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The Journal of Immunology, 2002, 169: 4370-4378.
Copyright © 2002 by The American Association of Immunologists

Src Homology 2 Domain-Containing Inositol Polyphosphate Phosphatase Regulates NF-{kappa}B-Mediated Gene Transcription by Phagocytic Fc{gamma}Rs in Human Myeloid Cells1

Susheela Tridandapani2, Yijie Wang, Clay B. Marsh and Clark L. Anderson

Department of Internal Medicine, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fc{gamma}R-mediated phagocytosis is accompanied by the generation of tissue-damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering Fc{gamma}R on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic Fc{gamma}R activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-Fc{gamma}R and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-Fc{gamma}R, using cell systems that lack Fc{gamma}RIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing Fc{gamma}RIIa in vivo; and third, phosphorylation of SHIP by Fc{gamma}RIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-Fc{gamma}R down-regulates NF-{kappa}B-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM-Fc{gamma}R serves to regulate gene expression during the phagocytic process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages, playing an important role in the adaptive immune response, phagocytose infectious particles via Fc{gamma}R and complement receptors (1). Because phagocytosis is often accompanied by the generation of tissue-damaging products such as reactive oxygen species and inflammatory cytokines, a tight regulation of this phagocytic process would seem mandatory. Although much is known about the molecular events leading to the activation process initiated by Fc{gamma}R, it is not clear how this activation process is regulated.

Macrophages express three classes of Fc{gamma}R: I, II, and III (2). Although the expression of Fc{gamma}RI and III is common to both murine and human hematopoietic cells, the expression of Fc{gamma}RII is different between the two species. Thus, while human cells express two functionally different forms of Fc{gamma}RII (i.e., IIa and IIb, products of two separate genes), mouse cells express Fc{gamma}RIIb but not Fc{gamma}RIIa, having no gene for Fc{gamma}RIIa. Fc{gamma}RI, III, and IIa are activating receptors associated with immunoreceptor tyrosine-based activation motifs (ITAM)3 that are present either within the cytoplasmic tail of the receptor as in Fc{gamma}RIIa (3) or within the associated low-m.w. FcR{gamma} subunit (4). In contrast, Fc{gamma}RIIb is an inhibitory receptor that has in its cytoplasmic tail an immunoreceptor tyrosine-based inhibition motif (ITIM). The extracellular domains of Fc{gamma}RIIa and IIb are virtually identical, a fact that has complicated the detection and study of Fc{gamma}RIIb in human macrophages. However, recent studies using novel reagents established conclusively the presence of Fc{gamma}RIIb protein in human macrophages (5, 6). Fc{gamma}RIIb serves to down-regulate phagocytosis of IgG-coated particles, as demonstrated in several studies (7, 8), using as its effector molecule Src homology (SH)2 domain-containing inositol polyphosphate phosphatase (SHIP). We and others have well characterized the molecular details of SHIP association with Fc{gamma}RIIb (9, 10, 11).

SHIP, expressed exclusively in hematopoietic cells, is a multidomain cytosolic protein, which contains not only an inositol phosphatase domain but also several protein interaction domains (12, 13) including 1) a SH2 domain that associates with high affinity to the phosphorylated ITIM of Fc{gamma}RIIb (9, 10, 11) and with phosphorylated Shc (13, 14); 2) a proline-rich domain that constitutively associates with the SH3 domain of the adapter protein Grb2 (13, 15); and 3) multiple sites of tyrosine phosphorylation that are involved in SHIP association with Shc (14, 16, 17). SHIP hydrolyzes phosphatidylinositol-3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase, which is required for the activation of several enzymes that contain pleckstrin homology domains, such as Bruton’s tyrosine kinase (18), involved in intracellular calcium mobilization; Akt (19, 20), an enzyme involved in cell survival; and Vav (21), the guanine nucleotide exchange factor for the Rho family of GTPases. The role of SHIP as an inhibitory protein working via the ITIM-bearing Fc{gamma}RIIb as well as through ITAM-bearing receptors is well established both in vitro using cell lines and in vivo using SHIP-deficient mice. Thus, the presence of SHIP attenuates B cell receptor-mediated activation and Fc{epsilon}RI-mediated activation when Fc{gamma}RIIb is coclustered with these receptors (11, 15, 19, 20, 22, 23, 24). Likewise, Greenberg et al. (25) demonstrated that SHIP-deficient murine macrophages display enhanced phagocytic efficiency in comparison to the wild-type macrophages. Paradoxically, the inhibitory influence of SHIP is exerted even when activating receptors alone are clustered, without invoking Fc{gamma}RIIb, as is elegantly established in genetically altered mice and in avian B cell lines (23, 24, 26, 27), indicating that SHIP serves as a modulator of activation events independently of its being recruited by Fc{gamma}RIIb. Although the molecular mechanism of SHIP association with the ITIM is well explored, it is unclear how SHIP associates with ITAMs and becomes phosphorylated by ITAM-bearing receptors. Likewise, it is unclear whether the activation of SHIP by ITAM-Fc{gamma}R has any functional consequence.

