From the
Molecular, Cellular, and Developmental Biology Program and the ¶Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Dorothy M. Davis Heart and Lung Institute, James Cancer Hospital and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210
Received for publication, March 21, 2003 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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Human monocytes and macrophages express three classes of FcR (2). Fc
RI, Fc
RIIa, and Fc
RIII are activating receptors that are associated with an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Fc
RIIb is an inhibitory receptor that bears in its cytoplasmic tail a tyrosine-based inhibitory motif that predominantly recruits negative regulatory phosphatases and causes down-regulation of activation events. In addition to the regulatory role of Fc
RIIb, recent studies have revealed that the Fc
R ITAMs are often capable of simultaneously activating positive and negative regulatory proteins such that the final biologic response is tempered. We (3) and others (4) have demonstrated that Fc
R ITAMs are capable of recruiting and activating the inositol phosphatase SHIP-1. Other studies have shown that the protein-tyrosine phosphatase SHP-1 (5) and the dual phosphatase PTEN (6) also serve to regulate Fc
R-mediated macrophage functions. Thus, it is clear that Fc
R-mediated activation of macrophages is subject to multiple levels of regulation, which are not fully understood. In this report, we present molecular details of a novel regulatory influence exerted by the recently identified inositol phosphatase SHIP-2.
FcR clustering in monocytes and macrophages initiates a biochemical cascade that begins with the activation of the Src kinases that phosphorylate the ITAMs of Fc
R (7). Once phosphorylated, the ITAMs serve as docking sites for several signaling enzymes and enzyme-adapter complexes, including the Syk tyrosine kinase (8); the p85 subunit of phosphatidylinositol 3-kinase (9); and the Ras adapter molecule Shc (3, 10), which serves to recruit the Grb2-Sos complex, leading to activation of the Ras pathway. Association of phosphatidylinositol 3-kinase with the ITAM places the enzyme in proximity with its lipid substrates, resulting in the generation of the important lipid second messenger PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 is critical for the activation of PH domain-containing enzymes such as Btk (11); the Tec family tyrosine kinase involved in intracellular calcium mobilization, Vav (12); the guanine nucleotide exchange factor for Rac; and Akt, the serine/threonine kinase that is involved in cell survival (13, 14) and in the activation of NF
B (15). The hydrolysis of PtdIns-3,4,5-P3 by SHIP-1 has been demonstrated to down-regulate the activity of the above PH domain-containing enzymes and the downstream functional outcomes (11, 13, 14).
SHIP-2 is an inositol 5'-phosphatase that has high level homology to SHIP-1 in its catalytic region (1618). The molecules are largely divergent in the C-terminal region, consisting of a proline-rich domain that associates with unique SH3 domain-containing proteins (19). In addition, whereas SHIP-1 has two tyrosine residues in the C terminus that conform to an NPXY motif shown to bind phosphotyrosine-binding domains upon phosphorylation, SHIP-2 has only one NPXY motif (20). Thus, while enzymatically similar, these two enzymes likely differ in functions that are related to their protein interactions via the C-terminal region. These two molecules also differ in their expression patterns: SHIP-1 is expressed predominantly in hematopoietic cells, whereas SHIP-2 is much more ubiquitously expressed (20). Recent studies have revealed a role for SHIP-2 in regulating insulin receptor signaling (2123). Other studies have demonstrated a role for SHIP-2 in mediating the inhibitory effect of FcRIIb in B cells (21, 24, 25). However, the expression and function of SHIP-2 in monocytes and macrophages are not known.
