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The Journal of Immunology, 2003, 171: 4227-4234.
Copyright © 2003 by The American Association of Immunologists

Dual Phosphorylation of Phosphoinositide 3-Kinase Adaptor Grb2-Associated Binder 2 Is Responsible for Superoxide Formation Synergistically Stimulated by Fc{gamma} and Formyl-Methionyl-Leucyl-Phenylalanine Receptors in Differentiated THP-1 Cells 1

Haruka Momose2, Hiroshi Kurosu2, Noriko Tsujimoto, Kenji Kontani, Kyoko Tsujita, Hiroshi Nishina and Toshiaki Katada3

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as Fc{gamma}RII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in Fc{gamma}RII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of Fc{gamma}RII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fc{gamma} and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils or polymorphonuclear leukocytes move and orientate themselves in gradients of chemotactic substances released from invading microorganisms. Stimulation of chemotactic receptors by formyl peptides such as fMLP induces neutrophil migration and other responses, including the generation of reactive oxygen species and the release of lysosomal enzymes, which are essential for host defense. At site of infection, Fc{gamma}R, which recognize Fc portion of IgG, play an important role in host defense, together with the chemotactic receptor. Foreign organisms are covered with host IgG, and the extracellular immunocomplexes are rapidly phagocytized through Fc{gamma} receptors and finally digested by fusion with intracellular lysosomes (see Refs.1, 2, 3 for review).

We have previously reported that fMLP-induced superoxide formation was markedly enhanced by Fc{gamma}RII cross-linking in differentiated THP-1 cells and that this response was completely abolished by treatment with wortmannin, a specific inhibitor of phosphoinositide (PI) 4 3-kinase (4, 5, 6). Furthermore, accumulation of the PI 3-kinase product phosphatidylinositol 3, 4, 5-triphosphate (PIP3) in response to fMLP is enhanced by the simultaneous stimulation of Fc{gamma}RII. It is, therefore, conceivable that the activation of PI 3-kinase plays a crucial role in enhanced superoxide generation in neutrophil-type THP-1 cells.

PI 3-kinase catalyzes phosphorylation of inositol phospholipids at the 3-position of inositol ring. It can be classified into three groups (class I, II, and III) according to the structure and substrate specificity, and only the class I PI 3-kinase family can generate PIP3. Furthermore, the class I PI 3-kinase is comprised of two types in terms of mode of activation in mammalian cells: one (class Ia) is stimulated by receptors linked to Tyr kinase ({alpha}, {beta}, and {delta} types), and the other (class Ib) is activated by G protein-coupled receptors ({gamma} type). The PI 3-kinase {alpha}, {beta}, and {delta} are heterodimers consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85); the p85 subunit possesses two Src homology 2 (SH2) domains. The interaction of Tyr-phosphorylated receptors or adaptors with the SH2 domains of p85 is a dominant step for activating the enzyme (see Refs. 7 and 8 for review).

We also reported that the class Ia PI 3-kinase consisting of p110{beta} and p85 has unique properties in terms of the regulation of its lipid kinase activity (9, 10). The p110{beta}/p85 isoform can be synergistically activated by a Tyr-phosphorylated peptide and the {beta}{gamma} subunits of G proteins. Such synergistic activation was, however, not observed in other class Ia PI 3-kinases (10). Thus, the p110{beta}/p85 isoform appears to be responsible for the synergistic activation by two different types of membrane receptors, one possessing Tyr kinase activity and the other activating trimeric GTP-binding proteins (9). Subsequently, these unique properties observed in the p110{beta}/p85 isoform were also confirmed by the study of Maier et al. (11). As described above, cellular responses stimulated by both Fc{gamma} and fMLP receptors, such as enhanced superoxide formation, appear to be associated with the synergistic activation of p110{beta}/p85 PI 3-kinase. However, the precise mechanisms underlying this priming process are presently unclear.

In this study, we examined the activation mechanism of p110{beta}/p85 PI 3-kinase from the point of its associated molecules. We found that Grb2-associated binder 2 (Gab2) and c-Cbl (Casitas B-lineage lymphoma) function as Tyr-phosphorylated adaptors for PI 3-kinase in Fc{gamma}RII-stimulated THP-1 cells and that the adaptor Gab2 is further phosphorylated on Ser/Thr residue(s) by extracellular signal-regulated kinase (ERK) upon the stimulation with fMLP. Interestingly, this dual phosphorylation on Gab2 appears to be responsible for the enhanced superoxide formation in neutrophil-like THP-1 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Anti-p110{beta}, anti-Cbl, and anti-ERK2 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p85 and anti-Gab2 (anti-Gab2/C) Abs were purchased from Seikagaku Kogyo (Tokyo, Japan) and Upstate Biotechnology (Lake Placid, NY), respectively. Another anti-Gab2 Ab (anti-Gab2/P) was obtained by immunizing rabbit with keyhole limpet hemocyanin-conjugated peptide (CVRQSSEPSKGAKL) corresponding to the C terminus of human Gab2. Anti-Fc{gamma}RII (CD32) IV.3 (subclass IgG2b; 025-101) and F(ab')2 of goat anti-mouse IgG (201-1806) were purchased from Medarex (Annandale, NJ) and Kirkegaard & Perry Laboratories (Gaithersburg, MD), respectively. An anti-phospho-Tyr Ab (AB1600) was obtained from Chemicon International (Temecula, CA). Sepharose 4B and protein A-Sepharose 4-FF were from Pharmacia Biotech (Uppsala, Sweden). PMA, fMLP, staurosporine, myelin basic protein (MBP), and protein phosphatase 2A (PP2A) were purchased from Sigma-Aldrich (St. Louis, MO). A mitogen-activated protein kinase kinase inhibitor, PD98059, was purchased from Calbiochem (San Diego, CA). [{gamma}-32P]ATP and 125I-labeled protein A were purchased from DuPont NEN (Boston, MA). HRP-labeled anti-rabbit IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents were of analytical grade and were obtained from commercial sources.