We and others (28, 29) earlier reported the activation and association of SHIP with ITAM-Fc{gamma}R in human monocytes. In light of our current knowledge that Fc{gamma}RIIb protein is present in human monocytes, we undertook to test whether the previously reported ITAM-induced SHIP activation could be explained by the presence of trace amounts of intact Ab in the F(ab')2 fractions of our activating Abs that could have involved Fc{gamma}RIIb coclustering. Therefore, we analyzed the ability of ITAM-Fc{gamma}R to induce SHIP phosphorylation in the absence of any functional Fc{gamma}RIIb. We report here, using COS-7 transfection models that lack endogenous SHIP and FcRs, and knockout mouse models that lack Fc{gamma}RIIb expression, that the ITAM-Fc{gamma}R are indeed capable of inducing SHIP phosphorylation. Coimmunoprecipitation experiments revealed an in vivo association of SHIP with Fc{gamma}RIIa, an interaction that might occur either directly or via an adapter molecule. Using transfection systems in which dominant negative forms of the SHIP-associated adapters, Grb2 and Shc, were overexpressed we demonstrate that, while SHIP is capable of a direct interaction in vitro with the ITAM of Fc{gamma}RIIa, the association of SHIP with Fc{gamma}RIIa in vivo is indirect and requires the presence of Shc. We extend these studies to provide evidence that SHIP activation serves to down-regulate gene transcription induced by the engagement of phagocytic Fc{gamma}R in human myeloid cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Abs, and reagents

THP-1 human monocytic cells and COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). P388D1 mouse macrophage cells stably expressing human Fc{gamma}RIIa were a kind gift from Dr. J. Edberg (University of Alabama, Birmingham, AL). P388D1 and COS-7 cells were maintained in DMEM supplemented with 10% FBS. THP-1 cells were maintained in RPMI supplemented with 10% FBS. F(ab')2 of anti-Fc{gamma}RI mAb 32.2 and Fab of anti-Fc{gamma}RIIa Ab IV.3 were obtained from Medarex (Annandale, NJ). Rabbit polyclonal anti-Shc, anti-extracellular signal-regulated kinase (Erk), and anti-phosphotyrosine Ab 4G10 were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-SHIP Ab was a generous gift from Dr. K. M. Coggeshall (Oklahoma Medical Research Foundation, Oklahoma City, OK). Anti-human CD32 and anti-mouse CD16/32 were purchased from BD PharMingen (San Diego, CA). The NF-{kappa}B-luciferase reporter plasmid was purchased from Stratagene (La Jolla, CA) and the plasmid pEGFP was purchased from Clontech Laboratories (Palo Alto, CA).

Culture of murine bone marrow-derived macrophages (BMM)

Strain-matched wild-type and Fc{gamma}RII-deficient mice were purchased from Taconic Farms (Germantown, NY). BMM were derived as previously described (30). Briefly, bone marrow cells were cultured in RPMI containing 5% FBS and supplemented with 50 ng/ml CSF-1 for 5 days. The BMMs were dissociated from the plates with Cell Dissociation Buffer (Life Technologies, Rockville, MD) and activated by clustering Fc{gamma}R with mAb 2.4G2 (BD PharMingen) followed by F(ab')2 of goat anti-rat IgG secondary Ab (Pierce, Rockford IL).

Immunoprecipitation and Western blotting

Cell activation, immunoprecipitation, and Western blotting were performed as previously described (30). Briefly, THP-1 cells and transfected COS-7 cells were activated by clustering Fc{gamma}RI and or Fc{gamma}RIIa with mAb 32.2 and mAb IV.3 Fab followed by goat anti-mouse Ig (GAM) secondary Ab. Resting and activated cells were lysed in TN1 buffer (50 mM Tris (pH 8), 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 Ab of interest and protein G-agarose beads (Life Technologies) or GAM covalently linked to Sepharose, depending on the Ab. 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% bromophenol blue, and 5% 2-ME) for 5 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the Ab of interest and appropriate HRP-conjugated secondary Abs. The filters were then developed by ECL.

Immunoblot data quantitation

The ECL signal was quantitated using a scanner and a densitometry program (Scion Image; National Institutes of Health). To quantitate the phosphotyrosine signal in the activated samples, we first subtracted background, normalized the signal to the amount of precipitated protein, and plotted the values obtained by subtracting the value in unstimulated samples, as previously described (30).

Far Western assay

Far Western assays were performed as previously described (9). Briefly, immunoprecipitated proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then probed with 1 µM biotinylated, doubly phosphorylated ITAM peptide from Fc{gamma}RIIa (described in Ref. 9) or pITIM from Fc{gamma}RIIb (described in Ref. 28) overnight at 4°C. The blots were subsequently probed with HRP-conjugated streptavidin (Pierce) and developed by ECL. Bands were quantitated as described above.

