Cbl functions downstream of Src kinases in FcRI ... - Semantic Scholar

Cbl functions downstream of Src kinases in FcgRI signaling in primary human macrophages Anat Erdreich-Epstein,* Ming Liu,* Anita M. Kant,* Kayvon D. Izadi,* Jan A. Nolta,† and Donald L. Durden* *Neil Bogart Memorial Laboratories, Department of Pediatrics, Division of Hematology-Oncology, and †Division of Research Immunology and Bone Marrow Transplantation, Childrens Hospital Los Angeles Research Institute, University of Southern California School of Medicine, California

Abstract: Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated in response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck. A few reports describe Cbl interactions in primary human hematopoietic cells. We show evidence that Cbl participates in signaling initiated by FcgRI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway. FcgRI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein. Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38kDa band identified as the CrkL adaptor protein. CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after FcgRI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after FcgRI stimulation and was associated with both Cbl and Grb2. Grb2 and Cbl binding to SLP-76 were inducible after FcgRI stimulation of the macrophages. Nck was inducibly bound to Cbl after FcgRI stimulation, whereas Grb2 was constitutively associated with it. Shc was also inducibly tyrosine phosphorylated and bound to Grb2 after FcgRI stimulation of the macrophages. PP1, a specific inhibitor of Src kinases, inhibited the FcgRI-induced respiratory burst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL. These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and the association of Cbl with CrkL, SLP-76, and Nck in FcgRI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity. J. Leukoc. Biol. 65: 523–534; 1999. Key Words: adaptor protein · CrkL · cross-linking · ITAM · SLP-76 · Nck

INTRODUCTION Signal transduction events initiated by stimulation of Fc receptors are important to understanding processes such as immune reactions, inflammation, autoimmunity, and leukemic transformation. FcgRI, the high-affinity Fc receptor for monomeric IgG (CD64), is a member of the immunoglobulin gene superfamily, which includes the T cell receptor, the B cell receptor, and Fc receptors such as the high-affinity receptors for IgA (FcaR) and IgE (FceR) [1, 2]. Fc receptors are unique in that they do not possess intrinsic kinase activity, but mediate downstream signaling events through a conserved stretch of amino acids, the immunoreceptor tyrosine-based activation motif (ITAM), which resides in the cytoplasmic region of the associated g-chains [3–7]. Fc receptor activation is associated with rapid tyrosine phosphorylation of multiple cellular proteins including the adaptor protein, Cbl, which is complexed with the g-subunit of FcgRI [8–12]. The c-cbl proto-oncogene product was first identified as the cellular homolog of the viral transforming protein of the murine Cas NS-1 retrovirus [13, 14]. The endogenous molecule, mammalian Cbl, is a widely expressed cytoplasmic complex adaptor protein that becomes tyrosine phosphorylated in response to events such as cytokine stimulation [8, 15–23], T cell or B cell receptor activation [24–29], and Fc receptor activation [11, 12, 24]. By virtue of its proline-rich regions and its phosphotyrosine-binding (PTB) domain Cbl binds kinases and adaptor proteins containing SH2 or SH3 domains (e.g., Nck, Grb2, Crk, CrkL, Shc, Syk, Fyn, and Lyn) in both hematopoietic as well as non-hematopoietic cells [8, 9, 11, 12, 15, 20, 24, 30–37]. The SH2- and SH3-containing CrkL adaptor protein

Abbreviations: BMHM, bone marrow-derived human macrophages; BSA, bovine serum albumin; FcR, Fc receptor; PMA, phorbol myristate acetate; SH domain, src homology domain; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; U937IF, interferon-g-differentiated U937 cells; PTB, phosphotyrosine binding; M-CSF, macrophage colony-stimulating factor; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; MM, macrophage medium; IMDM, Iscove’s modified Dulbecco’s medium; FITC, fluorescein isothiocyanate; HBSS, Hanks’ balanced salt solution; EGF, epidermal growth factor. Correspondence: Donald L. Durden, Division of Pediatric HematologyOncology, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Mailstop No. 57, Los Angeles, CA 90027. E-mail: [email protected] Received August 11, 1998; revised October 30, 1998; accepted November 14, 1998.

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binds Cbl in transformed cells [20, 24, 32, 38–40], as well as in normal T cells [24]. CrkL is constitutively tyrosine phosphorylated in BCR/Abl and v-Abl-transformed cells [41–45], but not in non-BCR/Abl-expressing human hematopoietic cells [41] and normal platelets [45]. CrkL can be inducibly phosphorylated in human papilloma virus-transformed mammary cells in response to epidermal growth factor (EGF) [20] and in normal human platelets stimulated by thrombopoietin [45]. The physiological importance of Cbl is exemplified in mouse osteoclasts, where Cbl functions in bone resorption downstream of c-Src [46] and in T cell anergy, where it shields CrkL and Grb2 from interacting with C3G and SOS [12, 47]. Recent experiments in Cbl-deficient mice further demonstrate that Cbl is critical in normal hematopoiesis and control of cellular proliferation [48]. Since its cloning, Cbl has been studied mostly in cell lines [9, 15, 23, 24, 31–35, 38, 49], with only a few reports describing Cbl interactions in non-human [8, 46, 50] or human [20, 24] primary cells. Although several groups have published reports on Cbl interactions in Fc receptor signaling [8–10], little has been published about the function of Cbl following specific activation of the high-affinity FcgRI receptor for monomeric IgG (CD64) and, to date, this interaction has not been studied in detail in primary human macrophages. In addition, although the CrkL-Cbl interaction has been studied in transformed cells [11, 20, 32, 38–40, 51] and in responses to EGF [20, 52] and T cell receptor activation [24, 47], little is known about the CrkL-Cbl association in Fc receptor signal transduction in normal hematopoietic cells. In this report we describe molecular interactions of the Cbl adaptor protein in response to specific FcgRI stimulation in a pure population of human bone marrow-derived macrophages. Macrophages were derived from the nonadherent CD342 cells from human bone marrow, which were expanded and differentiated in macrophage colony-stimulating factor (M-CSF). We show that Cbl was rapidly and transiently tyrosine phosphorylated in response to FcgRI cross-linking on the bone marrowderived primary human macrophages (BMHM). Immunoprecipitated Cbl was complexed with other tyrosine-phosphorylated proteins after FcgRI stimulation, the most prominent of which was p38, which we show to be to CrkL. Tyrosine-phosphorylated Cbl bound inducibly to CrkL and to the recently characterized SLP-76, both of which were inducibly tyrosine phosphorylated in the FcgRI-stimulated macrophages. The Cbl-CrkL interaction involved most of the cellular tyrosine-phosphorylated Cbl and CrkL, which constituted only a small fraction of the total cellular Cbl and CrkL pool. The Nck adaptor protein, which in cell lines is constitutively complexed with Cbl, was also complexed with Cbl in our unstimulated macrophages, and the interaction was inducible above baseline after FcgRI crosslinking. Both Grb2 and tyrosine-phosphorylated Cbl were associated with tyrosine-phosphorylated SLP-76 in resting BMHM and showed an enhancement of the interaction on FcgRI stimulation. Shc, which became robustly tyrosine phosphorylated in FcgRI-stimulated cells, inducibly bound to Grb2 after activation. Grb2 was constitutively bound to Cbl in the human macrophages and the complex did not undergo change after FcgRI cross-linking. The Src family kinase inhibitor, PP1, inhibited the FcgRI-induced generation of superoxide, while 524