Cell culture and differentiation

Human myeloid THP-1 cells were grown in RPMI 1640 containing 10% FBS, 100 IU/ml of penicillin, 100 µg/ml of streptomycin, and 0.6 mg/ml of glutamine at 37°C in 95% air and 5% CO2. THP-1 cells were caused to differentiate into neutrophil-like cells by treatment with 0.5 mM dibutyryl cAMP for 3 days.

Assay for in vitro Tyr kinase activity

Lysates from the differentiated THP-1 cells were immunoprecipitated with various Abs specific for a certain type of Tyr kinases, and the in vitro Tyr kinase assay was performed with enolase as a substrate by the method described previously (12).

Immunoprecipitation and immunoblotting

The differentiated THP-1 cells were washed with ice-cold PBS and a suspension buffer consisting of 10 mM HEPES-NaOH (pH 7.4), 136 mM NaCl, 4.9 mM KCl, 5.5 mM glucose, and 0.2% (w/v) BSA, and incubated in the suspension buffer containing 1 mM CaCl2 at a density of ~5 x 107 cells/ml. For stimulation of Fc{gamma}RII, the cells (1 x 107 cells/200 µl), after being held on ice for 25 min with 1 µg/ml of IV.3, were first incubated at 37°C for 5 min and further incubated for 2 min (or the indicated times) with 10 µg/ml of goat anti-mouse IgG (H + L) F(ab')2 for the receptor cross-linking. In the indicated experiments, the cells were simultaneously stimulated with 1 µM fMLP at 37°C.

Tyr-phosphorylated proteins in the cells were analyzed, as described previously (5, 6). Briefly, the reaction was terminated by adding the same volume (200 µl) of a lysis buffer consisting of 40 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 30 mM NaF, 2 mM Na3VO4, 20 mM Na4P2O7, 4 mM EDTA, 2% Nonidet P-40, and 10 µg/ml of aprotinin. The cell extract was immunoprecipitated with 1 µg of anti-p110{beta} (or 4 µg of anti-Gab2/P or Gab2/C) and protein A-Sepharose resin. The precipitated proteins were subjected to SDS-PAGE (8% acrylamide), and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). In some experiments (see Fig. 4A), the immunocomplexes were diluted in a buffer consisting of 20 mM Tris-HCl (pH 7.4,) 50 mM NaCl, and 0.1% 2-ME, and incubated with 33 U of PP2A at 30°C for 30 min. The membrane, after being shaken in TBS (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 5% BSA, was incubated with the indicated Abs at room temperature for 1 h and further incubated with 125I-labeled protein A or HRP-conjugated anti-rabbit IgG. The radioactivity and peroxidase activity retained on the membrane were visualized with a Fuji BAS 1800 and a LAS 1000 bioimaging analyzer (Fuji Film, Tokyo, Japan), respectively.



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  		FIGURE 4. Characterization of the fMLP-induced modification of the PI 3-kinase adaptor Gab2. The differentiated THP-1 cells were stimulated at 37°C for 2 min with Fc{gamma}RII-CL and/or fMLP, as described in Fig. 1, and the cell lysates were immunoprecipitated with anti-p110{beta} Ab, separated by SDS-PAGE, and subjected to immunoblot analysis with anti-pY AB1600 Ab. A, The immunoprecipitated samples were incubated at 30°C for 30 min with (lanes 3 and 4) or without (lanes 1 and 2) 33 U of PP2A before the immunoblot analysis. B–D, the THP-1 cells were pretreated at 37°C with 50 ng/ml of pertussis toxin (PTX) for 4 h (B, lanes 4–6), 5 µM staurosporine (SP) for 10 min (C, lanes 4–6), or 1 µM wortmannin (WT) for 10 min (D, lanes 3 and 4), before the cell stimulation with Fc{gamma}RII-CL, fMLP, and/or 0.5 µM PMA.