Transfection of COS-7 cells

COS-7 cells were transfected as previously described (30). Briefly, cells were grown on culture dishes until they were 60–70% confluent. Two micrograms of cDNA for Fc{gamma}RI {alpha}-chain in pCEXV3 (kindly provided by Dr. J. Ravetch, Rockefeller University, New York, NY), 2 µg of {gamma} subunit cDNA in pSVL (a gift from Dr. J.-P. Kinet, Harvard Medical School, Boston, MA), 1 µg of SHIP cDNA in pcDNA3, 10 µg each of SHIP SH2 domain (kindly provided by Dr. K. M. Coggeshall), and Shc Y-F and Grb2 SH3 (a kind gift from Dr. B. Mayer, Harvard Medical School) mutant were mixed in various combinations with Lipofectamine 2000 reagent (Life Technologies). The DNA mix was added to cells in serum-free DMEM and incubated for 3 h at 37°C in a CO2 incubator. The medium was then replaced by DMEM supplemented with 10% FBS. The cells were harvested 48 h later and analyzed for expression of the transfected cDNAs by flow cytometry and Western blotting. Having ensured that the various transfectants expressed comparable levels of protein, we then examined SHIP phosphorylation in response to Fc{gamma}R clustering.

Transfection of THP-1 cells and luciferase assays

For analysis of SHIP influence on NF-{kappa}B transcriptional activity, THP-1 cells were transfected by electroporation (310 V, 950 uF; Gene Pulser II; Bio-Rad, Hercules, CA) with 20 µg of wild-type SHIP or SHIP SH2 domain in pCDNA3, 5 µg of NF-{kappa}B-luc plasmid, and 2 µg of pEGFP to normalize for transfection efficiency. Transfectants were harvested 24 h later and activated by clustering Fc{gamma}RI or Fc{gamma}RIIa by methods described above for 6 h at 37°C. The cells were lysed in 100 µl of cell culture lysis reagent (Promega, Madison, WI). Luciferase activity was measured using the Promega luciferase assay reagent. Data are represented as graphs indicating the fold induction of NF-{kappa}B activity in cells activated by clustering FcRs over those that were not activated. Data points are expressed as mean and SD of three independent experiments. Statistical analysis was performed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SHIP phosphorylation is induced by ITAM-Fc{gamma}R in the absence of Fc{gamma}RIIb

Our previous experiments indicated that clustering Fc{gamma}RIIa with mAb IV.3 Fab and GAM or Fc{gamma}RI with mAb 32.2 F(ab')2 and GAM induced SHIP phosphorylation (28). Subsequent work from our laboratory and others established that human monocytes and the monocyte-like cell lines THP-1 and U937 express functional Fc{gamma}RIIb (5, 6), raising the possibility that the SHIP phosphorylation observed in response to Fc{gamma}RIIa or Fc{gamma}RI clustering might be explained by the coligation of Fc{gamma}RIIb by contaminating intact IgG in the anti-Fc{gamma}R mAb preparations. Hence, we undertook to test whether ITAM-Fc{gamma}R can induce tyrosine phosphorylation of SHIP in the complete absence of Fc{gamma}RIIb. For this, we used three different approaches involving transiently transfected COS-7 cells, P388D1 mouse macrophage cells stably transfected to express human Fc{gamma}RIIa. and BMMs from Fc{gamma}RII-deficient mice.

First, to test whether Fc{gamma}RIIa can induce SHIP phosphorylation in the absence of Fc{gamma}RIIb, COS-7 cells, which lack endogenous expression of SHIP and FcRs, were transiently transfected to express a wild-type (Fig. 1GoA, lanes 3 and 4) or a tailless version Fc{gamma}RIIa (lanes 5 and 6) along with SHIP. Forty-eight hours after transfection cells were harvested and activated by clustering Fc{gamma}RIIa with IV.3 Fab and GAM. SHIP was immunoprecipitated from resting and activated cells and analyzed for tyrosine phosphorylation by Western blotting with anti-phosphotyrosine Abs. As the results indicate (Fig. 1GoA, upper panel), SHIP phosphorylation was induced by Fc{gamma}RIIa in a manner that is dependent on the expression of the ITAM present in the cytoplasmic tail of the receptor (lane 4 but not lane 6). The membrane was reprobed with anti-SHIP Ab to ensure comparable levels of SHIP expression and to affirm that the phosphorylation signal observed was not due to a difference in amounts of protein loaded in the different lanes. In additional experiments, P388D1 mouse macrophage cells stably transfected with human Fc{gamma}RIIa were activated by clustering Fc{gamma}RIIa. SHIP phosphorylation was assessed by Western blotting. As seen in Fig. 1GoB, upper panel, SHIP is tyrosine phosphorylated in cells activated by clustering Fc{gamma}RIIa (lane 2). Shown in Fig. 1GoB, lower panel, is a reprobe with anti-SHIP Ab to indicate equal loading of SHIP in all lanes.



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  		FIGURE 1. SHIP phosphorylation is induced by ITAM-Fc{gamma}R in transfected cells. A, Transiently transfected COS-7 cells were activated by clustering Fc{gamma}RIIa with IV.3 Fab and GAM for 3 min, followed by immunoprecipitation with anti-SHIP Ab. Tyrosine phosphorylation of SHIP was assessed by immunoblotting with anti-phosphotyrosine Ab (upper panel). The membrane was reprobed with anti-SHIP Ab to ensure equal loading (lower panel). B, P388D1 mouse macrophage cell line stably expressing Fc{gamma}RIIa was activated by clustering Fc{gamma}RIIa as indicated above, and SHIP phosphorylation was assessed. C, COS-7 cells were transiently transfected to express the proteins indicated. Cells were activated by clustering Fc{gamma}RI with 32.2 F(ab')2 and GAM, and phosphorylation of SHIP was assessed as above. These data are representative of three independent experiments. Molecular weights are indicated to the left of the panels.