preventing tyrosine phosphorylation of Cbl and the formation of a Cbl-Crkl complex. These results suggest that tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and Cbl association with CrkL, SLP-76, and Nck, function in FcgRI signaling in primary bone marrow-derived human macrophages. Our PP1 experiments demonstrate that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.

MATERIALS AND METHODS Antibodies and reagents Sodium orthovanadate, PAO, bovine serum albumin (BSA, Cohn fraction V), and all other chemicals were obtained from Sigma (St. Louis, MO). Pansorbin was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). Glutathioneconjugated Sepharose 4B beads were purchased from Pharmacia (Piscataway, NJ). FcgRI-specific monoclonal antibodies [32.2 and 22 F(Ab’)2 fragments or whole antibodies, and 197, an a-CD64 whole monoclonal antibody] were gifts from Medarex, Inc. (Annandale, NJ). Monoclonal anti-phosphotyrosine antibodies (G410) and a-Shc and a-Nck polyclonal antibodies were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). a-Cbl (SC117) and a-CrkL (SC319) polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). a-Grb2 and a-Shc monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). a-SLP-76 polyclonal antibodies were a gift from Gary A. Koretzky (University of Iowa, Iowa City, IA). Rabbit a-mouse F(Ab’)2 fragment and rabbit pre-immune IgG were purchased from Cappel Laboratories (Durham, NC). Enhanced chemiluminescence (ECL) reagents and horseradish peroxidase (HRP)-conjugated a-rabbit and a-mouse IgG for detection were purchased from Amersham Life Sciences (Arlington Heights, IL). Alkaline phosphatase-conjugated a-rabbit and a-mouse IgG for detection were purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). The Src-selective tyrosine kinase inhibitor, PP1 [4-amino-5(4-methylphenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], was obtained from Calbiochem (La Jolla, CA) [53].

Cell culture and macrophage preparation Filters used to remove bony spicules from marrow harvested for clinical indications were used as a source of macrophage progenitors. Use of the samples was approved by the Childrens Hospital Committee on Clinical Investigation. Cells were removed by flushing the filter well with phosphatebuffered saline (PBS, BioWhittaker, Walkersville, MD). Each sample was then treated with Ortho-mune Lysing Reagent (Becton Dickinson, San Jose, CA) to remove red blood cells. After this lysis step, leukocytes were washed, counted, and plated in 75-cm2 flasks at a concentration of 106/mL in macrophage medium (MM), with 15 mL cell suspension per flask. Macrophage medium was Iscove’s modified Dulbecco’s medium (IMDM, GIBCO) with 20% heatinactivated fetal bovine serum (Omega Scientific, Tarzana, CA), 2 mM L-glutamine, 100 U/mL penicillin, 10 µg/mL streptomycin (all from GIBCOBRL, Gaithersburg, MD), and 50 ng/mL recombinant human M-CSF (R & D Systems, Minneapolis, MN). The flasks were cultured flat in a 37°C incubator with 5% CO2 and full humidity for 2 h to overnight, to remove stromal fibroblasts. Nonadherent cells were then transferred to a clean flask, and the adherence process repeated three times during the first 48 h of culture. After the adherence steps, the nonadherent cells were cultured for 1 week under the same conditions. At the end of the first week of culture, the nonadherent cells were moved to a new flask in fresh MM, and the adherent layer was re-fed. After a total of 12 days of culture, nonadherent cells were removed from all flasks and the monolayers were flushed well with PBS. The adherent macrophage monolayers were re-fed MM and cultured an additional 1–2 days before harvest. After flushing away all nonadherent cells, macrophages were collected with a sterile cell scraper, counted by trypan blue exclusion, subjected to FACS analysis to determine the purity, and plated in 100-mm dishes at a concentration of 5 3 106 cells per plate in 10 mL of MM (for biochemical assays) or as specified (for measurement of superoxide production or phagocytosis). U937IF cells were grown and differentiated in interferon-g as described [11, 12].

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FACS analysis to determine purity of macrophage preparations

Characterization of primary BMHM monolayers

The fluorescein isothiocyanate (FITC)-conjugated antibodies used to test the purity of the macrophage preparations were as follows: HLE-1 (anti-CD45, Becton Dickinson), My9-RD1 (anti-CD33, Coulter, Hialeah, FL), HPCA2 (anti-CD34, Becton Dickinson), Leu-4 (anti-CD3, Becton Dickinson), Leu-3a (anti-CD4, Becton Dickinson), Leu-2a (anti-CD8, Becton Dickinson), Leu-12 (anti-CD19, Becton Dickinson), Leu-M7 (anti-CD13, Becton Dickinson), Leu-M3 (anti-CD14, Becton Dickinson), glycophorin A (Becton Dickinson), and Stro-1 (donated by Beverly Torok-Storb [54]). Cell suspensions were preincubated for 15 min on ice with a mixture of unconjugated mouse immunoglobulin (MsIgG, Coulter) and human immune globulins (USP, for intramuscular injection, Cutter Biological, Elkhart, IN) for blocking. Antibodies were added into the blocked tubes at the concentrations specified by the producer. After a 15-min antibody binding period on ice, cells were washed and fixed in 1% paraformaldehyde. Samples with the non-conjugated anti-CD64 [22 F(Ab’)2, anti-FcgRI, Medarex) were incubated with the primary antibody for 10 min on ice followed by a 15-min incubation with FITC-labeled goat anti-mouse IgG, followed by washing and fixing as above. Samples were acquired on a Becton Dickinson FACScan and analyzed using the CellQuest software package (Becton Dickinson).

