Signaling Immunoreceptor Tyrosine Activation Motif CBL-GRB2

CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling

This information is current as Rae Kil Park, Wade T. Kyono, Yenbou Liu and Donald L. Durden of September 26, 2021. J Immunol 1998; 160:5018-5027; ; http://www.jimmunol.org/content/160/10/5018 Downloaded from References This article cites 42 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/160/10/5018.full#ref-list-1

Why The JI? Submit online.

http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 26, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling1

Rae Kil Park,*† Wade T. Kyono,* Yenbou Liu,* and Donald L. Durden2*

In this study, we provide the ﬁrst evidence for role of the CBL adapter protein interaction in Fc␥RI receptor signal transduction. We study the Fc␥RI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-␥- differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc␥RI␥ of Fc␥RI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc␥RI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc␥RI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461–670) binds Downloaded from to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671–906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL- GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc␥RI signaling. The Journal of Immunology, 1998, 160: 5018–5027. http://www.jimmunol.org/ he cbl gene, originally described as the transforming gene with adapter proteins (e.g., GRB2, CRK, CRKL, NCK) that reg- of the Cas NS-1 murine retrovirus, induces pre-B cell ulate the guanine nucleotide exchange factors, “son of sevenless” T lymphomas and myeloid leukemias in mice (1, 2). Inter- (SOS) and C3G in mammalian cells following the activation of the estingly, the viral oncoprotein has lost a portion of its C terminus TCR (12–14). Marcilla et al. reported that stimulation of multiple that encodes the GRB2-SH3 binding site. The N terminus of the Fc␥R classes (Fc␥RI, Fc␥RII, and Fc␥RIII) in HL-60 cells with CBL protein is closely related to the SLI-1 gene product recently IgG/anti-IgG complexes induces the tyrosine phosphorylation of cloned in Caenorhabditis elegans, which is a putative negative CBL (9). Stimulation with these immune complexes results in the regulator of RAS in the LET-23 pathway (epidermal growth factor ␥ ␥ ␥

activation of Fc RI, Fc RIIA, and Fc RIII receptors, making it by guest on September 26, 2021 related) for vulval development (3). Several reports suggest that more difficult to interpret these results. Matsuo et al. and Tanaka et cbl p120 is involved in the regulation of small GTPases that are al. subsequently implicated CBL in Fc␥RII/III signaling in mac- activated by receptor protein tyrosine kinases (4–7). rophages and THP-1 cells, respectively (14, 15). The function of CBL is tyrosine phosphorylated following the activation of re- CBL tyrosine phosphorylation and/or the interaction of CBL with ceptors belonging to the Ig gene superfamily (TCR, B cell recep- adapter proteins as it relates to specific signaling through the ␥ tor, and Fc receptors). These multisubunit receptors signal Fc␥RI receptor in myeloid cells and the regulation of RAS have 3 through an immunoreceptor tyrosine activation motif (ITAM) not been thoroughly studied. (YXXLX6–8YXXL, consensus) (8–11). CBL is known to interact We investigated the role of the CBL adapter protein interaction following specific cross-linking of the Fc␥RI receptor in myeloid ␥ ␥ ␥ *Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, Children’s signaling. CBL is bound to the Fc RI subunit of the Fc RI re- Hospital Los Angeles Research Institute and University of Southern California School ceptor in myeloid cells, providing direct evidence that CBL is in- of Medicine, Norris Cancer Center, Los Angeles, CA 90027; and †Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan, volved in ITAM signaling. Our data demonstrate that CBL binds Korea in vitro to GRB2 and CRKL molecules via different domains of the Received for publication September 2, 1997. Accepted for publication January CBL C terminus. CBL is tyrosine phosphorylated after Fc␥RI 21, 1998. cross-linking, and this phosphorylation is associated with an al- The costs of publication of this article were defrayed in part by the payment of page tered CBL-GRB2 interaction. At the same time, the GRB2-SH2 charges. This article must therefore be hereby marked advertisement in accordance ␥ with 18 U.S.C. Section 1734 solely to indicate this fact. domain inducibly binds to SHC after Fc RI stimulation. These events are associated with the conversion of GDPras to GTPras 1 This work was partially supported by a grant from National Institutes of Health, RO1 CA 37256-01 to D.L.D. Work was performed in Neil Bogart Memorial Labo- (unpublished observation). Taken together, our data support a ratories, as supported by T. J. Martell Foundation for Leukemia, Cancer, and AIDS model by which the CBL-GRB2 interaction may modulate the in- Research. D.L.D. is supported by a Career Development Award from Children’s ␥ Hospital Los Angeles Research Institute and STOP Cancer Foundation and a grant teraction between GRB2 and SOS in Fc RI signaling in myeloid from Robert E. and May R. Wright Foundation through USC School of Medicine. cells. R.K.P. was supported by Wonkwang University in 1996. 2 Address correspondence and reprint requests to Dr. Donald L. Durden, Department of Pediatrics, Division of Hematology-Oncology (M/S #57), Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail address: Materials and Methods ddurden%[email protected] Antibodies 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine activation motif; ECL, enhanced chemiluminescence; GST, glutathione-S-transferase; IP, immunopre- The Fc␥RI␣-specific cross-linking Abs were generously provided by Me- cipitate; MAP, mitogen-activated protein; SOS, son of sevenless. darex (West Lebanon, NH). The mAb 197 and mAb 32.2 are specific for