 
Second, to assess whether Fc{gamma}RI can induce SHIP phosphorylation in the absence of Fc{gamma}RIIb, COS-7 cells were transiently transfected to express Fc{gamma}RI {alpha}-chain and SHIP (Fig. 1GoC, lanes 1 and 2) or Fc{gamma}RI {alpha}-chain, the {gamma} subunit, and SHIP (lanes 3 and 4). Cells were activated by clustering Fc{gamma}RI with F(ab')2 of mAb 32.2 and GAM, followed by immunoprecipitation with anti-SHIP Ab. SHIP phosphorylation was assessed as described above. Results indicated that indeed Fc{gamma}RI could induce SHIP phosphorylation when the ITAM-bearing {gamma} subunit was coexpressed (lane 3), but not when the Fc{gamma}RI {alpha}-chain was expressed alone (lane 2).

Third, we assessed the ability of murine ITAM-Fc{gamma}R to induce SHIP phosphorylation in the absence of Fc{gamma}RIIb. For this, BMMs derived from wild-type and Fc{gamma}RII-deficient mice were activated by clustering Fc{gamma}R with the mAb 2.4G2 that recognizes equally well the ITAM-bearing Fc{gamma}RIIIa and the ITIM-bearing Fc{gamma}RII. SHIP phosphorylation was assessed by Western blotting with anti-phosphotyrosine Ab (Fig. 2Go, upper panel). Results indicated that, while SHIP phosphorylation was maximally induced in cells expressing Fc{gamma}RII (Fig. 2Go, lane 2), Fc{gamma}RIIIa alone induced measurable tyrosine phosphorylation of SHIP (Fig. 2Go, lane 4). These data collectively provide compelling evidence that ITAM-Fc{gamma}R are capable of inducing SHIP phosphorylation in vivo.



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  		FIGURE 2. SHIP phosphorylation is induced by the ITAM-associated Fc{gamma}RIIIa in Fc{gamma}RII-deficient mice. BMMs derived from wild-type and Fc{gamma}RII-deficient mice were activated by clustering Fc{gamma}RIIIa/II with mAb 2.4G2 and mouse anti-rat IgG secondary Ab. SHIP proteins were immunoprecipitated from resting and activated cells and assayed for tyrosine phosphorylation by immunoblotting with anti-phosphotyrosine Ab (upper panel). The membrane was reprobed with anti-SHIP Ab to ensure equal loading in all lanes (middle panel). Phosphorylation of SHIP is expressed as fold increase over that seen in resting cells (lower panel). These results are representative of two independent experiments.

 
SHIP coimmunoprecipitates with endogenous Fc{gamma}RIIa

We and others have earlier reported that SHIP is capable of associating in vitro with phosphorylated ITAM peptides derived from Fc{gamma}RIIa (28), the {gamma} subunit of Fc{epsilon}RI, Fc{gamma}RI, and IIIa, and the {zeta} subunit of TCR (31). To test whether SHIP could associate in vivo with an ITAM, we performed coimmunoprecipitation experiments in THP-1 cells, testing here the ability of endogenous SHIP to interact with endogenous Fc{gamma}RIIa. For this, Fc{gamma}RIIa was immunoprecipitated from resting (Fig. 3GoA, upper panel, lane 1) and activated (lane 2) THP-1 cells that were activated by clustering Fc{gamma}RIIa receptors. Fc{gamma}RIIa immunoprecipitates were analyzed for the presence of SHIP in the resultant immune complex by Western blotting with anti-SHIP Ab. As seen in Fig. 3GoA, SHIP protein was detectable in Fc{gamma}RIIa immunoprecipitates only upon activation of the cells (lane 2). The same membrane was reprobed for Fc{gamma}RIIa (Fig. 3GoA, lower panel).



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  		FIGURE 3. SHIP associates with Fc{gamma}RIIa in vivo. A, Fc{gamma}RIIa was immunoprecipitated from resting and activated THP-1 cells that were activated by clustering Fc{gamma}RIIa (lanes 1 and 2). The membrane was probed with anti-SHIP Ab (upper panel) and subsequently with anti-Fc{gamma}RIIa Ab (lower panel). B, COS-7 cells were either transfected with vector alone or transfected with plasmids encoding Fc{gamma}RIIa and SHIP. Cells were activated by clustering Fc{gamma}RIIa followed by immunoprecipitation of Fc{gamma}RIIa. The membrane was probed with anti-phosphotyrosine (upper panel). The same membrane was subsequently reprobed with anti-SHIP (middle panel), and with Ab260 to detect the presence of Fc{gamma}RIIa in the immunoprecipitates (lower panel).