In this study, we first demonstrate that SHIP-2 was expressed in human alveolar macrophages, but was almost undetectable in peripheral blood monocytes derived from the same donors. Interestingly, expression of SHIP-2 in PBMs was induced by bacterial LPS in a dose-dependent manner. Second, FcRIIa clustering in the human myeloid cell line THP-1 induced tyrosine phosphorylation of SHIP-2, suggesting that SHIP-2 may play a role in Fc
R-mediated function. Analyzing the functional consequence of SHIP-2 in Fc
RIIa signaling, we report that overexpression of wild-type SHIP-2 (but not catalytically deficient SHIP-2) completely abrogated NF
B-dependent gene transcription in response to Fc
RIIa clustering in THP-1 cells. Transient cotransfection experiments in COS-7 cells demonstrated that wild-type SHIP-2 down-regulated Fc
RIIa-induced Akt phosphorylation. Additional experiments analyzing the molecular mechanism of SHIP-2 activation by Fc
RIIa demonstrated that the SH2 domain of SHIP-2 was necessary for optimal association of SHIP-2 with Fc
RIIa and for optimal tyrosine phosphorylation of SHIP-2. Finally, we also demonstrate that Fc
RI clustering resulted in phosphorylation of SHIP-2, suggesting that SHIP-2 may regulate both Fc
RIIa- and Fc
RI-mediated myeloid cell function.
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EXPERIMENTAL PROCEDURES |
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Isolation of PBMsCD14-positive PBMs were isolated as described previously (26). Briefly, peripheral blood mononuclear cells were first isolated by density gradient centrifugation over Histopaque (Sigma). Monocytes were then purified from the peripheral blood mononuclear cells by negative selection using the MACs monocyte isolation kit (Miltenyi Biotech). For this, peripheral blood mononuclear cells were first treated with FcR blocking reagent (human IgG), followed by a hapten/antibody mixture (mixture of hapten-conjugated monoclonal anti-CD3, anti-CD7, anti-CD19, anti-CD45RA, anti-CD56, and anti-IgE antibodies). The labeled cells were further treated with MACS anti-hapten magnetic microbeads conjugated to monoclonal anti-hapten antibody. The cells were then passed over a MACS column, and the effluent was collected as the negative fraction representing enriched monocytes.
Preparation of Human Alveolar MacrophagesMacrophages were obtained from healthy donors by bronchoalveolar lavage. Cells were washed twice with phosphate-buffered saline, counted, and analyzed by Diff-Quick staining for purity. Cell preparations were >95% positive for macrophages.
Immunoprecipitation and Western BlottingTHP-1 cells and transfected COS-7 cells were activated by clustering FcRI and or Fc
RIIa with monoclonal antibody 197 or Fab fragments of antibody IV.3 and goat F(ab')2 anti-mouse Ig secondary antibody. Resting and activated cells were lysed in buffer A (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, and 10 µg/ml each aprotinin and leupeptin), and post-nuclear lysates were incubated overnight with the antibody of interest and protein G-agarose beads (Invitrogen) or goat F(ab')2 anti-mouse Ig covalently linked to Sepharose, depending on the antibody. Immune complexes bound to beads were washed with buffer A and boiled in SDS sample buffer (60 mM Tris (pH 6.8), 2.3% SDS, 10% glycerol, 0.01% bromphenol blue, and 2% 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.
Immunoblot Data QuantitationThe ECL signal was quantitated using a scanner and a densitometry program (Scion Image). To quantitate the phosphorylation signals in the activated samples, we first subtracted background, normalized the phosphorylation signal to the amount of total precipitated protein, and plotted the values obtained by expressing them as -fold increase over the values in unstimulated samples.