 
Preparation of recombinant proteins and affinity resin

The aa sequence 312–725 of PI 3-kinase p85{alpha} subunit, which contains two SH2 domains, was produced in Escherichia coli as a GST-fused form and conjugated with activated Thiol-Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer’s instruction. THP-1 cells were stimulated, as described above, and the reaction was terminated by adding a buffer consisting of 4% SDS, 200 mM NaCl, 60 mM Tris-HCl (pH 7.4), 20 mM DTT, 5 mM Na3VO4, 8 µM leupeptin, and 0.8 µg/ml of pepstatin A. The cell extract was boiled for 5 min and treated with 30 mM N-ethylmaleimide. After 13-fold dilution with a buffer consisting of 1% Triton X-100, 80 mM NaCl, 30 mM Tris-HCl (pH 7.4), 0.5 mM Na3VO4, 5 µM leupeptin, and 0.5 µg/ml of pepstatin A, the cell extract was applied to the affinity resin. Proteins bound to the resin were eluted with a buffer consisting of 4% SDS, 1 mM EDTA, 1 mM Na3VO4, 10% glycerol, and 100 mM Tris-HCl (pH 6.8). Phosphorylation of the eluted proteins was visualized according to the method described above.

Transfection of Gab2 mutant

The cDNA encoding a dominant-negative human Gab2 mutant (Y452/476/584F), whose Tyr residues contained in the putative p85-binding sites were all replaced by Phe, was constructed by Kunkel method. The Y-to-F mutant and wild-type Gab2 were cloned into mammalian expression vector pCMV5 and introduced into THP-1 cells by electroporation.

In vitro kinase and in-gel kinase assays

COS7 cells were transfected with pcDNA3 vector encoding Flag-tagged Gab2 by electroporation. The Gab2 protein was immunoprecipitated with anti-Flag affinity gel (M2) and eluted with 1 mg/ml of Flag peptide. Active form of ERK2 was prepared from the differentiated THP-1 cells that had been stimulated with fMLP by immunoprecipitation using an anti-ERK2 Ab. The resultant resin containing the activated ERK2 was suspended in a kinase buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, and 5 µCi [{gamma}-32P]ATP. The solution was mixed with Flag-tagged Gab2 and incubated at 30°C for 30 min. Phosphorylated proteins were analyzed by SDS-PAGE (8% acrylamide), and the radioactivity was visualized with the bioimaging analyzer.

For in-gel kinase assay, the cell extract was separated by SDS-PAGE (10% acrylamide and 0.5 mg/ml of MBP), and SDS was removed by washing the gel twice with 125 ml of propanol solution (50 mM Tris-HCl, pH 8.0, and 20% 2-propanol) for 30 min. The gel, after being washed twice with 125 ml of buffer A (50 mM Tris-HCl, pH 8.0, and 5 mM 2-ME) for 30 min at room temperature, was denatured twice with 125 ml of buffer A containing 6 M guanidine hydrochloride for 30 min at room temperature and renatured with 100 ml of buffer A containing 0.04% (w/v) of Tween 40 for 16 h at 4°C. The gel was further washed twice with the same buffer for 30 min and incubated in buffer B (40 mM HEPES-NaOH, pH 7.5, 0.1 mM EGTA, 20 mM MgCl2, and 2 mM DTT) for 1 h at 25°C. Phosphorylation of MBP was conducted by incubating the gel in buffer B containing 25 µM ATP and 2.5 µCi/ml of [{gamma}-32P]ATP at 25°C for 1 h. After incubation, the gel was washed with a buffer consisting of 5% TCA and 1% sodium pyrophosphate. The radioactivity was visualized with the bioimaging analyzer.

Assays for superoxide formation and PIP3 production in THP-1 cells

Superoxide formation was determined by measuring the reduction of cytochrome c, as described previously (5). Briefly, the differentiated THP-1 cells were suspended in Hank’s solution containing 10 mM HEPES-NaOH (pH 7.4), 1 mg/ml of BSA, and 1.2 mg/ml of cytochrome c. An aliquot (180 µl, 5 x 106 cells/ml) was first incubated at 37°C for 5 min with or without superoxide dismutase (170 U/ml) and further incubated with 20 µl of the indicated reagents at 37°C for the indicated times. The reaction was terminated by adding 10 µl of 40 mM N-ethylmaleimide, and the reduced cytochrome c was determined by spectroscopic analysis at 550 nm.

PIP3 production was measured according to the method described by Okada et al. (9). The THP-1 cells were suspended at the density of 4 x 107 cells/ml in a labeling medium consisting of 10 mM HEPES-NaOH (pH 7.4), 136 mM NaCl, 4.9 mM KCl, and 5.5 mM glucose, and incubated at 37°C for 30 min with 500 µCi/ml of 32Pi. The radiolabeled cells were washed twice with the same medium and resuspended at 5 x 106 cells/ml in the medium containing 1 mM CaCl2. Aliquots (400 µl, 2 x 106 cells/ml) were preincubated for 5 min and further incubated with the indicated reagents. The reaction was terminated by the addition of 1.55 ml of chloroform/methanol/8% HClO4 (50:100:5). Then 0.5 ml each of chloroform and 8% HClO4 were added to the mixture, and the lipid phase was separated by centrifugation. The lipid phase was washed with chloroform-saturated 1 M NaCl containing 1% HClO4 and dried. The extracted lipid was dissolved in 20 µl of chloroform/methanol (95:5) and spotted on a TLC plate (Silica gel 60; Merck, West Point, PA). Before spotting, the plate was once developed with methanol/water (2:3) containing 1.2% potassium oxalate and preactivated by heating at 110°C for 20 min. The plate was developed in chloroform/methanol/acetone/acetic acid/water (80:30:26:24:14), and radioactivities were visualized with the bioimaging analyzer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhancement of fMLP-induced superoxide formation by Fc{gamma}RII cross-linking requires Tyr kinase-dependent PIP3 formation