FcgRI cross-linking and the respiratory burst assay FcgRI cross-linking in the macrophages and U937IF cells was performed as previously described for U937IF cells [11, 55] with the following modifications for BMHM. Briefly, 10-cm plates (two plates, 107 cells/sample) were washed twice with ice-cold Hanks’ balanced salt solution (HBSS), incubated with primary antibody [a-FcgRI monoclonal, 32.2, 22, or 197, F(Ab’)2 or whole antibody, on ice for 30 min], pre-warmed to 37°C, and cross-linked with rabbit a-mouse antibody [F(Ab’)2 fragment] at 37°C. At the completion of stimulation the plates were transferred to ice and the medium was rapidly aspirated. Cell lysates were prepared by the addition of ice-cold lysis buffer [11, 55] and prompt scraping of the cells with a rubber policeman. The remainder of the procedure was identical to that described for U937IF [11, 55]. The respiratory burst (e.g., generation of superoxide anions) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c as described previously [11, 56, 57].

Immunoprecipitation and immunoblotting Immunoprecipitation and immunoblotting were performed as described [11, 55]. Controls for nonspecific immunoprecipitation included in each experiment were incubated with rabbit IgG. Proteins were transferred to nitrocellulose filters (0.8 mA-h/cm2 over 2 h) using a dry transfer system (Ellard, Inc., Seattle, WA), as described [58]. After reacting membranes with monoclonal a-phosphotyrosine antibody, membranes could be directly reacted overnight with specific polyclonal antibodies without detection of any residual signal from the first reaction with the monoclonal antibody. If, on the other hand, the second antibody was also monoclonal, the membranes were stripped in 100 mM 2-b-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mM Tris-HCl, pH 6.7, at 50°C for 30 min with gentle rocking, followed by washing and re-blocking as above before incubation with the detecting antibody.

RESULTS One of the major proteins phosphorylated in hematopoietic cells after ITAM stimulation is Cbl, which is associated with Src family kinases and SH3- and SH2-containing adaptor proteins [8, 9, 11, 12, 33]. We sought to further elucidate the specific role of Src kinases in Cbl phosphorylation after specific stimulation of the FcgRI receptor in primary human myeloid cells and to determine the role of Src in the control of NADPH oxidase activation in myeloid cell signaling.

Cells recovered from the screens used to filter bone marrow after harvest were used as a source of monocyte/macrophage progenitors. The macrophages were expanded from the plasticnonadherent fraction in medium containing fetal calf serum and M-CSF. Monolayers of differentiated macrophages with greater than 95% purity were generated and were used in the experiments described below. Characterization of the purity of the primary human macrophage preparations was determined by antibody reactivity to cell surface determinants through the use of FACS analysis. Samples of macrophage preparations were tested for purity by incubation with antibodies that would detect cell surface markers on contaminating cells of other lineages. The monolayers did not contain cells expressing CD3 (T cells), CD19 (B cells), CD13 (granulocytes), glycophorin A (erythroid lineage), or Stro-1 (stromal fibroblasts), as shown in Table 1. At least 95% of the cells in each preparation were mature macrophages, as determined by expression of the CD14 antigen. The remaining cells were primarily comprised of monocyte/macrophage progenitors that expressed CD33, and had not yet induced expression of CD14 (Table 1). The BMHM displayed FcgRIdependent physiological responses such as phagocytosis of IgG-coated sheep red blood cells (data not shown) and production of superoxide in response to immune complex (BSA-aBSA) stimulation [4.9 6 0.4 nmol superoxide (SD) per 106 cells over 30 min]. In addition, the macrophages also produced superoxide in response to stimulation with 1 µM phorbol myristate acetate (PMA; 12.1 nmol superoxide/30 min/106 cells), further supporting their identity as functional macrophages.

Cbl is tyrosine phosphorylated in response to FcgRI stimulation in primary human macrophages Rapid and transient tyrosine phosphorylation of multiple cellular proteins was observed following specific cross-linking of FcgRI in the BMHM (Fig. 1A). As demonstrated in Figure 1A, the general pattern of tyrosine-phosphorylated proteins in BMHM cell lysates was similar to data obtained in the human myeloid cell lines THP-1 and interferon-g-differentiated U937 TABLE 1.

BMHM are a Homogeneous Population of Cells that Express FcgRI % BMHM that expressed this marker

Marker

Ascites (negative control) CD14 CD64 (FcgRI) CD341/CD141 CD331/CD142 CD13 CD3 CD19 Glycophorin A Stro-1

0.02 99.5 98.75 1.43 0.5 0.00 0.00 0.00 0.00 0.03

Cell types that are typically positive for this marker

None Macrophages Myeloid cells Myeloid progenitors Myeloid progenitors Granulocytes T cells B cells Erythrocytes Stromal cells

Typical cell-surface determinant (CD) expression on macrophages differentiated ex vivo was examined by FACS analysis as described in Materials and Methods.

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molecules are highly overexpressed and show increased phosphorylation in interferon-g-differentiated U937 cells [56, 59]. Anti-FcgRI or rabbit a-mouse antibodies alone did not induce the tyrosine phosphorylation response to FcgRI cross-linking in the macrophages (Fig. 1A, compare lanes 1–3 and lanes 4–6). As can be seen from Figure 1, although most of the phosphoproteins on the blot appear similar, some differ between the three cell types (e.g., p52, p60, p65). This demonstrates that the primary BMHM and the myelomonocytic cell lines U937IF and THP-1 undergo a similar, albeit not identical, pattern of tyrosine phosphorylation after FcgRI stimulation. The Cbl adaptor protein is a major substrate for tyrosine kinases in several myeloid cell lines and in mouse peritoneal macrophages after stimulation of Fcg receptors [8–12]. Antiphosphotyrosine blotting of Cbl immunoprecipitates from BMHM shows that Cbl was rapidly and transiently tyrosine phosphorylated on specific ligation of the FcgRI receptor (Fig. 1, B and C). The maximal level of tyrosine phosphorylation occurred 3 min after the start of stimulation and declined thereafter (Fig. 1B). Thus, Cbl tyrosine phosphorylation after FcgRI stimulation follows a similar pattern to that seen in myeloid cells lines [9, 11, 12].