␥ ␣ Ј the Fc RI subunit; mAb 32.2 is a F(ab )2 fragment of IgG. The cross- Ј linking Ab was a rabbit anti-mouse F(ab )2 fragment purchased from Or- ganon Teknika (West Chester, PA). Anti-CBL Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine, anti- SHC Abs, and anti-CRKL antisera were purchased from Upstate Biotech- nology (Lake Placid, NY), and the anti-GRB2 mAb (G16720) was obtained from Transduction Laboratories (Lexington, KY). GRB2 immunoprecipitations were performed with polyclonal anti-GRB2 (C-231) against residues 195–217 of human GRB2 molecule from Santa Cruz Bio- technology. The anti-␥ subunit (Fc␥RI␥) antisera 5927 was prepared in our laboratory, as described (16, 17), and the 4D8 anti-␥ mAb was generously provided by J. Kochan (Hoffman-La Roche, Nutley, NJ) (18). Preimmune immunoprecipitations were performed with an equal amount of purified rabbit IgG. Differentiation and stimulation of U937 cells U937 cells were maintained in RPMI 1640 with 10% FCS and differentiated with 250 U/ml human rIFN-␥ (obtained from Genentech, San Fran- cisco, CA) for 4 days (termed U937IF cells). U937IF cells were cultured at a concentration of 5 ϫ 105 cells/ml, and the medium was replenished with fresh IFN-␥ (250 U/ml) every 2 days, as described (16, 19). At the time of performing cross-linking experiments, the U937IF cells are 48 h from the Downloaded from addition of fresh IFN-␥. Flow-cytometric analysis of U937IF cells demonstrated the expression of the Fc␥RI and Fc␥RII receptors on these cells (data not shown). For stimulation of Fc␥RI receptors on U937IF cells, cells were washed twice in cold HBSS and adjusted to a concentration of 4 ϫ 107 cells/ml; 0.5-ml aliquots were incubated on ice for 30 min with anti- Fc␥RI Abs (0.25 ␮g/sample). Cross-linking Abs used in Figure 1 were the 32.2 (F(abЈ) fragment) and 197 (whole IgG) anti-Fc␥RI mAbs. Experi- 2 http://www.jimmunol.org/ ments shown in Figures 2, 3, 6, and 7 were conducted with the 32.2 Ј (F(ab )2 fragment), and Figures 4 and 5 were performed with 197 Ab cross-linking, as described (19). In both 32.2 and 197 cross-linking exper- ␮ Ј iments, we added 10 g/ml rabbit anti-mouse (F(ab )2 fragment) Ab at 37°C for different times. Stimulated cells were cooled rapidly with cold HBSS and centrifuged at 500 ϫ g for 5 min in a cold centrifuge. The cell pellet was lysed with 800 ␮l of Triton X-100 extraction buffer (EB buffer) on ice for 30 min or resuspended in 25 ␮lof1ϫ sample buffer per 1 ϫ 106 FIGURE 1. CBL is tyrosine phosphorylated and associates with phos- cells for whole cell lysates. phorylated proteins following Fc␥RI cross-linking. U937IF cells, differentiated in 250 U/ml of IFN-␥ for 4 days, were stimulated with mAbs against Immunoprecipitation by guest on September 26, 2021 the Fc␥RI receptor (16). A, Anti-phosphotyrosine blot of CBL immuno- Cell lysates were prepared in a lysis buffer (EB) containing 1% Triton precipitation (SC-117). Resting U937IF cells, lane 2; U937IF cells stim- X-100, 10 mM Tris, pH 7.6, 50 mM NaCl, 0.1% BSA, 1 mM PMSF, 1% ulated with Fc␥RI cross-linking using the 32.2 (F(abЈ) ) mAb directed ␮ 2 aprotinin, 5 mM EDTA, 50 mM NaF, 0.1% 2-ME, 5 M phenylarsine against the Fc␥RI receptor for 1 and 5 min, lanes 3 and 4, respectively. ␮ oxide, and 100 M vanadate. Lysates were cleared by centrifugation at Lanes 5 and 6 represent stimulation with the 197 mAb (whole IgG) di- 15,000 ϫ g for 45 min at 4°C. For precipitation of specific protein, we rected against the Fc␥RI receptor. Lane 7 is a whole cell lysate prepared added 3 to 10 ␮l of the appropriate Ab to clarified cell lysates. After ϫ 6 ␥ incubation on ice for 2 h, 100 ␮l of a 10% suspension of Formalin-fixed from 1 10 Fc RI-stimulated U937IF cells. Lane 1 represents an immuno- Staphylococcus aureus was added to the immunoprecipitate (IP) and in- precipitation performed with preimmune antisera. B, Anti-CBL immuno- cubated on ice for 1 h. The absorbed immune complexes were washed three blot of anti-CBL immunoprecipitates (SC-117). Lanes are identical to A. times in EB buffer and resuspended with 25 ␮lof1ϫ sample buffer. After boiling at 98°C for 5 min, samples were resolved by SDS-PAGE. Electrophoresis and immunoblotting into pGEX2T vector for expression in Escherichia coli. Preparation of GST-GRB2 fusion constructs were as previously described by Lioubin et Immunoprecipitates were resolved on 10 or 15% acrylamide, 0.193% of al. (20). We performed DNA sequence analysis to confirm the identity and bisacrylamide gels by SDS-PAGE. Proteins were transferred to nitrocel- fidelity of the N-terminal GST-GRB2-SH3, C-terminal GRB2-SH3 do- lulose membranes (1 mAh/cm2) using a dry transfer system (Ellard, Seat- main, GRB2-SH2 domain, and CBL C-terminal fusion constructs. GST tle, WA), as described (16). The blot was incubated with blocking solution fusion proteins were affinity purified from cell lysates of E. coli DH5␣ by (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5% powered milk) for 1 h adsorption to glutathione Sepharose beads. Sepharose-bound GST fusion at room temperature and then incubated with specific anti-phosphotyrosine, proteins were washed several times and stored Ϫ80°C. Sepharose-bound anti-CBL, anti-SHC, anti-GRB2, or anti-CRKL for2hatroom temperature fusion proteins were added to lysates (EB lysis conditions) of resting or with continuous agitation. After three washes in rinse solution (10 mM Fc␥RI-stimulated U937IF cells, incubated for1hat4degrees C. Beads Tris-HCl, pH 7.5, and 150 mM NaCl), the membranes were incubated at were then washed with EB buffer without vortexing, and bound proteins room temperature for 1 h with secondary Ab conjugated with horseradish were eluted with 2ϫ SDS sample buffer at 95°C, and resolved by SDS- peroxidase for enhanced chemoluminescence (ECL; Amersham, Arlington PAGE. The glutathione Sepharose-bound GST fusion protein (10 ␮g) was Heights, IL) or conjugated with alkaline phosphatase for colorimetric de- confirmed by Bradford protein assay and by performing SDS-PAGE on an velopment. To reprobe the membrane, we stripped membrane with 0.1 M aliquot of the Sepharose-bound GST fusion proteins eluted from the beads. glycine, pH 2.5, at room temperature for 30 min and then reblotted with Equivalent amounts of each GST fusion protein were used in each exper- primary Ab. imental group confirmed by Coomassie blue staining of the protein gels after transfer of protein. GST fusion protein experiments The C terminus of CBL was defined as the domain containing 11 PXXP Results motifs, as previously described (1, 2). We prepared GST fusion constructs CBL is tyrosine phosphorylated and associates with representing the entire C terminus of the CBL molecule (residues 461–906) phosphorylated proteins following Fc␥RI cross-linking or the proximal C terminus (residues 461–670) or the distal C terminus (residues 671–906). We subcloned these cDNAs into the pGEX using a Our results demonstrate that CBL is tyrosine phosphorylated in two-staged PCR reaction initially to clone into the pBS and subsequently resting U937IF cells (Fig. 1A, lane 2; Fig. 2A, lane 2; and Fig. 5A, 5020 CBL-GRB2 IN Fc␥RI SIGNALING