 
As an additional approach to determine whether SHIP-Fc{gamma}RIIa interaction occurs in vivo, COS-7 cells were transfected with empty vectors (Fig. 3GoB, upper panel, lanes 1 and 2) or with plasmids encoding Fc{gamma}RIIa and SHIP (lanes 3 and 4). Cells were activated by clustering Fc{gamma}RIIa (lanes 2 and 4). Fc{gamma}RIIa was immunoprecipitated and analyzed for association with SHIP by Western blotting with anti-phosphotyrosine Ab. As seen in Fig. 3GoB, upper panel, receptor clustering induced phosphorylation of Fc{gamma}RIIa and association with p145 SHIP. The membrane was subsequently reprobed with anti-SHIP (middle panel) and anti-Fc{gamma}RIIa (lower panel).

SHIP is capable of direct association with Fc{gamma}RIIa ITAM in vitro

To assess whether the association of SHIP with Fc{gamma}RIIa is direct or is mediated by an adapter protein we first performed a far Western analysis applying a phosphorylated Fc{gamma}RIIa ITAM peptide (pITAM) to SHIP immunoprecipitates on nitrocellulose membrane. As the results indicate (Fig. 4GoA, upper panel), pITAM bound SHIP (lane 1), Shc, and the p85 subunit of phosphatidylinositol 3-kinase (lanes 2 and 3) but not Grb2 (lane 4). The membrane was reprobed with anti-Grb2 Abs to indicate that Grb2 was indeed present on the membrane (lower panel, lanes 1 and 4) but the ITAM peptide was incapable of binding to it. This result is consistent with previous reports indicating that immunoreceptors, unlike growth factor receptors, do not bind Grb2 directly; rather, they do so through Shc (32). The anti-Grb2 reactive band in lane 1 indicates that Grb2 is present in the SHIP immunoprecipitate as previously reported (reviewed in Ref. 33). A parallel membrane probed with a phosphorylated ITIM peptide (pITIM) derived from Fc{gamma}RIIb indicated that the ITIM was capable of strong binding to SHIP but not with Shc, p85, or Grb2 (Fig. 4GoB). However, it is noteworthy that the direct interaction of pITAM with SHIP was severalfold weaker than that of pITIM. In contrast, the interaction of pITAM with Shc and p85 was much stronger than the interaction of pITAM-SHIP. These results prompted us to investigate the possibility that the in vivo association of SHIP with Fc{gamma}RIIa might be facilitated via adapter molecules rather than by a direct interaction between SHIP and the receptor.



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  		FIGURE 4. SHIP is capable of direct association with Fc{gamma}RIIa ITAM peptide. SHIP, Shc, p85, Grb2, and normal rabbit Ig immunoprecipitates from THP-1 cells were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with either a phosphorylated Fc{gamma}RIIa ITAM peptide (A, upper panel) or a phosphorylated Fc{gamma}RIIb ITIM peptide (B). The membrane in A was reprobed with anti-Grb2 Ab (A, lower panel).

 
Dominant negative Shc but not Grb2 attenuates SHIP tyrosine phosphorylation by Fc{gamma}RIIa

SHIP is reported to associate with at least two adapter molecules: Shc and Grb2 (reviewed in Ref. 33). The interaction of SHIP with Grb2 is constitutive and occurs via the Grb2 SH3 domain and the C-terminal proline-rich domain of SHIP. In contrast, the association of SHIP with Shc is activation induced, requiring tyrosine phosphorylation events and involving multiple interaction domains (34). Studies in B cells indicated that the SHIP SH2 domain can interact with the phosphotyrosines of Shc and in turn the Shc phosphotyrosine binding domain can interact with the phosphotyrosines of SHIP (14). To test whether the in vivo interaction of SHIP with Fc{gamma}RIIa is direct or whether it occurs via adapter proteins such as Shc and Grb2, we used a transient transfection system. Here we transfected into COS-7 cells plasmids encoding Fc{gamma}RIIa alone (Fig. 5GoA, lanes 1 and 2), Fc{gamma}RIIa and SHIP (lanes 3 and 4), Fc{gamma}RIIa, SHIP and a dominant negative Grb2 that is mutated in its 2 SH3 domains (lanes 5 and 6), or with Fc{gamma}RIIa, SHIP, and a dominant negative mutant of Shc (lanes 7 and 8). The Shc mutant has all three phosphorylatable tyrosine residues (Y239, Y240, and Y317) replaced by phenylalanine. Empty vectors were used to equalize amounts of DNA used for transfection. Transfected cells were harvested 48 h later and SHIP proteins were immunoprecipitated from resting cells and cells activated by clustering Fc{gamma}RIIa. Western blotting with anti-phosphotyrosine Ab (Fig. 5GoA, upper panel) indicated that SHIP phosphorylation is attenuated in cells overexpressing a dominant negative Shc (lane 8) but not in cells expressing dominant negative Grb2 (lane 6). These results are representative of four independent experiments. The membrane was reprobed with anti-SHIP Ab to ensure equal loading of SHIP in all lanes (lower panel). To demonstrate that the dominant negative Grb2 was indeed functional we analyzed Erk activation in the transfected COS-7 cells. As seen in Fig. 5GoB, Erk is heavily activated in these cells, as indicated by the gel shift, in all cases except when the mutant Grb2 is overexpressed. The activation of Erk seen here is constitutive (likely induced by growth factors in the serum) and is independent of Fc{gamma}RIIa clustering. Fig. 5GoC shows equal expression of Fc{gamma}RIIa in all transfectants and Fig. 5GoD demonstrates overexpression of transfected Shc. We conclude, based on the above data, that SHIP phosphorylation by Fc{gamma}RIIa requires Shc, suggesting that the association of SHIP with Fc{gamma}RIIa is via Shc.