TransfectionCOS-7 cells were transfected as described previously (3). Briefly, cells were grown on culture dishes to 6070% confluency. cDNA for FcRIIa in pCEXV3 (kindly provided by Dr. J. Ravetch, Rockefeller University, New York) and cDNAs for wild-type SHIP-2, the inactive SH2 domain mutant of SHIP-2, and the inactive catalytic domain mutant of SHIP-2 in pcDNA3 (generously provided by Dr. Shonna Moodie, Metabolex Inc., Hayward, CA) (27) were mixed in various combinations 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 medium was then replaced with 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. Having ensured that the various transfectants expressed comparable levels of protein, we then examined the ability of the wild-type and mutant SHIP-2 molecules to associate with the Fc
RIIa ITAM and to become tyrosine-phosphorylated. In other experiments, GST-Akt (a kind gift from Dr. R. B. Pearson, Peter MacCallum Cancer Institute, Melbourne, Australia) (28) was cotransfected with the above molecules (2 µg of Fc
RIIa, 5 µg of SHIP-2, and 1 µg of GST-Akt) to analyze the influence of SHIP-2 on Akt activation by Fc
RIIa.
Transfection of THP-1 Cells and Luciferase AssaysFor analysis of SHIP-2 influence on NFB transcriptional activity, THP-1 cells were transfected as described previously (3). Briefly, THP-1 cells were electroporated (310 V, 950 microfarads; Bio-Rad Gene Pulser II) with 5 µg of wild-type SHIP-2 or catalytically deficient SHIP-2 in pcDNA3, 1 µgof NF
B-luciferase plasmid, and 0.5 µg of pEGFP to normalize for transfection efficiency. Transfectants were harvested 24 h later and activated by clustering Fc
RIIa by the 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 percent induction of NF
B activity in cells activated by clustering Fc
RIIa over the activity in cells that were not activated. Data points are expressed as the mean ± S.D. of three independent experiments. Statistical analysis was performed by Student's t test.
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RESULTS |
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SHIP-2 Is Tyrosine-phosphorylated and Serves to Down-regulate FcRIIa-mediated FunctionTo test whether SHIP-2 is involved in Fc
R-mediated myeloid cell activation, SHIP-2 phosphorylation was assessed in THP-1 cells stimulated by clustering Fc
RIIa (Fig. 2A). For this, Fc
RIIa receptors were clustered using Fab fragments of monoclonal anti-Fc
RIIa antibody IV.3, followed by F(ab')2 fragments of goat anti-mouse IgG. SHIP-2 was immunoprecipitated from resting and activated cells with goat polyclonal anti-SHIP-2 antibody and analyzed by Western blotting with anti-phosphotyrosine antibody. The results indicate that Fc
RIIa clustering induced SHIP-2 phosphorylation within 1 min and that the phosphorylation signals peaked at
7 min and subsided by 30 min post-stimulation (Fig. 2A, upper panel). A reprobe of the same membrane with anti-SHIP-2 antibody revealed equal loading of SHIP-2 in all lanes. The seventh lane is a negative control immunoprecipitation with normal goat IgG (Fig. 2A, lower panel). In a second set of experiments, human PBMs cultured overnight with 100 ng/ml LPS were stimulated by clustering Fc
RIIa, and SHIP-2 phosphorylation was assessed by Western blotting (Fig. 2B). As shown in Fig. 2B (upper panel), SHIP-2 phosphorylation was induced by Fc
RIIa clustering in PBMs. Fig. 2B (lower panel) demonstrates equivalent loading of SHIP-2 in the two lanes.