CrkL is tyrosine phosphorylated in BMHM in response to FcgRI cross-linking

Fig. 1. Cbl is tyrosine phosphorylated after FcgRI stimulation of macrophages. (A) a-phosphotyrosine blot of whole cell lysates of BMHM, U937IF, and THP-1 cells (2.6 3 105 cell equivalents per lane) after FcgRI [32.2 F(Ab’)2] cross-linking. Cross-linking was done as described in Materials and Methods. Lane 1, BMHM incubated without any stimulating antibodies; lane 2, lysate from BMHM stimulated only with the rabbit a-mouse cross-linking antibody for 3 min; lanes 3–6, lysates of BMHM cross-linked for 0–10 min. Equivalent numbers of U937IF cells (lanes 6 and 7) and THP-1 cells (lanes 10–12) were similarly cross-linked for comparison. (B and C) BMHM were stimulated by FcgRI cross-linking (22, whole antibody) followed by immunoprecipitation with polyclonal a-Cbl antibody (107 cells/immunoprecipitation). No cross-linking antibody was added to lane 1. Lanes 2–5 were cross-linked for 1–30 min before Cbl immunoprecipitation. Lane 6 was cross-linked for 3 min but was subsequently immunoprecipitated with pre-immune rabbit IgG. Lane 7, lysate of FcgRI-stimulated U937IF cells (2.5 3 105 cell equivalents, positive lysate) stimulated for 1 min. (A) a-phosphotyrosine blot; (B) a-Cbl blot.

cells, with some variation (Fig. 1A and unpublished data). The most prominent FcgRI-inducible tyrosine phosphorylation in the macrophage lysates was of proteins with molecular mass of 120–130 kDa. As visualized by Coomassie blue staining (not shown), despite having an equal number of cells in each sample (2.6 3 105 cells/lane), the lanes containing the U937IF lysates (lanes 79) had significantly more total protein per lane than the lanes containing either BMHM (Fig. 1A, lanes 1–6) or THP-1 (lanes 10–12) cell lysates, accounting for the more pronounced phosphotyrosine signal in Figure 1A. Lanes with THP-1 lysates had an intermediate amount of protein per lane with an equivalent cell number (not shown). Relevant to the increased tyrosine phosphorylation observed in the U937IF cells (Fig. 1A, lanes 7–9) are previous data demonstrating that some signaling 526



Cbl, a complex adaptor protein, associates with some signaling molecules constitutively and with others it forms an inducible complex. CrkL, an adaptor that binds Cbl in several cell lines, is constitutively tyrosine phosphorylated in transformed cells expressing the BCR/ABL protein [41, 45, 60], where it demonstrates slower electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared to non-tyrosine-phosphorylated CrkL [41]. CrkL can be inducibly tyrosine phosphorylated in response to several stimuli in transformed cell lines [61–64], but only a few reports describe tyrosine phosphorylation of CrkL in normal cells [45, 60]. To assess the inducible phosphotyrosine pattern in the primary myeloid cells we immunoprecipitated CrkL from resting and FcgRI-stimulated BMHM (Fig. 2). CrkL immunoprecipitations of lysates of resting BMHM displayed low level tyrosine phosphorylation of CrkL (Fig. 2, C and D, lane 1), whereas FcgRI-stimulated BMHM displayed an intense FcgRI stimulation-induced tyrosine-phosphorylated band of 38 kDa (Fig. 2C, lanes 1 and 2). Reprobing of the blot with a-CrkL antibodies demonstrated several CrkL-reactive bands (Fig. 2D, lanes 1 and 2), which probably represent CrkL species of differing levels of tyrosine phosphorylation [41]. The tyrosinephosphorylated band in Figure 2C, lanes 1 and 2, aligned with one of the slower migrating CrkL-reactive bands (Fig. 2D, lanes 1 and 2). A fainter tyrosine-phosphorylated band was also visible on the blot below the major CrkL tyrosine-phosphorylated band and superimposed directly with the major CrkLreactive band (Fig. 2, C and D, lanes 1 and 2). By additionally probing CrkL immunoprecipitations with a monoclonal a-CrkL antibody we verified that indeed, as shown here, the immunoprecipitated CrkL was composed of two to four closely migrating bands and that the major tyrosine-phosphorylated CrkL species corresponded to a relatively less intense CrkL-reactive band http://www.jleukbio.org

Fig. 2. CrkL is tyrosine phosphorylated and associated with Cbl in BMHM following FcgRI cross-linking. BMHM (107 cells/immunoprecipitation) were either not stimulated or incubated with the 32.2 (FAb’)2 a-FcgRI antibody, cross-linked for 3 min, and then immunoprecipitated with a-CrkL (lanes 1 and 2) or aCbl (lanes 3 and 4). Lane 5, BMHMs stimulated for 3 min and immunoprecipitated with pre-immune rabbit IgG. Lanes 6 and 7, lysates of FcgRI-stimulated BMHM or U937IF cells, respectively (2.5 3 105 cells/lane). (A) a-phosphotyrosine blot; (B) a-Cbl blot; (C) a-CrkL blot; (D) a-phosphotyrosine blot.

that migrated more slowly than the major CrkL-reactive band (Fig. 2D and data not shown). Shorter exposures of these CrkL blots also demonstrated a stimulation-dependent increase in the intensity of the slower-migrating CrkL bands (data not shown). Thus, CrkL is strongly tyrosine phosphorylated in response to FcgRI stimulation in primary non-transformed BMHM.