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  		FIGURE 5. Overexpression of dominant negative Shc but not Grb2 attenuates SHIP phosphorylation by Fc{gamma}RIIa. COS-7 cells were transfected to express the proteins indicated. Forty-eight hours after transfection, cells were activated by clustering Fc{gamma}RIIa. A, SHIP proteins were immunoprecipitated and analyzed by Western blotting with anti-phosphotyrosine Ab (upper panel). The membrane was reprobed with anti-SHIP Ab (middle panel). SHIP phosphotyrosine signals were quantitated and expressed as fold increase of the activated cells over the phosphorylation in resting cells (lower panel). The graph represents the mean and SD from the means of two independent experiments. B, Whole cell lysates (WCL) from the same samples as above were probed with anti-Erk Ab to demonstrate the dominant negative effect of the transfected Grb2 mutant on the Ras signaling pathway in lanes 5 and 6. C, Whole cell lysates (WCL) from the transfectants were probed with anti-Fc{gamma}RIIa Ab to ensure expression of the transfected receptor. D, Whole cell lysates (WCL) were probed with anti-Shc Ab to demonstrate overexpression of Shc.

 
SHIP SH2 domain is necessary for SHIP phosphorylation by Fc{gamma}RIIa

The above data suggest a model in which Shc associates with the phosphorylated ITAM of Fc{gamma}RIIa, becomes phosphorylated on tyrosine residues, and serves as a docking site for SHIP SH2. The SHIP proteins that are thus transported to the membrane are then phosphorylated by the membrane-associated Src kinases, as previously reported (35). To test whether SHIP SH2 domain is involved in the translocation of SHIP to the receptor and the subsequent tyrosine phosphorylation of SHIP, COS-7 cells were transiently transfected to express SHIP alone (Fig. 6GoA, lanes 3 and 4), Fc{gamma}RIIa and SHIP (lanes 5 and 6), or Fc{gamma}RIIa, SHIP, and excess of SHIP SH2 domain (lanes 7 and 8). SHIP phosphorylation in response to Fc{gamma}RIIa clustering was assessed in the transfectants by Western blotting. Consistent with our model, overexpression of SHIP SH2 domain attenuated Fc{gamma}RIIa-induced SHIP phosphorylation (Fig. 6GoA, lane 8). The membrane was reprobed with anti-SHIP Ab to ensure that the lack of phosphorylation in lane 8 was not due to differences in the amount of SHIP protein present in the resting and activated lanes (Fig. 6GoA, lower panel). To exclude the possibility that the lack of SHIP phosphorylation in the SHIP SH2 overexpressing cells is not due to a lack of Fc{gamma}RIIa expression, whole cell lysates from the transfectants were analyzed by Western blotting for the presence of transfected Fc{gamma}RIIa (Fig. 6GoB). Results indicate that Fc{gamma}RIIa is indeed expressed in all cells transfected with a plasmid encoding Fc{gamma}RIIa (lanes 3 and 4, but not lanes 1 and 2); therefore, the lack of SHIP phosphorylation upon receptor clustering could be due only to the dominant negative effect of the overexpressed SHIP SH2 domain.



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  		FIGURE 6. Overexpression of SHIP SH2 abrogates SHIP phosphorylation by Fc{gamma}RIIa. COS-7 cells were transfected to express the proteins indicated. Cells were harvested 48 h posttransfection and activated by clustering Fc{gamma}RIIa. A, SHIP proteins were immunoprecipitated and analyzed by Western blotting with anti-phosphotyrosine Ab (upper panel). The membrane was reprobed with anti-SHIP Ab (middle panel). SHIP phosphotyrosine signals were quantitated and expressed as fold increase of the activated cells over the phosphorylation in resting cells (lower panel). The graph represents the mean and SD from the means of two independent experiments. B, Whole cell lysates (WCL) from the transfectants were probed with anti-Fc{gamma}RIIa Ab to ensure expression of the transfected receptor. The numbers below the panel indicate densitometry measurement of band intensity.

 
SHIP regulates NF-{kappa}B induction by ITAM-Fc{gamma}R

The functional consequence of SHIP activation by phagocytic Fc{gamma}R is not clear. Because inflammatory cytokine genes, such as IL-1, IL-8, and TNF-{alpha}, that are induced during phagocytosis require the activation of NF-{kappa}B (36), we asked whether SHIP activation would regulate NF-{kappa}B-dependent gene transcription by phagocytic FcRs. In these experiments we analyzed NF-{kappa}B-dependent transcription from a reporter plasmid encoding the luciferase gene in the presence of excess of wild-type SHIP or the dominant negative SHIP SH2 domain that abrogates association of SHIP with the ITAM, as we have demonstrated above. Thus, THP-1 cells were transiently cotransfected with NF-{kappa}B-luc plasmid and with plasmids encoding wild-type SHIP or SHIP SH2 domain. The transfected cells were activated by clustering either Fc{gamma}RI (Fig. 7GoA) or Fc{gamma}RIIa (Fig. 7GoB) for 6 h at 37°C by methods described above. Transcription of the luciferase gene was measured by a luciferase enzyme assay, as previously described (36). Results are expressed as fold increase in luciferase activity in cells activated by clustering FcRs over the activity in resting cells. Data indicate that NF-{kappa}B-dependent transcription of the luciferase gene occurs upon FcR clustering (~3-fold induction). However, overexpression of wild-type SHIP blocks the induction of gene transcription. Importantly, gene transcription is greatly enhanced in the presence of a dominant negative SHIP SH2 domain (~6-fold induction), suggesting that SHIP serves to down-regulate NF-{kappa}B-dependent gene transcription by phagocytic Fc{gamma}R.