become markedly phosphorylated upon Fc␥RI stimulation (unpublished observation) (21). The CBL immunoprecipitates contain a prominent 76-kDa tyrosine-phosphorylated protein noted to associate with CBL under conditions of Fc␥RI stimulation (Fig. 1A, lanes 4–6). Anti-CBL immunoblots confirm that equivalent amounts of CBL are immunoprecipitated (Fig. 1B, lanes 2–6; Fig. 2B, lanes 2–8; and Fig. 5B, lanes 2–4) and confirm the marked mobility shift of CBL following Fc␥RI stimulation. Preimmune Ab does not immunoprecipitate CBL or the tyrosine-phosphorylated pp76 or pp120 molecules (Fig. 1, A and B, lane 1). Upon Fc␥RI cross-linking, CBL is observed to undergo a mobility shift on SDS-PAGE (Fig. 1, A and B, lanes 3–6). The upper CBL isoform appears as a tyrosine-phosphorylated band only in stimulated cell lysates, suggesting that the shift is driven by the phosphorylation of the CBL protein. The kinetics of CBL tyrosine phosphorylation differs with the two different Fc␥RI-specific mAbs (32.2 and 197) used to cross-link the receptor (Fig. 1A, compare lanes 3–4 with 5–6). CBL is more extensively tyrosine phosphorylated Downloaded from 1 min following 197 stimulation as compared with stimulation with 32.2 cross-linking (Fig. 1A, compare lane 3 with 5). Our observation that the 197 mAb is a stronger stimulus for CBL tyrosine phosphorylation is consistent with previous reports from our lab showing that the 197 Ab is a more potent activator of SYK, SHC, GRB2, RAF-1, and MAP kinase (21, 22). The effect of 197 Ab could be via a dual binding of 197 to the Fc binding region of http://www.jimmunol.org/ ␥ ␣ Ј Fc RI subunit as well as binding through F(ab )2 region of 197 to another epitope of Fc␥RI␣. This may increase the efficiency of cross-linking, but should remain relatively Fc␥RI specific, as Fc␥RI is the only Fc␥ receptor with affinity for binding monomeric IgG (23, 24). The Fc␥RI-induced CBL mobility shift seen on anti- CBL immunoblots does not differ under conditions of 32.2 or 197 cross-linking, demonstrating that the altered mobility of CBL on

SDS-PAGE does not correlate with the simultaneous extent or ki- by guest on September 26, 2021 netics of tyrosine phosphorylation (Fig. 1B, compare lanes 3–6). From these data, we conclude that the mobility shift in CBL is unrelated to its tyrosine phosphorylation state in myeloid cells. Lane 7 of Figure 1B represents whole cell lysate of U937IF cells (1 ϫ 106 cell equivalents of protein). The CBL band is not appar- ent in lane 7 due to the short exposure time used to clearly demonstrate the CBL mobility shift in lanes 3 to 6. From these data, we conclude that the mobility shift in CBL is unrelated to its induced tyrosine phosphorylation state in myeloid cells. ␥ ␥ FIGURE 2. Coimmunoprecipitation of Fc RI subunit with CBL in We then explored in more detail the kinetics of CBL phosphor- U937IF cells. We studied the tyrosine phosphorylation of CBL in U937IF ylation following Fc␥RI stimulation (Fig. 2A). The tyrosine phos- cells under conditions of Fc␥RI stimulation. A, Anti-phosphotyrosine im- phorylation of CBL is tightly controlled in U937IF following munoblot performed on CBL immunoprecipitates from U937IF cells ␥ (lanes 1–8). Lane 1, Preimmune immunoprecipitations; lane 2, CBL IP Fc RI stimulation (Fig. 2A, lanes 4–8). PMA stimulation of from resting U937 cells. U937IF cells stimulated with PMA (1 ␮g/ml), U937IF cells induced a mobility shift in CBL similar to that in- lane 3;Fc␥RI stimulation (32.2 mAb) of U937IF cells for 1, 5, 15, 30, and duced by Fc␥RI stimulation, in the absence of tyrosine phosphor- 60 min, lanes 4 to 8, respectively. B, Anti-CBL immunoblot performed on ylation (Fig. 2, A and B, lane 3). PMA stimulation induced the same CBL IP after stripping with 0.1 M glycine, pH 4. The characteristic dephosphorylation of CBL coincident with a pronounced mobility mobility shift in CBL is shown by arrows. The lanes are as described in A. shift. In contrast, Fc␥RI stimulation of U937IF cells induces a C, Anti-Fc␥RI␥ immunoblot performed on CBL IP. We used the 5927 rapid tyrosine phosphorylation of CBL (Fig. 2A, lanes 2–8) with ␥ ␥ ␥ anti- antisera to probe the anti-CBL IP for Fc RI , as described (16). We complete tyrosine dephosphorylation observed 15 min after recep- show the characteristic mobility shift in the Fc␥RI␥ subunit (␥ and ␥ 0 1 tor activation (Fig. 2A, lane 6). The CBL mobility shift is maximal subunit) occurring after PMA and Fc␥RI stimulation (16) (lanes 3 and 4). at 15 min and disappears 30 to 60 min following Fc␥RI stimulation The lanes are as described in A. (Fig. 2B, lanes 6–8). ␥ ␥ lane 2) and that Fc␥RI cross-linking induces the augmented ty- CBL is bound to Fc RI subunit in U937IF cells rosine phosphorylation of CBL (Fig. 1A, lanes 3–6; Fig. 2A, lanes Anti-CBL immunoprecipitations performed on U937IF cell lysates 4–8; and Fig. 5A, lanes 3 and 4). CBL tyrosine phosphorylation is were probed with anti-␥ subunit antisera (Fig. 2C). We detected rapid following Fc␥RI cross-linking, occurring 1 min after stimu- the presence of the Fc␥RI␥ subunit, an ITAM-containing receptor lation using two different Fc␥RI-speciﬁc mAbs (Fig. 1A, lanes 3 subunit, in CBL immunoprecipitates (Fig. 2C, lanes 2–8). Fc␥RI␥ and 5). Other GRB2-binding proteins (e.g., SLP-76, VAV, and subunit protein is detected readily in CBL immunoprecipitates SHC) are not tyrosine phosphorylated in resting U937IF cells, and from resting, PMA-, and Fc␥RI-stimulated cells (Fig. 2C, lanes The Journal of Immunology 5021