CrkL associates with phosphorylated Cbl in response to FcgRI cross-linking in the human macrophages Several groups have shown that in cells expressing BCR/Abl or v-Abl the CrkL adaptor protein is tyrosine phosphorylated and constitutively associated with Cbl [32, 38, 39]. CrkL-Cbl interactions have also been described after T and B cell receptor activation [24, 40, 47] and after stimulation by EGF [20]. To examine whether the Cbl-CrkL interaction could be induced in our non-transformed primary macrophages in response to FcgRI stimulation, we examined the above CrkL immunoprecipitations of FcgRI-stimulated and unstimulated BMHM lysates for co-precipitated Cbl (Fig. 2, lanes 1 and 2). The CrkL immunoprecipitations of lysates of FcgRI-stimulated

BMHM co-precipitated an induced tyrosine-phosphorylated band of 120 kDa, in the area where Cbl normally migrates (Fig. 2A lanes 1 and 2, compare to lanes 3 and 4). Reprobing of the blots with specific a-Cbl antibodies demonstrated that the p120 tyrosine-phosphorylated band superimposed on the Cblreactive band (Fig. 2, A and B, lanes 1 and 2), migrated with the same electrophoretic mobility as Cbl from Cbl immunoprecipitation (Fig. 2B, lanes 3 and 4), and was inducibly complexed with CrkL after FcgRI stimulation (Fig. 2B, lanes 1 and 2). Reciprocal Cbl immunoprecipitation demonstrated a FcgRIinduced interaction of CrkL with Cbl (Fig. 2D, lanes 3 and 4), complementing the results seen in the CrkL immunoprecipitation. To verify that indeed the tyrosine phosphorylation of this pp120 was primarily on Cbl, we immunodepleted the lysates of Cbl by performing a Cbl immunoprecipitation before the CrkL immunoprecipitation. As a result, a large proportion of the phosphorylated pp120 and the corresponding Cbl-specific band were depleted by the Cbl pre-clearing, demonstrating that indeed most of the pp120 associated with CrkL was comprised of Cbl (data not shown). The reciprocal immunodepletion (CrkL pre-clearing followed by a Cbl immunoprecipitation) demonstrated similar depletion of the Cbl-associated CrkL and its corresponding phosphotyrosine band (data not shown). A larger protein of about 140 kDa was consistently found to coprecipitate with CrkL (Fig. 2A, lane 2). Although the BMHM express p130Cas, the 140-kDa protein migrates in a position higher than that of both Cbl and p130Cas (not shown), and may thus represent another CrkL-associated protein. This 140-kDa tyrosine-phosphorylated band was not affected by the Cbl pre-clearing, indicating that although it complexed with CrkL, it was distinct and independent of the Cbl-CrkL complex (data not shown). Immunoprecipitations with rabbit IgG did not contain specific Cbl, CrkL, or phosphotyrosine-reactive bands (Fig. 2, lane 5). Thus, CrkL and Cbl are inducibly complexed in response to FcgRI cross-linking in primary BMHM. The reciprocal Cbl and CrkL immunoprecipitations allowed us to estimate the proportion of the total cellular Cbl, which is associated with CrkL and vice versa in FcgRI-stimulated BMHM (Fig. 2). The Cbl immunoprecipitations contained a significantly larger amount of Cbl than that found in the CrkL immunoprecipitations of FcgRI-stimulated BMHM (Fig. 2B, compare lanes 2 and 4). This demonstrates that only a small amount of the total cellular Cbl is complexed with CrkL after FcgRI stimulation. It is interesting to note that the relatively smaller amount of Cbl in the CrkL immunoprecipitation is highly tyrosine phosphorylated, to a degree comparable to the tyrosine phosphorylation of Cbl in the Cbl immunoprecipitations, which contain a much higher amount of Cbl (Fig. 2, A and B, lanes 2 and 4, and shorter exposures, not shown). Similar results were obtained in multiple experiments in the BMHMs as well as in myeloid cell lines (data not shown). This suggests that only a small proportion of the total cellular Cbl and CrkL are tyrosine phosphorylated after FcgRI stimulation, and that these phosphorylated Cbl and CrkL were tyrosine phosphorylated to a high degree, possibly on multiple sites. Based on the similar amounts of tyrosine-phosphorylated Cbl and CrkL in the respective Cbl and CrkL immunoprecipitations, and the large differences in total Cbl and CrkL in the same immunoprecipita-



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tions, it seems that the Cbl-CrkL interaction occurs predominantly between the highly tyrosine-phosphorylated protein species. Further support of the phosphotyrosine dependence of the Cbl-CrkL interaction was shown by potato-acid phosphatase treatment of FcgRI-stimulated U937IF cells, which completely abrogated the Cbl-CrkL interaction, whereas increasing tyrosine phosphorylation by pretreating the cells with the phosphatase inhibitors PAO and vanadate enhanced it [11, and data not shown]. We conclude that a large proportion of the highly tyrosine-phosphorylated Cbl and CrkL, which comprise only a small proportion of the total cellular Cbl and CrkL pool, complex with each other after FcgRI stimulation in primary BMHM.

