84: 1753-1759Crossref PubMed Google Scholar). More recent studies have identified an LTL motif in the cytoplasmic domain of FcγRIIa that is involved in the formation of phagolysosomes (46Worth R.G. Mayo-Bond L. Kim M.K. van de Winkel J.G. Todd III, R.F. Petty H.R. Schreiber A.D. Blood. 2001; 98: 3429-3434Crossref PubMed Scopus (25) Google Scholar, 47Worth R.G. Kim M.K. Kindzelskii A.L. Petty H.R. Schreiber A.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4533-4538Crossref PubMed Scopus (42) Google Scholar). Thus the cytoplasmic domain of FcγRIIa is made up of a complex set of signaling motifs that are not yet fully explored. Once FcγRIIa receptors are clustered the Src family of tyrosine kinases phosphorylate tyrosine residues in the cytoplasmic domain of FcγRIIa (6Cooney D.S. Phee H. Jacob A. Coggeshall K.M. J. Immunol. 2001; 167: 844-854Crossref PubMed Scopus (50) Google Scholar). Phosphorylation of the ITAM promotes recruitment and activation of Syk, followed by the phosphorylation of multiple cytosolic signaling proteins. Unlike its T cell homolog ZAP-70 that requires both of its tandem SH2 domains to be engaged by phosphorylated ITAMs to be activated, single SH2 domain engagement is sufficient for Syk activation (48Futterer K. Wong J. Grucza R.A. Chan A.C. Waksman G. J. Mol. Biol. 1998; 281: 523-537Crossref PubMed Scopus (168) Google Scholar). Accordingly, the results shown in Fig. 3 demonstrate that the COOH-terminal ITAM tyrosine of FcγRIIa is necessary and sufficient for Syk association. Interestingly, there was constitutive Syk association with Y252F FcγRIIa, at a time when no tyrosine phosphorylation of the receptor was detectable (Fig. 4B). These results suggest that perhaps mutation of tyrosine 252 might lead to a non-SH2-dependent association of Syk with Y252F FcγRIIa. Additional studies are needed to define the nature of this novel interaction. Recent studies have revealed that FcγRIIa clustering not only initiates activating events, but it also induces negative regulatory events such that the resultant biologic outcome is tempered. Thus, FcγRIIa recruits the inositol phosphatases SHIP-1 and SHIP-2 to modulate signaling events (19Tridandapani S. Wang Y. Marsh C.B. Anderson C.L. J. Immunol. 2000; 169: 4370-4378Crossref Scopus (50) Google Scholar, 20Nakamura K. Malykhin A. Coggeshall K.M. Blood. 2002; 100: 3374-3382Crossref PubMed Scopus (79) Google Scholar, 21Pengal R.A. Ganesan L.P. Fang H. Marsh C.B. Anderson C.L. Tridandapani S. J. Biol. Chem. 2003; 278: 22657-22663Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In a transfected COS-7 fibroblast model the protein-tyrosine phosphatase SHP-1 has also been shown to regulate FcγRIIa-mediated phagocytosis (22Kant A.M. De P. Peng X. Yi T. Rawlings D.J. Kim J.S. Durden D.L. Blood. 2002; 100: 1852-1859Crossref PubMed Google Scholar). Our current studies extend these latter findings to demonstrate that in myeloid cells SHP-1 translocates to the membrane, associates with the phosphorylated NH2-terminal ITAM tyrosine of FcγRIIa, and regulates FcγRIIa-mediated signaling. Together, these observations suggest that signal transduction from FcγRIIa is internally regulated by both positive and negative signaling enzymes. SHP-1 was initially thought to be the effector molecule of FcγRIIb-mediated inhibition (49D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-296Crossref PubMed Scopus (507) Google Scholar). However, later studies using chimeric FcγRIIb receptors and SHP-1-deficient cells demonstrated that SHP-1 is not required for FcγRIIb function (50Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 51Nadler M.J.S. Chen B. Anderson J.S. Wortis H.H. Neel B.G. J. Biol. Chem. 1997; 272: 20038-20043Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), but that SHP-1 works in concert with other ITIM-bearing receptors such as the KIRs, gp49B, PIR-B etc. (52Pani G. Siminovitch K.A. Clin. Immunol. Immunopathol. 1997; 84: 1-16Crossref PubMed Scopus (20) Google Scholar), to mediate its inhibitory function. Our current observations of SHP-1 association with FcγRIIa ITAM are novel, and are consistent with earlier findings that SHP-1 association with immune receptors occurs in the absence of involvement of the ITIM-bearing FcγRIIb (53Phee H. Rodgers W. Coggeshall K.M. Mol. Cell. Biol. 2001; 21: 8615-8625Crossref PubMed Scopus (47) Google Scholar). Activation of SHP-1 enzyme requires the engagement of its NH2-terminal SH2 domain with phosphotyrosines to relieve the intramolecular constraint placed on the phosphatase domain (26Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Consistent with this notion, our results suggest that the engagement of SHP-1 by the NH2-terminal ITAM tyrosine of FcγRIIa leads to the activation of SHP-1. Other studies have reported a secondary mechanism of SHP-1 activation involving phosphorylation of SHP-1 on its COOH-terminal located tyrosine residues (29Zhang Z. Shen k. Lu W. Cole P.A. J. Biol. Chem. 2002; 278: 4668-4674Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In our experiments, although tyrosine phosphorylation of SHP-1 was detectable after FcγRIIa clustering (Fig. 1), the level of phosphorylation is weak suggesting that it is likely that the primary mechanism of SHP-1 activation is mediated by its association with FcγRIIa. Additional studies are required to assess whether the low level phosphorylation of SHP-1 contributes to activation of the enzyme. The identification of specific substrates of SHP-1 has been aided by the use of substrate-trapping mutant forms of SHP-1 and the SHP-1-deficient motheaten animals. Consistent with earlier observations in other cell systems, we have observed association of SHP-1 with Syk, p85, and p62dok. These findings suggest that the above molecules may be dephosphorylated by SHP-1 resulting in down-regulation of the related signaling pathways. In accordance with this notion, our data indicate that SHP-1 down-regulates NFκB-dependent gene transcription in myeloid cells stimulated by clustering FcγRIIa. NFκB activation has been shown to be important for FcγR-induced transcription of inflammatory cytokine genes such as interleukin-1, tumor necrosis factor-α, and interleukin-8 (54Sanchez-Mejorada G. Rosales C. J. Biol. Chem. 1998; 273: 27610-27619Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus the current study establishes a role for SHP-1 in modulating the production of inflammatory cytokines during immune complex clearance. We thank Mark Kotur and Alan Bakaletz for assistance with confocal microscopy and J. Parker-Barnes for assistance with GFP-SHP.1 constructs.