FIGURE 3. Immunoreactivity of the anti-␥ Abs. A, Anti-␥ immunoblot of anti-␥ immunoprecipitations performed on U937IF cells using two different anti-␥ subunit Abs, 5927 and 4D8. Synthetic peptides were used to identify the binding sites for these Abs. Pep- tides 1 and/or 2 at 10 ␮g/ml were added to the U937IF cell lysates 30 min before the addition of the primary anti-␥ Abs. Immunoprecipitation of the ␥ subunit was performed as described (16, 18). Lanes 1 to 4 represent 5927 IP, and lanes 5 to 8 represent 4D8 immunoprecipitation. Lanes 1 and 5 served as positive con- Downloaded from trols (no peptide added); lanes 2 and 6, lysate preincubated with peptide 1; lanes 3 and 7, preincubated with peptide 2; lanes 4 and 8, preincubated with both peptides 1 and 2. Proteins were resolved on SDS- PAGE, transferred to nitrocellulose, and blotted with the 5927 anti-␥ antisera. B, A schematic representa- tion of the Fc␥RI receptor noncovalently associated http://www.jimmunol.org/ with the ␥ subunit homodimer containing two ITAM subdomains, (ITAM) 1 and 2. We show the region of the cytoplasmic tail of the ␥ subunit corresponding to peptides 1 and 2 and designated the ITAM 4D8 peptide 1 (SDGVYTGLSTR) and ITAM 5927 peptide 2 (NQETYETLKHEKPPQ) to which the 4D8 and 5927 bind, respectively. An alignment of the intracytoplas- mic portions of the ␥ subunit of Fc␥RI and ␨ subunit of TCR/CD3 complex is illustrated. by guest on September 26, 2021

2–8) (16, 18, 23, 24). We previously reported that Fc␥RI stimu- in all experiments performed (Fig. 2C, lane 8). We did not detect lation induces a mobility shift on SDS-PAGE in the Fc␥RI␥ sub- CBL or Fc␥RI␥ in preimmune immunoprecipitations performed ␥ ␥ unit, forming 0 and 1 bands (16). Phosphoamino acid analysis on the same lysates (Fig. 2, A and C, lane 1). These IPs showed ␥ ␥ ␥ demonstrated that this 0/ 1 pattern is due to the Fc RI-induced strong background signal when probed with goat anti-rabbit sec- ␥ tyrosine and serine/threonine phosphorylation of the 1 protein ondary (Fig. 1B, lane 1; Fig. 2B, lanes 1 and 9); however, at ␥ (16, 17). In particular, the 1 isoform is predominantly serine phos- multiple exposure times using ECL we did not observe a CBL- phorylated upon Fc␥RI stimulation in U937IF cells (16). The char- specific band in these blots nor did we observe the coprecipitation ␥ ␥ ␥ ␥ ␥ ␥ acteristic 0 and 1 bands of Fc RI on SDS-PAGE previously of Fc RI (Fig. 2C, lane 1). reported to occur after PMA and Fc␥RI stimulation were clearly Similar results were obtained when we performed anti-␥ subunit observed in the CBL IPs (Fig. 2C, lanes 3 and 4). The pattern of immunoprecipitations using the 4D8 anti-␥ mAb and probed these ␥ ␥ 0 and 1 observed to coimmunoprecipitate with CBL in both blots for the CBL protein (Fig. 4A, lanes 1–3). We previously resting and Fc␥RI-stimulated U937IF cells (Fig. 2C, lanes 2–7)is reported that the 4D8 anti-␥ subunit Ab coimmunoprecipitates similar to the pattern observed in our anti-␥ subunit IPs performed SYK and Fc␥RI␥ (24). We used a series of Fc␥RI␥-specific pep- on Fc␥RI-stimulated cells, as previously described (16). Impor- tides to determine the binding specificity of the 4D8 mAb (Fig. 3, tantly, the detection of the coimmunoprecipitating Fc␥RI␥ protein A and B). We discovered that the 4D8 mAb binds to a defined shown in Figure 2C, lanes 2 to 8, is blocked completely by pre- peptide within the Fc␥RI␥ protein (Fig. 3, A and B) (SDGVYT- incubation of the immunoblotting antisera (5927) with a Fc␥RI␥- GLSTR) and that this peptide blocks the immunoprecipitation of ␥ specific peptide (NQETYETLKHEKPPQ) (Fig. 3A, lane 2), con- by 4D8 and not IP by 5927 antisera (Fig. 3A, compare lane 7 to 3). firming the identity of the coprecipitating ␥ subunit protein (16). Using this information, we designed a separate set of experiments The decreased quantity of Fc␥RI␥ bound to CBL at 60 min after to determine the specificity of the Fc␥RI␥-CBL interaction in receptor stimulation shown in Figure 2C is not a consistent finding U937IF cells (Fig. 4C). Immunoprecipitation performed with 4D8 5022 CBL-GRB2 IN Fc␥RI SIGNALING