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Having ensured that SHIP-2 is phosphorylated during FcRIIa signaling, we next assessed whether SHIP-2 influences Fc
RIIa-mediated functional outcomes. We (3) and others (10) have previously reported that Fc
R clustering induces NF
B-mediated gene transcription, which is down-regulated by the inositol phosphatase SHIP-1. Because SHIP-1 and SHIP-2 are both capable of hydrolyzing PtdIns-3,4,5-P3 and influence downstream events, we analyzed whether SHIP-2 plays a role in regulating NF
B-mediated gene transcription. In these experiments, we analyzed NF
B-dependent transcription from a reporter plasmid encoding the luciferase gene in the presence of excess wild-type SHIP-2 or the catalytically deficient SHIP-2 mutant D608A. Thus, THP-1 cells were transiently cotransfected with the NF
B-luciferase plasmid and with plasmids encoding wild-type SHIP-2 or catalytically deficient SHIP-2. The transfected cells were activated by clustering Fc
RIIa for 5 h at 37 °C. Transcription of the luciferase gene was measured by a luciferase enzyme assay as described above. The results are expressed as percent increase in luciferase activity in cells activated by clustering Fc
RIIa over the activity in resting cells (Fig. 2C). The data indicate that NF
B-dependent transcription of the luciferase gene occurred upon Fc
RIIa clustering. However, overexpression of wild-type SHIP-2 blocked the induction of gene transcription (p = 0.027, presence of exogenous wild-type SHIP-2 versus pcDNA3 empty vector). Importantly, gene transcription was significantly enhanced in the presence of dominant-negative, catalytically deficient SHIP-2 (p = 0.006, presence of exogenous catalytically deficient SHIP-2 versus pcDNA3 empty vector), suggesting that SHIP-2 serves to down-regulate NF
B-dependent gene transcription by Fc
RIIa.
Recent studies have demonstrated an upstream role for Akt in NFB activation (15). Because Akt is a PH domain-containing enzyme whose activity is regulated by PtdIns-3,4,5-P3, a substrate of SHIP-2, we next analyzed Akt activation in the presence of overexpressed wild-type SHIP-2 or catalytically deficient SHIP-2. For these experiments, COS-7 fibroblasts were used to achieve high levels of transfection. Thus, COS-7 cells were transiently transfected to express epitope (GST)-tagged Akt along with Fc
RIIa (COS-7 cells do not express any endogenous Fc
R) and either wild-type SHIP-2 or the catalytically deficient SHIP-2 mutant D608A. The transfected cells were harvested 24 h later and activated by clustering Fc
RIIa by the methods described above. Whole cell lysates from resting and activated cells were separated by SDS-PAGE and analyzed by Western blotting with anti-phosphoserine Akt antibody (Fig. 3A, upper panel). GST-Akt migrated at
95 kDa in comparison with endogenous Akt, which migrated at 65 kDa. The slower migration of GST-Akt allowed for analysis of the effect of overexpression of the SHIP-2 proteins on cotransfected GST-Akt. The membrane was reprobed with anti-Akt antibody to detect the total amount of GST-Akt present in each lane (Fig. 3A, middle panel). Fig. 3A demonstrates that GST-Akt was serine-phosphorylated upon Fc
RIIa clustering (fourth lane) in comparison with the resting sample (third lane). Overexpression of wild-type SHIP-2 down-regulated Fc
RIIa-induced Akt phosphorylation (compare the fifth and sixth lanes). In contrast, overexpression of catalytically deficient SHIP-2 resulted in enhanced phosphorylation of Akt in response to Fc
RIIa clustering (eighth lane), suggesting that catalytically deficient SHIP-2 functions in a dominant-negative manner, overcoming the inhibition of Akt by endogenous SHIP-2 (fourth lane). Akt phosphorylation was quantitated by normalizing phospho-Akt (GST) signals to total Akt (GST) signals as described under "Experimental Procedures." The results are expressed as -fold increase in Akt phosphorylation in activated cells in comparison with that in resting cells. Fig. 3A (lower panel) represents the mean ± S.D. of values obtained from three independent experiments. The inhibition of Akt phosphorylation by wild-type SHIP-2 was statistically significant (p = 0.02). To ensure that transfected Fc
RIIa was expressed to comparable levels in the different transfectants, WCLs were probed with anti-Fc
RIIa antibody (Fig. 3B). Likewise, expression of Xpress-tagged wild-type and catalytically deficient SHIP-2 was comparable in the transfected cells as shown in Fig. 3C. These data suggest that SHIP-2 down-regulates Fc
RIIa-induced Akt phosphorylation.