We first examined which subtypes of Fc{gamma}RII were expressed in THP-1 cells by means of RT-PCR (data not shown). Nondifferentiated THP-1 cells contained both IIA and IIC types of Fc{gamma}R. When the cells were differentiated into neutrophil-like cells by dibutyryl cAMP, there was a marked increase in the level of the type IIC receptor. However, neither nondifferentiated nor differentiated THP-1 cells contained the type IIB receptor. Thus, the neutrophil-like THP-1 cells used in the present study were revealed to possess only the ITAM-containing types of Fc{gamma}R, IIA and IIC.

We previously showed that fMLP-induced superoxide formation was markedly enhanced by Fc{gamma}RII stimulation in the differentiated THP-1 cells in a manner associated with the elevation of PI 3-kinase product PIP3 (4, 5, 6). Moreover, the enhanced superoxide formation was completely abolished by treatment of the cells with the PI 3-kinase inhibitor wortmannin. These results indicate that Fc{gamma}RII-dependent activation of the lipid kinase is involved in the cell response. We thus investigated the effect of PP2, a Src-type Tyr kinase inhibitor, on superoxide generation and PIP3 formation in the neutrophil-like THP-1 cells. As shown in Fig. 1A, superoxide formation was synergistically stimulated by Fc{gamma}RII cross-linking plus fMLP (left columns), and this response was progressively inhibited as the concentration of PP2 was increased (right panel). Similar inhibition was also observed in the cell response induced by Fc{gamma} alone. However, the Tyr kinase inhibitor did not exert its influence on the action of fMLP alone. We also measured the intracellular accumulation of PIP3 in the differentiated THP-1 cells under the same experimental conditions. There were further increases in PIP3 formation upon the stimulation of Fc{gamma}RII both in the presence and absence of fMLP (Fig. 1B, left columns). Interestingly, PP2 completely inhibited the action of Fc{gamma}RII, but not the fMLP-induced PIP3 formation (Fig. 1B, right columns).



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  		FIGURE 1. Enhancement of fMLP-induced superoxide formation by Fc{gamma}RII cross-linking requires Tyr kinase-dependent PIP3 formation in differentiated THP-1 cells. A, Differentiated THP-1 cells that had been treated with 1 µg/ml of the anti-Fc{gamma}RII mAb (IV.3) were incubated at 37°C for 10 min (A) or 2 min (B) with 10 µg/ml of the goat anti-mouse IgG F(ab')2 for cross-linking of the receptor (Fc{gamma}RII-CL) in the presence and/or absence of 1 µM fMLP. The incubation mixture also contained 20 µM (B, right columns) or the indicated concentrations (A, right panel) of PP2. Superoxide formation (A) and PIP3 production (B) in the cells were measured, as described in Materials and Methods. C, Lysates were prepared from the stimulated cells and immunoprecipitated with the indicated Abs specific for their Tyr kinases. In vitro Tyr kinase assay was performed, as described in Materials and Methods. The insets show 32P-phosphorylated enolase (upper lanes) and precipitated kinases (lower lanes) that were detected by their specific Abs. Bars indicate the SEs of means. Asterisks and NS indicate that the two paired values are statistically significant (p < 0.05) or not, respectively.

 
We also examined which member of Src-type Tyr kinases is responsible for the Fc{gamma}RII-dependent signaling in the cells. For the analysis, the cell lysates were immunoprecipitated with various Abs specific for a certain type of Tyr kinases and subjected to in vitro Tyr kinase assay with enolase as a substrate. As shown in Fig. 1C, Fyn, but not Lyn or Src, appeared to be activated by Fc{gamma}RII cross-linking. We could not detect any activation of Lck or Fgr-type Tyr kinase (data not shown). These results suggest that PI 3-kinase is activated by Fc{gamma}RII-linked Fyn Tyr kinase and that the lipid kinase activation is especially important for the synergistic superoxide formation observed with Fc{gamma}RII cross-linking plus fMLP.

Identification of PI 3-kinase adaptors in Fc{gamma}RII-stimulated THP-1 cells

To understand the molecular mechanism of this synergistic activation, we next investigated Tyr-phosphorylated proteins associated with the class I p110{beta}/p85 PI 3-kinase. The differentiated THP-1 cells were stimulated with Fc{gamma}RII cross-linking, and PI 3-kinase-associated proteins were immunoprecipitated with an Ab raised against the p110{beta} catalytic subunit. The precipitated proteins were separated by SDS-PAGE and immunoblotted with an anti-phospho-Tyr Ab. As shown in Fig. 2A (lanes 1–6), 120-kDa (p120) and 100-kDa (p100) proteins were Tyr phosphorylated in the Fc{gamma}RII-stimulated cells. When the cells were simultaneously stimulated with fMLP (Fig. 2A, lanes 7–12), a time-dependent mobility shift of p100 was clearly observed without a significant change in the profile of p120. We further examined whether the Tyr-phosphorylated proteins were capable of interacting with PI 3-kinase through SH2 domains of p85 using affinity resin conjugated with GST-fused p85{alpha} SH2 (the aa sequence 312–725). As shown in Fig. 2B (lane 2), both the Tyr-phosphorylated p120 and p100 were capable of binding to the SH2 domains. Based on their molecular weights and immunoreactivity, p120 and p100 could be identified as c-Cbl and Gab2, respectively (Fig. 2C). These results indicate that p120/c-Cbl and p100/Gab2 function as adaptor molecules for the p110/p85 PI 3-kinase in the Fc{gamma}RII-stimulated THP-1 cells.