CBL protein (Fig. 4C, lane 5). The reasons for this result are unclear and under active investigation. The addition of peptide (NQETYETLKHEKPPQ) in lane 3 was observed to increase the coprecipitation of CBL and Fc␥RI␥ (Fig. 4C, lane 3). The mechanism for this augmented CBL-␥ coprecipitation in vitro is unclear. The preimmune lane in Figure 4C shows high background at the exposure used to demonstrate both CBL-speciﬁc bands in lanes 1 to 6; at earlier exposures we observe no CBL-speciﬁc bands in these preimmune IPs using the ECL system. The reciprocal IP of CBL and Fc␥RI␥ (Figs. 2 and 4), combined with data from the peptide block experiments (Figs. 3 and 4), provide convincing evidence that CBL and Fc␥RI␥ form a complex in vivo in myeloid cells.

The CBL-GRB2 interaction is modulated during Fc␥RI stimulation Immunoprecipitation of CBL from resting and Fc␥RI-stimulated

cells (Fig. 5, A and B) conﬁrmed the tyrosine phosphorylation of Downloaded from CBL and the constitutive CBL-GRB2 association (Fig. 5C). In this and other experiments, we observe a small decrease in the amount of GRB2 protein bound to CBL under conditions of Fc␥RI stimulation at 1 and 5 min following receptor cross-linking (Fig. 5C, compare lanes 2–4; Fig. 6, lanes 1 and 2). The negative immu-

noblot in Figure 5, lane 5, occurs as result of short exposure time http://www.jimmunol.org/ used to see mobility shift in CBL; at longer exposure, the positive control lysates show distinct CBL bands. We were interested in defining the modules of GRB2 (SH3 and SH2 domains) that as- ␥ FIGURE 4. Coimmunoprecipitation of CBL with subunit. A, Anti-cbl sociate with CBL under conditions of rest vs Fc␥RI stimulation immunoblot performed on anti-Fc␥RI␥ (4D8) and anti-cbl immunoprecipi- (Fig. 5, A–D). GST fusion protein constructs representing the N- tates. Lane 1 represents anti-␥ IP performed with the 4D8 mAb on resting U937IF cells. Lanes 2 and 3 represent 4D8 IP of cells stimulated with and C-terminal GRB2-SH3 domains and the GRB2-SH2 domain ␥ Ј were used to characterize the in vitro binding of CBL to GRB2 Fc RI cross-linking (32.2, F(ab )2) for 1 and 5 min, respectively (18). Lanes 4 and 5 are cbl immunoprecipitations performed on resting and during Fc␥RI stimulation of U937IF cells (Fig. 5D). Both the C- Fc␥RI-stimulated U937IF cells (5-min stimulation). Lane 6 represents terminal GRB2-SH3 domain and the GRB2-SH2 domain bind by guest on September 26, 2021 whole cell lysate of U937IF cells stimulated with Fc␥RI cross-linking. B, CBL in resting cell lysates (Fig. 5D). Upon Fc␥RI stimulation, the Anti-Fc␥RI␥ subunit immunoblot (5927 antisera) performed on anti-␥ IP C-terminal GRB2-SH3 domain no longer binds CBL present in the (4D8 Ab) and anti-cbl immunoprecipitates. Lanes are as described in Fig- U937IF cell lysates. The tyrosine-phosphorylated CBL remained ure 2A. C, Specificity of Fc␥RI␥-cbl interaction. C represents separate exclusively bound to the GRB2-SH2 domain in the Fc␥RI-stimu- experiment performed. An anti-cbl immunoblot was performed on anti- lated myeloid cells (Fig. 5D). In these experiments, the N-terminal Fc␥RI␥ subunit immunoprecipitated with 4D8 Ab. Peptide-blocking ex- GRB2-SH3 domain was noted to bind a small quantity of the SOS periments were performed as described in Figure 3A to confirm the specificity of cbl-Fc␥RI␥ coimmunoprecipitation. We show a preimmune molecule, but did not bind detectable levels of CBL in resting or immunoprecipitation performed on U937IF cells after Fc␥RI stimulation stimulated cell lysates (Fig. 5D). Lane 5 of Figure 5, A and B, 6 and a 4D8 anti-Fc␥RI␥ IP performed on resting U937IF cells (NS). Lanes represents U937IF cell lysates from 1 ϫ 10 cell equivalents as 6 1 to 6 are immunoprecipitations performed using the 4D8 mAb. We pre- compared with 20 ϫ 10 cell equivalents loaded per lane in the incubated the lysates with different peptides (lanes 1–6) (10 ␮g/ml) CBL IP (lanes 1–4). before performing anti-␥ IP to assess the specificity of the cbl-␥ interaction. Lane 1, peptide KAAEITSYE; lane 2, KSDGVYTGLSTR; lane 3, CBL-GRB2 and SOS-GRB2 protein complexes are distinct in NQETYETLKHEKPPQ; lane 4, NQETY(PO )ETLKHEKPPQ; lane 5, 4 U937IF cells KSDGVY(PO4)TGLSTRNQETYETLKHEKPPQ; and lane 6, ETLKHE KPPQ. Following 4D8 immunoprecipitation, proteins were resolved on Based on the data shown in Figure 5D, we postulate that CBL SDS-PAGE and immunoblotted with anti-cbl Ab. tyrosine phosphorylation modulates the CBL-GRB2 interaction in vivo and that the physical interaction between CBL and GRB2 may regulate the capacity of GRB2 to bind SOS. We then asked Ab coimmunoprecipitates CBL and the ␥ subunit under condi- whether the CBL-GRB2 protein-protein complexes are distinct tions of rest or Fc␥RI stimulation. The addition of the 4D8-specific from GRB2-SOS complexes in myeloid cells (Fig. 6). CBL im- peptide (SDGVYTGLSTR) to cell lysates completely abrogates munoprecipitates were noted to contain GRB2, but no detectable the coimmunoprecipitation of both CBL bands (Fig. 4C, lane 2) SOS (Fig. 6, lanes 1 and 2). In contrast, GRB2 IPs contain SOS and the ␥ subunit (Fig. 3A, lane 7). Other peptides, lanes 3 to 6, and minimal CBL (Fig. 6, lanes 5 and 6), and SOS IPs contain corresponding to regions of Fc␥RI␥ that do not affect 4D8 im- GRB2, but no CBL (Fig. 6, lanes 3 and 4). The quantity of GRB2 munoprecipitation of ␥ (Fig. 3A, lane 2) do not affect the copre- bound to CBL decreases following Fc␥RI stimulation (10–15% cipitation of CBL (Fig. 4C, lanes 1 and 4–6). The peptide change) (Fig. 5C, lanes 3 and 4; Fig. 6, lanes 1 and 2), and CBL