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SHIP-2 Associates with the Phosphorylated ITAM of FcRIIaSHIP-2 is a cytoplasmic protein whose activity is regulated by subcellular localization, i.e. SHIP-2 must translocate to the membrane to access its lipid substrates (20). We therefore tested whether Fc
RIIa provides a docking site for SHIP-2 in the following experiments. First, we used biotinylated peptides derived from the ITAM of Fc
RIIa that were either non-phosphorylated or doubly phosphorylated at the tyrosine residues. These peptides were applied to THP-1 lysates, and the peptide-bound material was analyzed by Western blotting with anti-SHIP-2 antibody. As shown in Fig. 4A (upper panel), SHIP-2 associated with the doubly phosphorylated Fc
RIIa ITAM peptide, but not with the non-phosphorylated peptide. A WCL from THP-1 cells is shown in the third lane. To ensure that the binding of SHIP-2 to the phosphorylated ITAM peptide was specific, the same membrane was reprobed with anti-Syk antibody (Fig. 4A, lower panel). As previously reported, the phosphorylated ITAM of Fc
RIIa bound Syk (9).
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In a second set of experiments, we investigated whether native FcRIIa associates with SHIP-2 upon receptor clustering. Here, THP-1 cells were activated by clustering Fc
RIIa for 5 min. Fc
RIIa was immunoprecipitated from resting and activated cells, separated by SDS-PAGE, and analyzed for the presence of SHIP-2 by Western blotting. The results show that SHIP-2 was capable of associating with Fc
RIIa in an activation-dependent manner (Fig. 4B, upper panel). A reprobe of the same membrane indicated equal loading of Fc
RIIa in both resting and activated samples. Taken together, the results suggest that SHIP-2 translocates to the membrane upon Fc
RIIa clustering by associating with the phosphorylated ITAM of Fc
RIIa.
The SH2 Domain of SHIP-2 Is Required for Association with the FcRIIa ITAMThe above finding that SHIP-2 associates with the phosphorylated ITAM of Fc
RIIa suggests that the interaction may be mediated via the SH2 domain of SHIP-2. To formally test this possibility, COS-7 fibroblasts were transfected to express epitope (Xpress)-tagged wild-type SHIP-2, R47L SHIP-2 (SH2 domain mutant), or D608A SHIP-2 (catalytically deficient mutant). The tagged SHIP-2 proteins were assessed for their ability to bind the Fc
RIIa ITAM in a peptide binding assay. Thus, the peptide-bound material was separated by SDS-PAGE and immunoblotted with anti-Xpress antibody. The results indicate that the SH2 domain mutant of SHIP-2 was unable to associate optimally with the phosphopeptide (Fig. 5A, third lane). In contrast, both wild-type SHIP-2 and catalytically deficient SHIP-2 were able to efficiently bind the phosphorylated ITAM peptide (second and fourth lanes). No signal was seen in the first lane, as these cells were not transfected with the Xpress-tagged SHIP-2 proteins. To ensure that the lack of binding observed with the SH2 domain mutant was not due to lack of expression of the transfected protein, parallel immunoprecipitations were performed with anti-Xpress antibody and analyzed by Western blotting with anti-SHIP-2 antibody (Fig. 5B). Fig. 5C is a quantitative measure of the percent of Xpress-tagged SHIP-2 that bound to the phosphorylated ITAM of Fc
RIIa. The data represent the mean ± S.D. of values obtained from three independent experiments.