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  		FIGURE 2. Stimulation of Fc{gamma}RII induces Tyr phosphorylation of c-Cbl and Gab2 in differentiated THP-1 cells. The differentiated THP-1 cells were stimulated at 37°C for 2 min with Fc{gamma}RII-CL and/or fMLP, as described in Fig. 1. A, The cell lysates were immunoprecipitated with an anti-p110{beta} Ab, separated by SDS-PAGE, and subjected to immunoblot analysis with the anti-pY AB1600, as described in Materials and Methods. B, Proteins bound to the SH2 domains of p85 in the cell lysate (Fc{gamma}RII-CL) were affinity purified by means of Sepharose resin conjugated with GST alone (lane 1) or GST-PI 3-kinase (p85/SH2) (lane 2), as described in Materials and Methods. C, The immunoblot analysis was performed with anti-Cbl (left) and anti-Gab2/C (right) Abs. The m.w. markers used were obtained from Bio-Rad and are indicated in kilodaltons.

 
Modification of the PI 3-kinase adaptor Gab2 by fMLP receptor stimulation

Because the PI 3-kinase adaptor Gab2 displayed its unique properties upon fMLP receptor stimulation, we further investigated the properties of Gab2 in the differentiated THP-1 cells. The cells stimulated with Fc{gamma}RII and/or fMLP receptors were lysed and immunoprecipitated with the anti-Gab2/P, and the precipitated proteins were analyzed by immunoblot using anti-Gab2/C and anti-phospho-Tyr (AB1600) Abs. As shown in Fig. 3, stimulation by Fc{gamma}RII induced slightly, but significantly the mobility shift (A, lane 2) and Tyr phosphorylation (B, lane 2) of Gab2. In contrast, stimulation by fMLP induced more apparent mobility shift (A, lane 3), but no Tyr phosphorylation (B, lane 3) of Gab2. Costimulation with Fc{gamma}RII and fMLP receptors enhanced the mobility shift (A, lane 4) and Tyr phosphorylation (B, lane 4) of Gab2. As expected, the p85-regulatory and the p110{beta}-catalytic subunits of PI 3-kinase could be more immunoprecipitated with Gab2 when Tyr phosphorylation of the adaptor was further enhanced by costimulation with fMLP (data not shown). Furthermore, Gab2-associated PI 3-kinase activity observed upon Fc{gamma}RII cross-linking was certainly enhanced by the costimulation. These results indicate that fMLP induces certain modification of Gab2 in a manner different from Fc{gamma}RII-induced Tyr phosphorylation, and that this modification is responsible for the enhanced PI 3-kinase activity.



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  		FIGURE 3. Stimulation of G protein-coupled fMLP receptor induces modification of the PI 3-kinase adaptor Gab2 that is responsible for the synergistic superoxide formation. The differentiated THP-1 cells were stimulated at 37°C for 2 min with Fc{gamma}RII-CL and/or fMLP, as described in Fig. 1. The cell lysate was immunoprecipitated with anti-Gab2/P, separated by SDS-PAGE, and subjected to immunoblot analysis with anti-Gab2/C (A) and anti-pY AB1600 (B) Abs. C, THP-1 cells were transfected with wild-type Gab2 (WT) or its mutant (Mu) and caused to differentiation into the mid stage of neutrophil-like cells. Superoxide formation in response to Fc{gamma}RII-CL and/or fMLP was measured, as described in Materials and Methods.

 
The contribution of Tyr phosphorylation of the PI 3-kinase adaptor Gab2 to superoxide formation was partly confirmed by transfection experiments with a dominant-negative Gab2 mutant (Y452/476/584F), whose putative Tyr residues contained in the binding sites for p85 were all replaced by Phe. For this analysis, THP-1 cells were transiently transfected with the Gab2 mutant (or its wild type) and caused to differentiate into the mid stage of neutrophil-like cells by 1-day culture, because further incubation inhibited the protein expression, and because the prior transfection reduced the differentiation. Although the cell differentiation and transfection efficiency (~50%) were limited under the present conditions, superoxide formation in response to Fc{gamma}RII cross-linking plus fMLP was significantly attenuated in the Gab2 mutant-transfected cells in comparison with mock- or wild-type Gab2-transfected cells (Fig. 3C). Thus, the PI 3-kinase adaptor Gab2 appeared to be responsible, if not all, for the synergistic superoxide formation induced by the two different types of receptors.