KSDGVY(PO4)TGLSTRNQETYETLKHEKPPQ, which is syn- IPs contain several-fold greater amounts of GRB2 as compared thetically phosphorylated on the ﬁrst tyrosine of the ITAM, does with amount of CBL detected in GRB2 IP, suggesting that most of not block the immunoprecipitation of ␥ subunit by the 4D8 Ab and GRB2 in the cell is not bound to CBL. Although the CBL and SOS correspondingly fails to block the coimmunoprecipitation of the IP brought down similar amounts of GRB2, we were unable to The Journal of Immunology 5023 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Modulation of CBL-GRB2 interaction by Fc␥RI stimulation. A, CBL immunoprecipitates blotted with anti-phosphotyrosine Ab. Lysates prepared from resting or Fc␥RI-stimulated U937IF cells were immunoprecipitated with polyclonal anti-CBL Ab (Santa Cruz; SC-117) and immunoblotted with an anti-phosphotyrosine Ab (PY72). Lane 2 represents CBL immunoprecipitated from resting U937IF cells. Lanes 3 and 4 represent CBL IP from U937IF cells stimulated with Fc␥RI cross-linking for 1 and 5 min, respectively. Lane 5 is a whole cell lysate prepared from Fc␥RI-stimulated U937IF cells. B, Anti-CBL immunoblot of CBL IP. Lysates prepared from resting or Fc␥RI-stimulated U937IF cells were immunoprecipitated with anti-CBL antisera and immunoblotted with anti-CBL antisera. Lanes are as designated in A. C, Anti-GRB2 immunoblot of CBL IP. Lysates prepared from resting or Fc␥RI-stimulated U937IF cells were immunoprecipitated with anti-CBL antisera and immunoblotted with anti-GRB2 antisera (monoclonal anti-GRB2; Transduction Labs). Lanes are as designated in A. D, Binding of GST-GRB2 fusion proteins to CBL. cDNAs corresponding to the C-terminal GRB2-SH3 domain, the N-terminal GRB2-SH3, or the GRB2-SH2 domain were cloned into pGEX bacterial expression vector, as described. Equivalent quantity (10 ␮g) of different Sepharose-bound GST fusion proteins was used representing GST alone; GST-GRB2-CSH2, GRB2 SH2 domain; GST-GRB2-CSH3, C-terminal GRB2 SH3 domain; GST-GRB2NSH3, N-terminal GRB2 SH3 were incubated with U937IF cell lysates prepared from resting (NS) or Fc␥RI cross-linked cells (197 Ab). Bound proteins were resolved on SDS-PAGE and blotted with anti-CBL antisera.

detect evidence of SOS and CBL in the same protein-protein com- Differential binding of CBL C terminus to GRB2 and CRKL plex (Fig. 6, compare lanes 1 and 2 with 3 and 4). The lack of adapter molecules detection of CBL coimmunoprecipitating with the anti-GRB2 antisera (Fig. 6, lanes 5 and 6) could be explained by the immuno- Previous reports suggested that CBL can bind to CRKL and GRB2 reactivity of the polyclonal anti-GRB2 antisera used to IP GRB2 in hemopoietic cells (25). To deﬁne the region of CBL-mediating (C-231 binds to residues 197–217 in the C terminus of GRB2, the constitutive binding of these adapter proteins in myeloid cell ly- region mediating the GRB2-CBL interaction shown in Fig. 5D). sates, we divided the C terminus of CBL into two proline-rich 5024 CBL-GRB2 IN Fc␥RI SIGNALING

rosine phosphorylation of SHC and the interaction of tyrosine- phosphorylated SHC with the GRB2-SH2 domain (21) (data not shown). Lane 9 is a whole cell lysate of U937IF cells stimulated with Fc␥RI cross-linking used to test the integrity of the immunoblots.

Discussion The identiﬁcation and characterization of substrates for protein tyrosine kinases activated by ITAM-linked receptors will enhance our understanding of the signaling events that occur following engagement of these receptors. Evidence from the study of hemopoietic cells supports a role for CBL as a substrate for nonreceptor protein tyrosine kinases in Fc, B cell receptor, TCR, and integrin receptor signaling (8–11, 14, 26–28). Ota and Samelson reported in rat basophilic leukemia cells evidence that CBL N terminus may antagonize the activation of SYK kinase in the Fc⑀RI signaling pathway (27). Matsuo et al. reported that cross-linking Fc␥RII

receptor in THP-1 cells induces the tyrosine phosphorylation of Downloaded from CBL, and Tanaka et al. implicated CBL in murine Fc␥RII/Fc␥RIII signaling in macrophages (14, 15). There are no reports of CBL FIGURE 6. The CBL-GRB2 and SOS-GRB2 complexes are distinct in tyrosine phosphorylation following Fc␥RI stimulation. In this U937IF cells. We performed anti-CBL (lanes 1 and 2), anti-SOS (lanes 3 6 work, we provide the ﬁrst experiments implicating CBL and the and 4), and anti-GRB2 (lanes 5 and 6) immunoprecipitations on 20 ϫ 10 ␥ Ϯ ␥ ␥ CBL-GRB2 interaction in Fc RI signaling. CBL is tyrosine U937IF cells stimulation with Fc RI cross-linking (using anti-Fc RI ␥ Ј phosphorylated in resting IFN- -differentiated U937 cells and http://www.jimmunol.org/ mAb, 32.2 F(ab )2). These immunoprecipitates were resolved by SDS- PAGE and immunoblotted as shown. Lane 7 represents a whole cell lysate undergoes increased tyrosine phosphorylation (10-fold increase) of 1 ϫ 106 U937IF cells. Position of SOS, CBL, and GRB2 molecules is indicated by arrows.