SLP-76 becomes tyrosine phosphorylated and associated with Cbl in response to FcgRI stimulation of human macrophages One of the proteins to be tyrosine phosphorylated in the human macrophages following FcgRI cross-linking was a 76-kDa protein (Fig. 1A). The SH2-containing Grb2-binding protein, SLP-76, associates with a 120-kDa tyrosine-phosphorylated protein in Jurkat cells and in FceRI-stimulated RBL-2H3 mast cell analogs [65, 66], suggesting that this may represent an interaction between SLP-76 and the 120-kDa adaptor protein Cbl. Data from our laboratory show that Cbl interacts with SLP-76 in U937IF cells in response to FcgRI stimulation [67]. To date, a Cbl-SLP-76 complex has not been described in primary hematopoietic cells. To delineate the nature of the SLP-76-Cbl interaction in the primary human macrophages we performed SLP-76 immunoprecipitations in FcgRI-stimulated BMHM cells (Fig. 3). SLP-76 was modestly tyrosine phosphorylated in resting human macrophages, but showed a dramatic increase in tyrosine phosphorylation following FcgRI crosslinking (Fig. 3A). Tyrosine phosphorylation of SLP-76 reached a maximum after 10 min and was linked with the inducible association of multiple tyrosine-phosphorylated proteins with the immunoprecipitated SLP-76 (Fig. 3A). Grb2, one of the adaptor proteins known to bind SLP-76 [65], showed baseline binding to SLP-76 at rest, which increased in parallel to the time course of increasing tyrosine phosphorylation of SLP-76 (Fig. 3, A and C). Tyrosine-phosphorylated Cbl was also bound to SLP-76 at low levels in resting cells and showed increased association with it following its tyrosine phosphorylation and the phosphorylation of SLP-76 (Fig. 3, A and B). Hybridization of the blot with a-SLP-76 antibodies showed that equal amounts of SLP-76 were immunoprecipitated in this experiment (except in lane 4, in which slightly smaller amounts of SLP-76 are apparent). Incubation of the macrophages with a-FcgRI antibody alone did not result in increased tyrosine phosphorylation or changes in the associated proteins in the resting macrophages (Fig. 3, compare lanes 1 and 6). Nonspecific control immunoprecipitation of FcgRI-stimulated macrophage lysate with pre-immune rabbit IgG confirmed that the observed associations were indeed specific (Fig. 3, lane 7). We conclude that in BMHM SLP-76 is tyrosine phosphorylated in response to FcgRI stimulation and inducibly associates with Cbl and Grb2.

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Fig. 3. Cbl and Grb2 are inducibly complexed with SLP-76 after FcgRI stimulation of macrophages. BMHM were stimulated by FcgRI cross-linking [22 F(Ab’)2] for the indicated time followed by immunoprecipitation with polyclonal a-SLP-76 antibody (107 cells/immunoprecipitation). Lane 1 was incubated only with the primary antibody but was not cross-linked. Lanes 2–5 were cross-linked for 1–20 min. Lane 6, a-SLP-76 immunoprecipitation of macrophages that were incubated without any antibody. Lane 7 was crosslinked for 3 min but was immunoprecipitated with pre-immune rabbit IgG. Lane 8 is lysate of U937IF cells stimulated for 1 min (2.5 3 105 cell equivalents, positive lysate). (A) a-phosphotyrosine blot; (B) a-Cbl blot; (C) a-SLP-76 blot; (D) a-Grb2 blot.

The adaptor proteins Nck and Grb2 are associated with Cbl in FcgRI-stimulated human macrophages Following stimulation of Fcg receptors and the ensuing tyrosine phosphorylation of Cbl, Cbl becomes complexed with several kinases and adaptor proteins [8–11]. To examine which additional proteins were associated with Cbl in the BMHM, we stimulated BMHM by cross-linking of the FcgRI receptor followed by immunoprecipitation of Cbl from the cell lysates and subsequent Western blotting (Figs. 4 and 5). Figure 4 depicts a Cbl immunoprecipitate that demonstrates the rapid and transient tyrosine phosphorylation of Cbl and the association of several tyrosine-phosphorylated proteins after cross-linking of the FcgRI receptor with a specific a-FcgRI monoclonal antibody (Fig. 4A). A small amount of Nck (46 kDa) bound to Cbl at rest (Fig. 4C, lane 1). After FcgRI stimulation


Shc becomes tyrosine phosphorylated in response to FcgRI stimulation of human macrophages In U937IF cells Shc becomes tyrosine phosphorylated and complexed with Cbl after FcgRI stimulation [11, 12, 55]. We therefore examined BMHM for Shc-Cbl interactions. FcgRI stimulation of the BMHM resulted in increased tyrosine phosphorylation of Shc, as can be seen from a-Shc immunoprecipitations of FcgRI-stimulated and unstimulated macrophages (Fig. 6, A and B). Both the 52- and 46-kDa isoforms were phosphorylated at rest and showed increased tyrosine phosphorylation after FcgRI cross-linking (Fig. 6A, lanes 1–4). Grb2 interacted with Shc in a stimulation-inducible manner in the human macrophages, demonstrating a low level baseline association that increased with the increased tyrosine phosphorylation of Shc after FcgRI receptor stimulation (Fig. 6C). Despite this robust activation of Shc and the induced Grb2-Shc interaction, we could not demonstrate co-immunoprecipitation of Shc with Cbl or Cbl with Shc in the BMHM in multiple experiments (not shown), suggesting that this association is either below our level of detection or is not present in the macrophages. From this we conclude that Shc is tyrosine phosphorylated and activated after FcgRI stimulation in the BMHM and that in its activated state it recruits Grb2.

Src kinase inhibitor abrogates FcgRI-induced Cbl phosphorylation and NADPH oxidase activity in BMHM Hanke et al. reported the development of a new class of protein tyrosine kinase inhibitors, PP1 and PP2, which are specific for

Fig. 4. Nck is complexed with Cbl after FcgRI stimulation of human macrophages. BMHM were stimulated by FcgRI cross-linking [32.2 F(Ab’)2] followed by immunoprecipitation with polyclonal a-Cbl antibody (107 cells/ immunoprecipitation). Lane 1 (no stimulation) was incubated only with the primary antibody but was not cross-linked. Lanes 2–4 were cross-linked for 1–10 min. Lane 5 was cross-linked for 3 min but was immunoprecipitated with pre-immune rabbit IgG. Lane 7 is lysate of U937IF cells (2.5 3 105 cell equivalents, positive lysate) stimulated for 1 min. (A) a-phosphotyrosine blot; (B) a-Cbl blot; (C) a-Nck blot.

of the macrophages the amount of Nck that bound to Cbl increased (Fig. 4C, lanes 3 and 4). Similar immunoprecipitations in FcgRI-stimulated U937IF cells also showed Nck bound to Cbl but the Cbl-Nck interaction in the U937IF cells was always constitutive and did not change after FcgRI stimulation (data not shown). As seen from the a-Cbl immunoprecipitation shown in Figure 5, Grb2 was constitutively associated with Cbl, without significant change in the stoichiometry of the Grb2 associated with Cbl after FcgRI stimulation. The amount of Grb2 bound to Cbl did not change after tyrosine phosphorylation of Cbl or its subsequent dephosphorylation after up to 30 min of stimulation (Fig. 5C).