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  		FIGURE 7. SHIP down-regulates NF-{kappa}B-dependent gene transcription mediated by phagocytic Fc{gamma}R. THP-1 cells were transfected with the plasmids indicated. Transfectants were activated by clustering either Fc{gamma}RI or Fc{gamma}RIIa with mAb 32.2 F(ab')2 plus GAM or IV.3 Fab plus GAM, respectively, for 6 h. Cells were then lysed and analyzed for luciferase activity. Data are expressed as fold increase of luciferase activity in the activated cells over that in resting cells. The graphs represent the mean and SD of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IgG-coated infectious particles engage both activating and inhibitory Fc{gamma}R expressed on the cell surface of macrophages/monocytes, leading to the activation of multiple signaling pathways that result in the ingesting, transport to the lysosome, and subsequent degradation of the infectious particle by lysosomal enzymes. Because the process of phagocytosis results in the production of tissue-damaging products such as reactive oxygen and nitrogen species, inflammatory cytokines, and lysosomal enzymes, a tight control on the magnitude of the phagocytic response is essential. Regulation of Fc{gamma}R-mediated phagocytosis is achieved at several levels. First, the ratio of activating and inhibiting Fc{gamma}R on the cell surface determines the magnitude of the phagocytic response. Thus, macrophages from mice deficient in Fc{gamma}RIIb expression display enhanced phagocytic capability in comparison to wild-type macrophages (25). Likewise, monocytes cultured in the presence of IL-4, which express more Fc{gamma}RIIb, are less efficient at phagocytosing IgG-coated particles than monocytes cultured in the absence of IL-4 (5, 6). Second, the expression and activation of SHIP influences phagocytic efficiency. Macrophages derived from SHIP-deficient mice are better able to phagocytose IgG-coated particles than are their wild-type counterparts (7). Because SHIP activation has been demonstrated to be mediated not only by the ITIM-bearing Fc{gamma}RIIb but also by ITAM-bearing receptors, it is a fascinating possibility that the levels of SHIP at the cell membrane are highly variable, thus providing a finely tuned regulatory mechanism for cellular responses such as phagocytosis.

The molecular mechanisms leading to the association of SHIP with the phosphorylated ITIM of Fc{gamma}RIIb and the subsequent phosphorylation of SHIP are well characterized (37, 38). SHIP is a constitutively active cytosolic enzyme that requires membrane translocation to gain access to its lipid substrates (35). Recruitment of SHIP to the membrane is also obligatory for tyrosine phosphorylation of SHIP. Thus, tyrosine phosphorylation of SHIP serves as a good indicator of membrane translocation of the enzyme. We and others have previously reported that SHIP phosphorylation is achieved when ITAM-Fc{gamma}R are clustered on monocytes. However, the molecular details of SHIP phosphorylation by activating receptors are poorly understood.

The results of this study indicate that SHIP phosphorylation by the ITAM-associated Fc{gamma}RIIa requires Shc phosphorylation, leading to the model presented in Fig. 8Go. Thus, clustering of Fc{gamma}RIIa leads to the activation of Src kinases that phosphorylate the tyrosines in the ITAM of the receptor. The phosphorylated ITAM serves as a binding site for the SH2 domain of Shc, which is itself subsequently phosphorylated on tyrosine residues. The direct association of Shc with ITAMs has previously been reported (32). The phosphotyrosine residues of Shc engage the SHIP SH2 domain, thus bringing SHIP to the membrane, where SHIP is phosphorylated by the membrane-associated Src kinases (35). Our previous findings that a phosphorylated ITAM peptide from Fc{gamma}RIIa is able to associate with SHIP in resting cell lysates where Shc is not tyrosine phosphorylated (28), as well as the Far Western analysis presented in Fig. 4Go, suggest a possible direct interaction of SHIP with Fc{gamma}RIIa ITAM. However, when this latter model was tested in an in vivo assay, presented in Fig. 5Go, it became very apparent that a direct interaction between SHIP and the Fc{gamma}RIIa ITAM is unlikely to occur in vivo. The latter may be due to the presence of other SH2 domain-containing molecules in the cell lysate that have a higher affinity for the ITAM than the SHIP SH2. Were it possible for SHIP to interact directly with Fc{gamma}RIIa, the overexpression of the Shc mutant would have had no influence on Fc{gamma}RIIa-induced SHIP phosphorylation; i.e., phosphorylation of SHIP in Fig. 5GoA, upper panel, lane 8, would have paralleled the phosphorylation of SHIP seen in lanes 4 and 6. A third potential model would present the following scenario: Shc associates with phosphorylated ITAM of Fc{gamma}RIIa, Shc becomes phosphorylated, and Grb2 SH2 domain associates with phospho-Shc and brings along SHIP, which is attached to the Grb2 SH3 domain. This last model is again unlikely to account for SHIP phosphorylation by Fc{gamma}RIIa, because a dominant negative double SH3 Grb2 mutant failed to influence SHIP phosphorylation (Fig. 5GoA, lane 6).