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The SH2 Domain of SHIP-2 Is Necessary for FcRIIa-induced SHIP-2 Tyrosine PhosphorylationAs an additional approach to determine the importance of the SH2 domain of SHIP-2 in its association with Fc
RIIa and thereby its translocation to the membrane, we next assessed whether SH2 domain mutants of SHIP-2 would become phosphorylated upon Fc
RIIa clustering. Here, COS-7 cells were transfected to express Fc
RIIa along with wild-type SHIP-2, R47L SHIP-2 (SH2 domain mutant), or D608A SHIP-2 (catalytically deficient mutant). SHIP-2 proteins were immunoprecipitated from resting and activated cells, which had been activated by clustering Fc
RIIa, using anti-Xpress antibody and analyzed by Western blotting with anti-phosphotyrosine antibody. The results indicate that, although both wild-type SHIP-2 and catalytically deficient SHIP-2 became tyrosine-phosphorylated upon Fc
RIIa clustering, the SH2 domain mutant of SHIP-2 failed to become phosphorylated (Fig. 6A, upper panel). A reprobe of the membrane with anti-SHIP-2 antibody showed the presence of SHIP-2 in all lanes (Fig. 6A, middle panel). The phosphorylation level of SHIP-2 was quantitated as described under "Experimental Procedures," normalized for the amount of total SHIP-2 present in each lane, and expressed as -fold increase in SHIP-2 phosphorylation in the activated samples over that in resting samples. The graph in Fig. 6A (lower panel) represents the mean ± S.D. of values obtained from three independent experiments. To ensure that the lack of phosphorylation of the SH2 domain mutant of SHIP-2 was not due to lack of expression of Fc
RIIa in these cells, WCLs from the transfectants were probed with anti-Fc
RIIa antibody (Fig. 6B). Taken together, these data indicate that the SH2 domain of SHIP-2 is necessary for the association of SHIP-2 with Fc
RIIa.
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SHIP-2 Is Phosphorylated by FcRI Clustering in THP-1 CellsIn additional experiments, THP-1 cells were activated by clustering Fc
RI with monoclonal antibody 197, followed by goat anti-mouse IgG. SHIP-2 phosphorylation was assessed by Western blotting with anti-phosphotyrosine antibody (Fig. 7A, upper panel). Fig. 7A (lower panel) is a reprobe with anti-SHIP-2 antibody to ensure equal loading of SHIP-2 in all lanes. The results indicate that SHIP-2 was indeed invoked upon Fc
RI clustering in THP-1 cells.
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Because our data indicated that SHIP-2 associated with the phosphorylated ITAM of FcRIIa, we next analyzed whether a similar association might exist between SHIP-2 and the phosphorylated ITAM of the Fc
R
-chain. For this, biotinylated peptides corresponding to the ITAM of the
-chain that were either non-phosphorylated or doubly phosphorylated were applied to THP-1 lysates. The peptide-bound material was analyzed by Western blotting with anti-SHIP-2 antibody. The results shown in Fig. 7B (upper panel) indicate that the phosphorylated ITAM of the
-chain was incapable of association with SHIP-2. To ensure that the phosphopeptide used in these experiments is functional, the same membrane was reprobed with anti-Syk antibody (Fig. 7B, lower panel). As shown, the phosphopeptide efficiently bound Syk, consistent with earlier reports (2). These data suggest that SHIP-2 translocation to the membrane in response to Fc
RI clustering might involve adapter molecules or membrane-associated proteins other than the
-chain. Further analysis is needed to resolve these issues.
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DISCUSSION |
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In this study, we have analyzed the molecular details of a novel level of regulation exerted by the inositol phosphatase SHIP-2. Our results indicate that SHIP-2 associates with the ITAM of FcRIIa via the SH2 domain of SHIP-2 and becomes tyrosine-phosphorylated. As in the case of SHIP-1, tyrosine phosphorylation of SHIP-2 does not influence SHIP-2 enzyme activity. However, because membrane translocation of SHIP-2 is necessary for SHIP-2 tyrosine phosphorylation by Src kinases (30) and because membrane translocation puts SHIP-2 in the proximity of its lipid substrates, tyrosine phosphorylation of SHIP-2 serves as a correlate of SHIP-2 activation. Our data also demonstrate that SHIP-2 serves to down-regulate Fc
RIIa-induced activation of Akt and NF
B-dependent gene transcription, as overexpression of wild-type SHIP-2 (but not catalytically deficient SHIP-2) almost completely abrogates these two events. Both SHIP-1 and SHIP-2 mediate their inhibitory effects via the SH2 domains, which are reported to have 54% homology. Thus, it must be noted that the enhanced activation of NF
B-dependent luciferase gene induction by the dominant-negative SHIP-2 mutant D608A used in the transfection studies in THP-1 cells (Fig. 2C) may be the result of competition of D608A SHIP-2 with both endogenous SHIP-1 and SHIP-2.