subdomains and prepared GST bacterial fusion protein constructs of each. Data from our GST-CBL pull-down experiments are

shown in Figure 7. Our GST fusion constructs consisted of the by guest on September 26, 2021 entire C terminus of CBL protein (residues 461–906), the proximal C terminus (residues 461–620, containing a PPVPPR consensus), and the distal C terminus (residues 621–906, containing 2 YXX- PXXP motifs). These fusion proteins were purified using glutathione Sepharose beads and used to characterize binding to the GRB2 and CRKL adapter proteins in U937IF cell lysates prepared from resting or Fc␥RI-stimulated cells. Equivalent amounts of GST (10 ␮g) or GST fusion proteins are incubated with cell lysates prepared from resting or Fc␥RI-stimulated U937IF cells, followed by adsorption of GST with glutathione Sepharose beads. Bound proteins are resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with specific antisera in Western blot analysis (immunoblot Ab shown on left border of Fig. 7). Figure 7 shows the immunoblot analysis for CBL, SHC, CRKL, and GRB2 of proteins that bind to GST vs GST-CBL fusion constructs under conditions of rest or Fc␥RI stimulation. We demonstrate that the proximal CBL C terminus, CBL (PC), binds the GRB2 molecule (Fig. 7, lanes 5 and 6), whereas the more distal CBL C terminus, CBL (DC), selectively binds to CRKL (Fig. 7, lanes 7 and 8). The GST protein alone did not bring down CBL, SHC, GRB2, or CRKL (Fig. 7, lanes 1 and 2), and CBL (PC) did not bind CRKL; the CBL (DC) FIGURE 7. Differential binding of CBL C terminus to GRB2 and did not bind GRB2. Interestingly, the presence of CRKL is re- CRKL adapter molecules. GST fusion constructs representing the entire C quired for the C terminus of CBL to bring down the endogenous terminus of CBL protein, CBL-C (residues 461–906); the proximal C ter- CBL molecule in these pull-down experiments, suggesting that minus, CBL-PC (residues 461–620); or the distal C terminus, CBL-DC ␮ CRKL may serve as the molecular bridge between GST-CBL (DC) (residues 621–906). Equivalent amounts (10 g) of GST alone (lanes 1 and 2), GST-CBL-C terminus (lanes 3 and 4), GST-CBL-PC (lanes 5 and 6), and endogenous CBL by virtue of its capacity to bind the CBL and GST-CBL-DC (lanes 7 and 8) were incubated with cell lysates pre- (DC) via CRKL-SH3 and endogenous CBL via the CRKL-SH2 pared from U937IF cells Ϯ stimulation by Fc␥RI cross-linking (32.2 domain. In contrast, the interaction of CBL with SHC only occurs mAb). Proteins bound to the GST fusion proteins were resolved by SDS- following Fc␥RI stimulation and is mediated via the GRB2 bind- PAGE and immunoblotted with specific antisera, as shown in left side of ing region of CBL (PC) region (Fig. 7, lanes 4 and 6). The SHC the panel. Lane 9 represents a whole cell lysate prepared from 1 ϫ 106 binding noted in Figure 7 correlates well with the kinetics of ty- U937IF cells stimulated with Fc␥RI cross-linking. The Journal of Immunology 5025 following Fc␥RI stimulation (Figs. 1A,2A, and 5A). In other ex- signaling (35). Ota and Samelson reported that CBL interacts with periments, we observe that other GRB2-binding proteins, SLP-76, the SYK kinase, alters SYK-␥ITAM signaling, and regulates VAV, and SHC, are only tyrosine phosphorylated upon Fc␥RI ␥ITAM function in myeloid cells (27). From these combined data, stimulation (21) (unpublished observation), suggesting a specific we conclude that CBL is associated with Fc␥RI␥ subunit and that role for the basal level of CBL tyrosine phosphorylation observed the CBL-GRB2 interaction functions in Fc␥RI signaling in my- in myeloid cells. This basal level of CBL phosphorylation is also eloid cells. Our preliminary experiment and the results of Ota et al. observed in primary cultures of human bone marrow-derived mac- (27) suggest that the CBL-Fc␥RI␥ interaction is not direct and that rophages and non-IFN-differentiated THP-1 myeloid cells (data an indirect Fc␥RI␥-SYK-CBL interaction exists in myeloid Fc␥RI not shown). We have also performed similar biochemical experi- signaling. ments in IFN-starved U937 and THP-1 cells and in primary bone The GRB2-SOS interaction is a critical event in the activation of marrow-derived human macrophages with similar results, suggest- RAS in many cell types (36–38). Previous studies from our lab- ing that the signaling events reported in this work are not the oratory implicated SHC, GRB2, RAF-1, and MAP kinase in Fc␥RI immediate consequence of IFN stimulation. Our data demonstrate signaling, suggesting a role for RAS in this signaling pathway that PMA induces a mobility shift in CBL under conditions in (21). In hemopoietic cells, four major GRB2-binding proteins un- which CBL is dephosphorylated (Fig. 2, A and B, lane 3). This dergo rapid tyrosine phosphorylation upon ITAM stimulation: 1) mobility shift is reversed 30 to 60 min following Fc␥RI stimula- CBL, 2) SLP-76, 3) LNK, and 4) VAV (33, 39–42). The role of tion (Fig. 2B, lanes 7 and 8). Other experiments show that potato these complex adapter proteins in ITAM signaling and the molec- acid phosphatase treatment of CBL IPs results in a dephosphory- ular consequences of their tyrosine phosphorylation and binding to Downloaded from lation of CBL and a concomitant loss of the mobility shift, and that GRB2 or other adapter proteins (CRK, CRKL, NCK, SHC) remain the omission of the tyrosine phosphatase inhibitors, phenylarsine to be determined. Buday et al. reported upon T cell activation, oxide (PAO) and vanadate, from the EB lysis buffer results in CBL rapidly dissociates from GRB2 and binds to CRKL (25). This tyrosine dephosphorylation of CBL with no alteration in the CBL work demonstrated that the capacity of CBL to bind to the N- and mobility shift (unpublished observation). These data are consistent C-terminal GRB2 SH3 domains in vitro is strongly reduced in with the data of Liu et al. demonstrating that phosphorylation of activated T cells. We performed experiments with GST fusion pro- two serine residues, S619 and S629, in the CBL C terminus serves teins representing different modular domains of the GRB2 mole- http://www.jimmunol.org/ as binding site for the 14-3-3␨ protein in T cells (26). We conclude cule to test the hypothesis that phosphorylation of CBL could alter that Fc␥RI receptor aggregation results in the tyrosine phosphor- its interaction with GRB2 following Fc␥RI stimulation in myeloid ylation of CBL in myeloid cells. Additional analysis will be re- cells (Fig. 5D). We observed at a very early time point following quired to prove that CBL is a substrate for serine/threonine kinases Fc␥RI stimulation, a qualitative change in the interaction between activated by Fc␥RI cross-linking. the domains of the GRB2 molecule and CBL in vitro (Fig. 5D). In We sought additional lines of evidence for role for CBL in U937IF cells, CBL is constitutively bound to GRB2 (Fig. 5C). Our Fc␥RI signaling. We surmised that if CBL is directly involved in in vitro data demonstrate that this interaction is mediated via the ␥ ␥