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  		FIGURE 8. Model for SHIP association with the Fc{gamma}RIIa ITAM. Fc{gamma}RIIa clustering leads to the activation of Src kinases that phosphorylate the ITAM in the receptor. Shc associates with the phosphorylated ITAM via the Shc SH2 domain and is subsequently phosphorylated on tyrosines 239, 240, and 317. The phosphotyrosines of Shc serve to dock the SHIP SH2 domain. The SHIP molecule that is thus localized in the membrane is phosphorylated by the Src kinases.

 
Although the model presented in Fig. 8Go accounts for our current observations, there are several intriguing questions about the signaling pathways that arise from this work that are likely to reveal novel mechanisms used for regulation of cell activation. For example, studies from our group and others previously reported that the SHIP SH2 domain and the Grb2 SH2 domain compete for the same phosphotyrosine residues on Shc (34, 39); however, surface plasmon resonance analysis revealed that the affinity of the Grb2 SH2 domain for phospho-Shc is higher than the affinity of SHIP SH2 domain for phospho-Shc (23). The higher affinity of Grb2 SH2 for phospho-Shc explains our earlier findings that SHIP phosphorylation achieved by clustering ITAM-Fc{gamma}R is severalfold lower than the SHIP phosphorylation that occurs when ITAM-Fc{gamma}R are coclustered with Fc{gamma}RIIB. However, it is not clear why the Grb2 molecules do not serve to adapt SHIP to Shc. Grb2 is reported to associate with a number of proline-rich domain-containing proteins such as the Ras guanine exchange factor Sos, SHIP, and Cbl, via the Grb2 SH3 domain. It is possible that the affinity of Grb2 SH3 binding with SHIP is not strong enough to compete with the other Grb2-binding partners. Alternately, it is possible that the numbers of molecules of SHIP are far fewer than the other Grb2-binding proteins. Further analysis is required to resolve these issues.

Based on the model shown in Fig. 8Go, we have interrupted SHIP function with a dominant negative SHIP SH2 domain in transiently transfected THP-1 cells and analyzed the outcome of SHIP function on Fc{gamma}R-mediated outcome. Our results indicate that SHIP regulates NF-{kappa}B-mediated gene transcription. These results are consistent with a recent report by Kalesnikoff et al. (40), which demonstrated a regulatory role for SHIP in IgE-mediated IL-6 production by inhibiting NF-{kappa}B activation. SHIP hydrolysis of phosphatidylinositol 3,4,5 trisphosphate leads to the down-regulation of a number of signaling pathways, including the activation of Akt, Erk, and p38, all of which are implicated in the activation of NF-{kappa}B (40).

In conclusion, in a study aimed at understanding regulation of Fc{gamma}R-mediated activation, we demonstrate here that ITAM-Fc{gamma}R are capable of inducing SHIP phosphorylation in myeloid cells. We have dissected the molecular events that lead to SHIP tyrosine phosphorylation by Fc{gamma}RIIa and report that the adapter molecule Shc, but not Grb2, is necessary for phosphorylation of SHIP by Fc{gamma}RIIa. These studies are the first to demonstrate that SHIP associates with and is recruited by native ITAM-bearing receptors independently of Fc{gamma}RIIb involvement. The functional consequence of SHIP activation by ITAM-Fc{gamma}R is not known. Using the information gained from the above experiments we have analyzed the influence of SHIP on gene transcription induced by phagocytic Fc{gamma}R under conditions that preclude the activation of SHIP by these receptors. Our data suggest that SHIP serves to down-regulate NF-{kappa}B-dependent gene transcription during the phagocytic process, thus providing a mechanism to limit the inflammatory processes associated with phagocytosis.


   Acknowledgments
 
We are grateful to Drs. Jeffrey Ravetch, Jean-Pierre Kinet, and Bruce Mayer for kindly providing the plasmids used in this work and Dr. Jeffrey Edberg for the P388D1 cells. We thank Dr. K. Mark Coggeshall for the many useful discussions on the subject and for his generosity with the reagents used in this study.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants AI50074-01A1, CA44983, HD35121, and HO38764. S.T. is supported by a Fellow Award from the Leukemia and Lymphoma Society of America. Back

2 Address correspondence and reprint requests to Dr. Susheela Tridandapani, Department of Internal Medicine, Ohio State University, Heart and Lung Research Institute, Room 405D, 473 West Twelfth Avenue, Columbus, OH 43210. E-mail address: tridandapani.2{at}osu.edu Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; SH, Src homology; GAM, goat anti-mouse Ig; SHIP, SH2 domain-containing inositol polyphosphate phosphatase; Erk, extracellular signal-regulated kinase; BMM, bone marrow-derived macrophage. Back

Received for publication April 22, 2002. Accepted for publication August 9, 2002.


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