Ser/Thr phosphorylation of the PI 3-kinase adaptor Gab2 by G protein-coupled fMLP receptor stimulation

To reveal the entity of the fMLP-induced modification of Gab2, the adaptor protein that had been immunoprecipitated with anti-p110{beta} Ab was treated with a protein phosphatase (PP2A) specific for Ser/Thr phosphorylation. As shown in Fig. 4A, PP2A treatment altered the mobility shift of Tyr-phosphorylated Gab2 induced by fMLP (compare lanes 1 and 3). Furthermore, PP2A changed the mobility shift of Tyr-phosphorylated Gab2 induced by Fc{gamma}RII stimulation (lanes 2 and 4). These results suggest that Gab2 is phosphorylated at Ser/Thr residues by the stimulation of fMLP receptors and Fc{gamma}RII, in addition to its Tyr phosphorylation by Fc{gamma}RII.

We next examined how fMLP receptor-dependent signaling pathways are related to the Ser/Thr phosphorylation of Gab2. Pertussis toxin, which inhibits the coupling of fMLP receptor to Gi-type G proteins, was first used for the analysis. Differentiated THP-1 cells that had been treated with pertussis toxin were stimulated with Fc{gamma} and/or fMLP, and the cell lysate was immunoprecipitated with anti-p110{beta} and immunoblotted with anti-phospho-Tyr (AB1600) Abs (Fig. 4B). The fMLP-induced mobility shift of Gab2 was almost completely abolished after the toxin treatment (compare lanes 3 and 6), indicating that activation of Gi is required for the Gab2 phosphorylation.

To identify the Ser/Thr kinase(s) involved in the fMLP-induced Gab2 phosphorylation, we examined effects of a protein kinase C activator (PMA) and a kinase inhibitor (staurosporine). Similar to the fMLP stimulation, PMA induced mobility shift of Gab2 (Fig. 4, B, lane 2, and C, lane 3), although this PMA-induced mobility shift was insensitive to pertussis toxin (Fig. 4B, lane 5). As expected, staurosporine inhibited the PMA-induced mobility shift of Gab2 (Fig. 4C, lane 6). However, the kinase C inhibitor failed to inhibit the action of fMLP (Fig. 4C, lane 5). These results indicate that neither protein kinase C nor a staurosporine-sensitive kinase(s) is involved in the fMLP-induced Ser/Thr phosphorylation of Gab2.

A recent report showed that another Ser/Thr kinase, protein kinase B, which is activated by the PI 3-kinase product PIP3, phosphorylates Gab2 at Ser 159 and negatively regulates heregulin-induced mitogenic signaling (13). In the report, heregulin-induced Tyr phosphorylation of Gab2 is enhanced by pretreatment with the specific PI 3-kinase inhibitor wortmannin. Therefore, we examined effect of wortmannin on the mobility shift of Gab2. There was decrease in the level of Tyr-phosphorylated Gab2 in cells treated with wortmannin (Fig. 4D, lanes 3 and 4). Furthermore, the action of fMLP was still clearly observed under the conditions that PI 3-kinase was inhibited by wortmannin. These results indicate that PI 3-kinase-dependent kinases including protein kinase B are not responsible for the fMLP-induced Ser/Thr phosphorylation of Gab2.

Involvement of ERK in the fMLP-induced Ser/Thr phosphorylation of Gab2

A recent report showed that Gab1, which is a member of Gab family, is phosphorylated, possibly at Thr476, by ERK after hepatocyte growth factor stimulation (14). Because Gab2 has a corresponding amino acid Ser491 to the Thr476 of Gab1, we examined whether ERK activation may be involved in the fMLP-induced Ser/Thr phosphorylation of Gab2. Differentiated THP-1 cells that had been treated with an ERK kinase (mitogen-activated protein kinase kinase) inhibitor, PD98059, were stimulated with Fc{gamma}RII cross-linking and fMLP, and the cell lysate was immunoprecipitated and immunoblotted by anti-Gab2 Ab. Interestingly, PD98059 significantly inhibited the fMLP-induced mobility shift of Gab2 (Fig. 5A, lanes 7 and 8). We next examined ERK activity by an in-gel kinase assay. Lysate prepared from the stimulated THP-1 cells was applied on SDS polyacrylamide gel containing the ERK substrate MBP, and in-gel phosphorylation was performed in the presence of [{gamma}-32P]ATP. As shown in Fig. 5B (lanes 3 and 4), there were phosphorylated bands at the positions of 42 kDa (ERK1) and 40 kDa (ERK2) upon the cell stimulation with fMLP. The fMLP-dependent activation of ERK1 and ERK2 was progressively inhibited as the concentration of PD98059 was increased (Fig. 5C). We also examined whether activated ERK in the fMLP-stimulated cells is capable of phosphorylating Gab2 in vitro using a recombinant Flag-tagged Gab2. As shown in Fig. 5D, recombinant Flag-tagged Gab2 was clearly phosphorylated by the activated ERK obtained from the fMLP-treated cells.