Fig. 5. Grb2 is constitutively complexed with Cbl in BMHM. BMHM were stimulated by FcgRI cross-linking (22, whole antibody) followed by immunoprecipitation with polyclonal a-Cbl antibody (107 cells/immunoprecipitation). Lane 1 was incubated in the absence of any antibodies. Lanes 2–5 were cross-linked for 1–30 min. Lane 6 was cross-linked for 3 min but was immunoprecipitated with pre-immune rabbit IgG. Lane 7 is lysate of BMHM (2 3 105 cell equivalents) stimulated for 1 min. (A) a-phosphotyrosine blot; (B) a-Cbl blot; (C) a-Grb2 blot.



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A

B

C

D Fig. 6. Shc is tyrosine phosphorylated and inducibly binds Grb2 after FcgRI stimulation in BMHM. BMHM were stimulated by FcgRI cross-linking [22, F(Ab’)2] followed by immunoprecipitation with polyclonal a-Shc antibody (107 cells/immunoprecipitation). Lane 1 was incubated in the absence of any antibodies. Lanes 2–4 were cross-linked for 1–10 min. Lane 5 was cross-linked for 3 min but was immunoprecipitated with pre-immune rabbit IgG. Lane 6 is lysate of U937IF cells (2.5 3 105 cell equivalents, positive lysate) stimulated for 1 min. (A) a-phosphotyrosine blot; (B) a-Shc blot; (C) a-Grb2 blot.

Src family kinases when applied to intact cells at micromolar concentrations [53]. Experiments performed with PP1 revealed that the FcgRI-induced tyrosine phosphorylation of Cbl was inhibited in the presence of 20 µM PP1 (Fig. 7A). This inhibition was observed both when cross-linking the cells with the specific FcgRI antibody, 32.2 F(ab’)2, as well as when using BSA-a-BSA insoluble immune complexes for stimulation (Fig. 7A). Anti-Cbl immunoblots confirmed that similar amounts of Cbl were precipitated in the controls and the PP1-treated lanes (Fig. 7B). Inhibition of the FcgRI-induced Cbl tyrosine phosphorylation was also seen at concentrations of PP1 as low as 5 µM (data not shown). The tyrosine phosphorylation-dependent interaction between Cbl and the CrkL adapter protein was markedly inhibited by PP1, whereas PP1 had no effect on the constitutive Cbl-Grb2 binding (Fig. 7, C and D, compare lanes 2 and 3 to 4 and 5). This demonstrates that Src kinases function upstream of Cbl, and their activation is necessary for the up-regulation of the Cbl-CrkL interaction in FcgRI-induced signaling in BMHM. The inhibition of Cbl tyrosine phosphorylation by PP1 correlated with marked inhibition of the Fcg receptor-induced superoxide response by this Src kinase inhibitor (Fig. 7E). Macrophages pretreated with PP1 (10 µM) showed 80% inhibition of the FcgR-induced NADPH oxidase activity (Fig. 7E). It is important to note that the induction of NADPH oxidase activity by activation of protein kinase C, using PMA,

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Fig. 7. Effect of the Src-kinase inhibitor, PP1, on Cbl phosphorylation and oxidant signaling. (A–D) BMHM (107 cells/sample) were preincubated in the presence of 20 µM PP1 or an equivalent volume of DMSO (D) followed by specific cross-linking of FcgRI (lanes 2 and 3) or stimulation with insoluble (BSA-a-BSA) immune complexes (lanes 4 and 5) for 3 min. Lane 1 is unstimulated control. Immunoprecipitation was performed with polyclonal Cbl antisera (lanes 1–5) or rabbit IgG (lane 6, control). Lane 7 is lysate of U937IF cells (2.5 3 105 cell equivalents, positive lysate) stimulated for 1 min. (A) a-phosphotyrosine blot; (B) a-Cbl blot; (C) a-Crkl blot; (D) a-Grb2 blot. (E) Respiratory burst response (superoxide production) of BMHMs (106 cells/ sample) after a 30-min incubation in the presence or absence of immune complexes (IC), with 10 µM PP1 (filled bars) or an equivalent volume of DMSO (stippled bars). Each bar depicts the means and standard deviations derived from three identical samples.


was not affected by PP1 (data not shown), a result that demonstrates specificity of this inhibitor for the Fc receptorinduced activation of the NADPH oxidase complex in these cells. From these data we conclude that myeloid Src kinases play an important role in the induction of the ITAM-induced respiratory burst in macrophages. In summary, our results show that BMHMs respond to FcgRI stimulation with vigorous tyrosine phosphorylation of multiple proteins, among them Cbl, CrkL, Shc, and SLP-76. Cbl forms inducible interactions with CrkL, SLP-76, and Nck, and maintains a constitutive interaction with Grb2. Shc is also inducibly tyrosine phosphorylated, forming inducible complexes with Grb2. Results obtained with the Src inhibitor, PP1, provide evidence that Cbl phosphorylation and the Cbl-CrkL interaction function in a Src kinase-dependent oxidant signaling pathway originating in FcgRI stimulation, and leading to activation of the NADPH oxidase complex.

DISCUSSION In this work we describe specific FcgRI-induced signaling events involving the Cbl adaptor protein in a pure population of primary nontransformed BMHMs. We show that Cbl became tyrosine phosphorylated and complexed with other proteins following cross-linking of FcgRI on the macrophages. Of the proteins complexed with Cbl in the BMHM we identified CrkL, SLP-76, Nck, and Grb2, but could not detect Shc. We provide evidence that FcgRI-induced Cbl phosphorylation and the Cbl-CrkL interaction function in a Src kinase-dependent oxidant signaling pathway originating in Fcg receptor stimulation, and leading to activation of the NADPH oxidase complex. We also report here for the first time tyrosine phosphorylation of CrkL in response to Fc receptor activation in normal nonleukemic primary human myeloid cells. FcgRI-induced Cbl tyrosine phosphorylation in the BMHM was rapid and transient, similar to that described in mouse peritoneal macrophages and human myeloid cell lines [8, 10–12] (Fig. 1). Several reports have shown Cbl to be associated with CrkL [20, 24, 32, 38–40]. This association is thought to be mediated via tyrosine phosphorylation of Cbl [38] and occurs in cells that express BCR/Abl or v-Abl [32, 38, 39] and in several other immortalized cell lines [20, 24, 40, 51, 68]. Cbl has also been shown to associate with CrkL in normal human T cells after stimulation of the T cell receptor [24]. Tyrosine phosphorylation of CrkL has also been described in transformed cell lines expressing activated Abl and in BCR/Abl-positive patient samples, where CrkL is constitutively tyrosine phosphorylated and exhibits a tyrosine phosphorylation-dependent retardation in its electrophoretic mobility [41, 42, 45]. In contrast, normal primary hematopoietic cells (human platelets and neutrophils and normal mouse bone marrow) only express the non-tyrosinephosphorylated, non-mobility-shifted form of CrkL [41, 45, 60]. To date, few reports have described tyrosine phosphorylation of CrkL in normal primary tissues [45, 60], implying that CrkL tyrosine phosphorylation may have an as yet undefined role in normal cells. The work presented here is the first description of an FcgRI-induced tyrosine phosphorylation of CrkL in normal