In contrast to the association of SHIP-2 with the FcRIIa ITAM, we were unable to detect any binding of SHIP-2 to the phosphorylated ITAM of the Fc
RI-associated
-chain, despite the fact that SHIP-2 was efficiently phosphorylated upon Fc
RI clustering (Fig. 7). Co-immunoprecipitation experiments revealed a very weak association of SHIP-2 with the
-chain that became apparent only when the Western blot was overexposed (data not shown). Based on the results of Fig. 7, we suggest that SHIP-2 association with the
-chain is likely mediated via adapter molecules or that the association may be weak and transient and is disrupted by the detergent-based lysis buffer. In a recent report, Mitchell and co-workers (31) demonstrated an association of SHIP-2 with the actin-binding protein filamin. This association is mediated via the C-terminal proline-rich domain of SHIP-2 and the SH3 domain of filamin. Because filamin is known to be associated with Fc
RI (reviewed in Ref. 32) in macrophages, it is possible that Fc
RI clustering recruits SHIP-2 via filamin. Nonetheless, our data indicate that Fc
RI clustering induces tyrosine phosphorylation of SHIP-2, suggesting a role for SHIP-2 in regulating Fc
RI-mediated events.
Interestingly, SHIP-2 expression in human PBMs is almost undetectable, but is induced upon activation of these cells with bacterial lipopolysaccharide. Similar findings were recently reported by Cambier and co-workers (24) in murine B cells. It is tempting to speculate that the induction of SHIP-2 by LPS is a mechanism employed by Gram-negative bacteria to suppress the host immune response so that the pathogen may evade the phagocytic machinery. Numerous earlier studies have reported that LPS attenuates FcR-mediated phagocytosis (33, 34). However, the molecular mechanism by which LPS mediates its suppressive effect has thus far remained elusive. Further studies are underway to investigate the details of LPS induction of SHIP-2 expression and the functional consequence on Fc
R-mediated phagocytosis.
Finally, is not clear why myeloid cells express two inositol 5'-phosphatases, SHIP-1 and SHIP-2. Our data suggest that these two molecules may not be coexpressed at all times. Furthermore, although the results of this study demonstrate overlapping functions for SHIP-1 and SHIP-2, i.e. inhibition of Akt and NFB activation, it is likely that these two phosphatases mediate additional and non-overlapping effects on signaling pathways. Indeed, previous studies have demonstrated that the C-terminal proline-rich regions of SHIP-1 and SHIP-2 are quite different, leading to association of a distinct sets of SH3 domain-containing molecules (19), which likely influence distinct signaling pathways. We are currently investigating the functional differences between SHIP-1 and SHIP-2 in Fc
R-mediated activation of monocytes and macrophages.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| Fellow of the Leukemia and Lymphoma Society. To whom correspondence should be addressed: Dept. of Internal Medicine, Ohio State University, Rm. 405B HLRI, 473 W. 12th Ave., Columbus, OH 43210. Tel.: 614-247-6768; Fax: 614-688-4662; E-mail: tridandapani.2{at}osu.edu.
1 The abbreviations used are: FcR, Fc
receptor; ITAM, immunoreceptor tyrosine-based activation motif; SH, Src homology; SHIP, SH2 domain-containing inositol phosphatase; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; PBMs, peripheral blood monocytes; LPS, lipopolysaccharide; GST, glutathione S-transferase; WCLs, whole cell lysates.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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