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  		FIGURE 5. fMLP-induced Ser/Thr phosphorylation of Gab2 is mediated through activation of ERK. The differentiated THP-1 cells were stimulated at 37°C for 2 min with Fc{gamma}RII-CL and/or fMLP, as described in Fig. 1, in the presence or absence of 75 µM PD98059. A, The cell lysate was immunoprecipitated with anti-Gab2/C Ab and subjected to immunoblot analysis with the same Ab. B and C, The cell lysates were subjected to an in-gel kinase assay with MBP, as described in Materials and Methods. Various concentrations of PD98059 were used in the stimulation of THP-1 cells with Fc{gamma}RII-CL and fMLP (C). D, An in vitro kinase assay was performed with a recombinant Flag-tagged Gab2 (lanes 1 and 2), as described in Materials and Methods. The cell lysate containing ERK was prepared from the cells that had been stimulated with fMLP (lanes 2 and 3) or not (lane 1).

 
Inhibition of the Ser/Thr phosphorylation of PI 3-kinase adaptor Gab2 affects superoxide formation and PIP3 production

We finally investigated whether the Ser/Thr phosphorylation of PI 3-kinase adaptor Gab2 exerts its influence on the lipid kinase activity and superoxide formation in the differentiated THP-1 cells. As shown in Fig. 6A, enhanced superoxide formation stimulated by Fc{gamma}RII cross-linking plus fMLP was inhibited significantly, but partially by PD98059 under the conditions that the ERK-dependent Ser/Thr phosphorylation of Gab2 was profoundly suppressed (see Fig. 5). It is worth noting that PD98059 did not affect superoxide formation induced by either Fc{gamma}RII cross-linking or fMLP. Interestingly, PD98059 also abolished the enhanced PIP3 formation induced by Fc{gamma} plus fMLP without significant changing in the stimulatory action of fMLP or Fc{gamma}RII cross-linking alone (Fig. 6B, right columns). These effects induced by the ERK inhibition were quite different from those observed with PP2 (see Fig. 1). Thus, the synergistic superoxide formation appeared to be mediated through the Tyr kinase-linked signaling pathway, which possibly involves the modification of PI 3-kinase adaptor Gab2 found in the present study. The partial inhibition observed with PD98059 suggests that there is another mechanism in the synergistic superoxide formation by the two different types of membrane receptors (see Discussion).



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  		FIGURE 6. Effects of PD98059 on superoxide formation and PI 3-kinase activity. The differentiated THP-1 cells were stimulated at 37°C for 10 min (A) or 2 min (B) with Fc{gamma}RII-CL and/or fMLP, as described in Fig. 1. The incubation mixture also contained 100 µM (B) or the indicated concentrations of PD98059 (A). Superoxide formation (A) and PIP3 production (B) in the stimulated THP-1 cells were measured, as described in Materials and Methods. B, Bars indicate the SEs of means. Asterisks and NS indicate that the two paired values are statistically significant (p < 0.05) or not, respectively.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously searched for various intracellular messengers that may be responsible for fMLP-induced superoxide formation in differentiated THP-1 cells and found that the activation of PI 3-kinase plays an important role in the cell function (4, 5). Interestingly, intracellular formation of PIP3 was significantly enhanced by the costimulation of Fc{gamma}RII and fMLP receptors in THP-1 cells (5, 6). Furthermore, we established that the class Ia PI 3-kinase p110{beta}/p85 could be synergistically activated by a Tyr-phosphorylated peptide and {beta}{gamma} subunits of G proteins (10) and showed that a specific PI 3-kinase inhibitor, wortmannin, completely inhibited the superoxide formation (5). However, it remained to be determined how p110{beta}/p85 is synergistically activated in intact cells. Our present study focusing on its adaptor molecules basically extends the above molecular mechanism and indicates a novel pathway leading to efficient PI 3-kinase activation. The new findings obtained in this study are summarized in Fig. 7.



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  		FIGURE 7. A schematic representation of signal transduction pathways involved in Fc{gamma}RII- and fMLP receptor-induced PIP3 production and superoxide formation. Superoxide formation by the stimulation of Fc{gamma}RII and fMLP receptors involves various signal transduction pathways, including: 1) the synergistic activation of p110{beta}/p85-type PI 3-kinase by the stimulation through p85/SH2 domains and p110{beta} by the adaptors and G protein {beta}{gamma} subunits, respectively (10 ); 2) the activation of p110{gamma}/p101-type PI 3-kinase by G protein {beta}{gamma} subunits (11 ); and 3) the activation of Gab2-associated PI 3-kinase through the Ser/Thr phosphorylation of the adaptor observed in the present study. See Discussion section for further explanation.

 
First, we identified Gab2 and c-Cbl as adaptor molecules for the class Ia PI 3-kinase in the Fc{gamma}RII-stimulated cells, because both proteins were Tyr phosphorylated and associated with the lipid kinase through SH2 domains of p85 (see Fig. 2). Second, the adaptor Gab2 was rapidly modified by Gi-coupled fMLP receptor stimulation in terms of its mobility shift on SDS-PAGE (see Fig. 4). Moreover, the mobility shift of Gab2 occurred without Tyr phosphorylation on the adaptor molecule. The entity of the Gab2 modification was proven to be phosphorylation at Ser/Thr residue(s), and Gi-dependent ERK activation appeared to be responsible for the Ser/Thr phosphorylation (see Figs. 4 and 5). Indeed, activated ERK obtained from fMLP-stimulated THP-1 cells directly phosphorylated Gab2 (see Fig. 5D). Although similar modification of Gab2 was observed with PMA in a manner sensitive to staurosporine (see Fig. 4, B and C), neither protein kinase C nor protein kinase B was involved in the fMLP-induced Ser/Thr phosphorylation of Gab2.