human macrophages. This suggests that, in addition to the recently described CrkL tyrosine phosphorylation following thrombopoietin stimulation in normal platelets [45], CrkL may also function in other hematopoietic pathways, mediating immune responses induced by activation of FcgRI. We present data demonstrating for the first time that induction of the Cbl-CrkL interaction requires activation of a Src kinase in myeloid cells (Fig. 7C). Whereas a significant amount of Cbl is tyrosine phosphorylated and associated with CrkL upon FcgRI stimulation in the U937IF and THP-1 cell lines (unpublished data), in the macrophages it is evident that only a small amount of the total cellular Cbl accounts for the prominently tyrosine phosphorylated Cbl band, and that it is this small amount of highly phosphorylated Cbl that participates in the interaction with CrkL (Fig. 3). This finding is in accordance with the preferential high-level phosphorylation of membrane-targeted Cbl seen after CSF-1 stimulation and integrin-mediated cell adhesion of macrophages [23, 69] and after lymphoid cell stimulation [70]. Thus, our data supports the concept that regulation of cellular responses by Cbl may couple Cbl phosphorylation to localization of activated complexes to the cell membrane. Experiments in our laboratory have recently identified the 120-kDa phosphorylated protein associated with SLP-76 in FcgRI-stimulated U937IF cells to be Cbl [67]. In the human macrophages Cbl and Grb2 associated with SLP-76 in a FcgRI stimulation-inducible manner (Fig. 3), suggesting that indeed, regulation of the Cbl-SLP-76 and SLP-76-Grb2 interaction may be important in biologically relevant events in non-transformed tissues and cells. These findings correlate well with recent data in our laboratory describing the SLP-76-Cbl interaction in the interferon-g-differentiated U937 myelomonocytic cell line [67]. Because the Cbl-Grb2 interaction is constitutive (Fig. 5), it is possible that both Cbl and Grb2 each bound directly to SLP-76, but it is also possible that one of them bound directly to SLP-76, and the other, by virtue of the constitutive Cbl-Grb2 interaction, bound indirectly to SLP-76. This could explain the parallel inducibility of both these interactions. In ongoing work in our laboratory we are currently trying to delineate the physiological significance of these inducible interactions. It is interesting to note that FceRI-dependent SLP-76 tyrosine phosphorylation requires Syk protein tyrosine kinase activation, placing SLP-76 downstream of Syk [66]. Syk has previously been shown to be involved in FcgRI signaling via its interaction with the ITAM-containing g-subunit of FcgRI [59] and to enhance Fc receptor-mediated phagocytosis [71]. Furthermore, Syk has been shown to associate with Cbl in the human erythroleukemia cell line HL60 [9] and in B cells [37], further supporting a potential role for the SLP-76-Cbl interaction in FcgRI signaling in normal hematopoietic cells. Nck, an adaptor protein that is comprised of three consecutive SH3 interaction domains and a carboxy-terminal SH2 domain, becomes phosphorylated in response to signaling by growth factors (e.g., EGF, platelet-derived growth factor, vascular endothelial cell growth factor) [72, 73] and the Fc receptors FceRI and FcgRII [74, 75]. Nck constitutively binds to Cbl both in vitro and in resting HL60 cells [33], as well as in U937IF cells stimulated with FcgRI or FcgRII [unpublished



531

data]. Here we showed that Nck was associated with Cbl in resting BMHM and that this interaction increased in response to FcgRI stimulation (Fig. 4). This is the first report of an inducible Cbl-Nck interaction in response to receptor stimulation in myeloid cells. We have shown that FcgRI stimulation results in a measurable respiratory burst in the BMHM as well as in U937IF cells [11, 57, 59]. Recent data from our laboratory also shows Cbl activation after similar stimulation [11, 12]. Because Nck is involved in signaling via p21-activated kinases (PAKs) [76, 77], which potentially link it to the small GTPases Rac and Cdc42, it is tempting to speculate that Cbl, via Nck, PAK, and Rac, may be involved in the regulation of the NADPH oxidase complex and motility responses in macrophages. Thus, this FcgRI-inducible Cbl-Nck interaction may represent a link between Fcg receptor activation and the respiratory burst and motility responses in myeloid cells. In conclusion, we showed that Cbl is a major substrate in FcgRI stimulation of normal human macrophages, and that it forms complexes with multiple adaptor proteins (CrkL, SLP-76, Nck, Grb2), some of which are activated in this pathway. Results obtained with the specific Src kinase inhibitor, PP1, provide the first evidence that Src kinases play a central role in macrophage oxidant signaling (Fig. 7, A–E). Our data further validate our use of myeloid cell lines (U937IF and THP-1) to study the molecular control of myeloid signaling events and suggest that PP1 may have anti-inflammatory potential in clinical applications.

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (RO1 CA7563701) and the American Cancer Society (RPG-98-244-01-LBC) to D.L.D., NIH grant NIDDK (RO1 DK 53041) to J.A.N. and a grant to the Neil Bogart Memorial Laboratories from the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research. D. L. D. was supported by the Robert E. and May R. Wright Foundation and is a recipient of the STOP Cancer Career Development Award. A. E. was supported by grants from the Concern Foundation and the Children’s Cancer Research Fund during this work. We thank Delia Ertl for her excellent technical assistance.

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