Gab2 belongs to Daughter OF SevenLes (DOS)/Gab family scaffolding adaptors that contain N-terminal pleckstrin homology domain and multiple Tyr residues, which serve as binding sites upon phosphorylation for many signal-relay molecules, including Grb2, SHP-2, and p85 of PI 3-kinase (13, 15, 16, 17, 18, 19), as confirmed in the present study. In addition to the Tyr phosphorylation sites, DOS/Gab family possesses many Ser/Thr phosphorylation sites for a number of protein kinases. Ser/Thr phosphorylation, as well as Tyr phosphorylation after receptor stimulation, is commonly used to regulate the adaptor function both positively and negatively (13, 14, 20, 21, 22, 23, 24). In this study, we found that Ser/Thr residue(s) of Gab2 was phosphorylated by Gi-coupled fMLP receptor stimulation and that ERK is involved in the Ser/Thr phosphorylation. Indeed, Gab2 has five Pro-X-(X)-Ser/Thr-Pro sequences that can be phosphorylated by ERK. The Ser491 within Pro-Met-Ser-Pro sequence on Gab2 may be phosphorylated by ERK, because another member of Gab family, Gab1, is phosphorylated at Thr476 within Pro-Met-Thr-Pro corresponding to the sequence of Gab2 (14). In addition, it is very likely that fMLP induces modification of Gab2 other than the ERK-dependent phosphorylation, because there was still significant difference in the mobility shift of Gab2 between the presence and absence of fMLP even after PP2A treatment (see Fig. 4A, lanes 3 and 4).

We have previously reported that stimulation of two different types of membrane receptors induces synergistic activation of PI 3-kinase, which leads to enhanced cell functions (5, 9, 25). The activation of Gi through the stimulation of G protein-coupled membrane receptors appears to be responsible for the enhanced PIP3 formation and cell functions possibly through the following three different mechanisms. The first one is dependent on the unique properties of class Ia PI 3-kinase consisting of p110{beta} and p85. The heterodimeric PI 3-kinase could be synergistically activated by a Tyr-phosphorylated peptide and {beta}{gamma} subunits resolved from Gi (9) (see Fig. 7, route 1). The target site of the {beta}{gamma} subunit-dependent activation is present in p110{beta}-catalytic subunit of PI 3-kinase rather than its regulatory p85 subunit, because other PI 3-kinase subtypes, p110{alpha}/p85 and p110{delta}/p85, were insensitive to the {beta}{gamma} subunits (10, 11). Second, {beta}{gamma} subunits of G protein are also capable of activating the p110{gamma}/p101-type PI 3-kinase (10, 11) (see Fig. 7, route 2). The p110{gamma}-catalytic subunit is a site for the stimulatory action of the {beta}{gamma} subunits, and p101 appears to be responsible for the substrate selectivity of the p110{gamma} type for PI-4, 5-bisphosphate (11). It has recently been reported that neutrophil functions induced by G protein-coupled receptors, such as fMLP-induced cell migration and respiratory burst, are severely impaired in mice lacking p110{gamma} (26, 27, 28). Thus, p110{gamma} PI 3-kinase appears to be a major subtype responsible for the neutrophil functions.

In addition to the above two mechanisms, we found in this study that dual-phosphorylated form of Gab2 may also serve as a cross talk molecule between the two distinct signals (see Fig. 7, route 3). The small, but significant enhancement of PIP3 formation, which was sensitive to the inhibition of Gab2 Ser/Thr phosphorylation by PD98059, appeared to be partly involved in the potentiation of superoxide formation (see Fig. 6). Thus, PIP3 formation at a locally targeting site within the cells may lead to the effective superoxide formation. It is well known that neutrophil functions are potentiated when the cells are also primed with inflammatory cytokines before fMLP stimulation (29, 30, 31, 32, 33). Among them, GM-CSF, which linked to tyrosine kinase activities, enhanced PIP3 and superoxide generation, which were both sensitive to wortmannin (31) and severely impaired in p85{alpha} knockout mice (33). Interestingly, PD98059 inhibited partially the enhanced superoxide formation in GM-CSF-primed human neutrophils (31). Therefore, in certain situations, the novel pathway revealed in this study may play an important physiological role in effective host defense in intact cells.


   Footnotes
 
1 This work was supported in part by research grants from the "Research for the Future" Program of the Japan Society for the Promotion of Science (JSPS-RFTF 96L00505), the Mitsubishi Foundation, and the Scientific Research Funds of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. Back

2 H.M. and H.K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Toshiaki Katada, Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan. E-mail address: katada{at}mol.f.u-tokyo.ac.jp Back

4 Abbreviations used in this paper: PI, phosphoinositide; Cbl, Casitas B-lineage lymphoma; ERK, extracellular signal-regulated kinase; Gab2, Grb2-associated binder 2; MBP, myelin basic protein; PIP3, phosphatidylinositol 3,4,5-triphosphate; PP2A, protein phosphatase 2A; SH2, Src homology 2. Back

Received for publication January 15, 2003. Accepted for publication August 14, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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