Fc RI signaling, the Fc RI receptor complex would contain sig- combined GRB2-SH3 and GRB2-SH2 domains (Fig. 5D). The by guest on September 26, 2021 nificant amounts of CBL protein. Our data demonstrate that ty- GST-GRB2 fusion constructs used in these experiments bind to a rosine-phosphorylated CBL is constitutively bound to Fc␥RI␥ sub- free pool of CBL not already complexed to GRB2 in the U937IF unit in resting and stimulated U937IF cells (Fig. 2, A and C, lanes lysates. Importantly, this pool of cellular CBL that binds to GRB2 2–8). The Fc␥RI␥-CBL interaction is confirmed using anti-␥ and in our in vitro experiments would not be the same species of CBL anti-CBL immunoprecipitations and peptides to block the immu- bound to the GRB2-SH3 and SH2 domains in vivo. Our results noprecipitation of ␥ (4D8 Ab) (Figs. 3 and 4) and the detection of therefore reflect an altered potential for interaction between CBL ␥ by 5927 antisera in anti-CBL IPs (Figs. 2–4). We used a series and GRB2 under conditions in which free CBL in the lysate be- of ␥-chain-specific peptides to determine the binding site for the comes tyrosine phosphorylated. Tyrosine phosphorylation of CBL anti-Fc␥RI␥ mAb (4D8) originally described by Schoeneich et al. is associated with the decrease in GRB2-SH3-CBL interaction, (Fig. 3) (18), and then used the 4D8-specific peptide to confirm the leaving CBL bound to the GRB2-SH2 domain (Fig. 5D). Ty- specificity of the ␥-CBL coprecipitation in U937IF cells (Figs. 3 rosine-phosphorylated CBL could be conformationally altered and 4). In several systems, the ITAM-containing receptor subunit such that it will not bind to the GRB2-SH3 domain. Alternatively, binds constitutively to the nonreceptor protein tyrosine kinase CBL tyrosine phosphorylation may result in the binding of CBL to SYK (29–31), suggesting that the constitutive CBL-␥ subunit in- another molecule, thereby preventing the CBL-GRB2-SH3 inter- teraction may be mediated by a multimeric protein complex con- action. The data reported by Buday et al. in T cells demonstrate taining the Fc␥RI␥ subunit, SYK and CBL. The constitutive as- that upon TCR activation, CBL is tyrosine phosphorylated and the sociation of CBL with Fc␥RI␥ in IFN-primed U937 cells is GRB2-SH3-CBL interaction is reduced dramatically at the same consistent with the data from our laboratory showing that SYK time that the CBL-CRKL complex is augmented (25). In our ex- binds to the Fc␥RI␥ subunit in a constitutive manner (22). Con- periments, the CBL-GRB2 interaction is qualitatively altered (Fig. stitutive binding of SYK to the ITAM motif occurs in both plate- 5D) with mild reduction in the total quantity of CBL-GRB2 bind- lets and B cells (31, 32). The data of Iwashima et al. demonstrate ing (Fig. 5C, lane 3; Fig. 6, lanes 1 and 2). that the tyrosine phosphorylation of SYK or ZAP-70 is not re- Other laboratories have observed the binding of CBL to GRB2- quired for their association with ITAM receptor subunits (30). The SH2 domain, but the mechanism and importance of this binding aggregation of the Fc␥RI receptor complex activates SYK kinase are not understood (5). Experiments performed in C. elegans sup- activity, which may result in the tyrosine phosphorylation of CBL port a model by which the sli-1 homologue of CBL modulates (22, 33). Lupher et al. reported that CBL contains a PTB motif signaling events through its direct interaction with the GRB2 mol- capable of an inducible binding to the tyrosine-phosphorylated ecule (3). The SLI-1 protein contains a conserved consensus ZAP-70 kinase in activated T cells (34). These data along with the GRB2-SH2 binding motif (YXNX) and directly binds the GRB2 report of Fournel et al., demonstrating that ZAP-70 and SYK can homologue in C. elegans. In contrast, mammalian CBL does not bind to CBL and that CBL is a substrate for SYK in COS cells, contain a direct binding site for the GRB2-SH2 domain and most support a potential interaction between SYK and CBL in Fc␥RI likely interacts via another phosphoprotein in our system. The 5026 CBL-GRB2 IN Fc␥RI SIGNALING

CBL, GRB2, and SOS in myeloid cells. It is of course possible that different lysis buffer conditions will support the detection of trimolecular complex between CBL, GRB2, and SOS in U937IF cells. It is also plausible that the CBL-GRB2 interaction could function in a parallel pathway for the activation of RAS via a nucleotide exchange factor other than SOS, or that the CBL- GRB2-SOS trimolecular complex is unstable and difﬁcult to detect in Fc␥RI signaling. These possibilities are currently under explo- ration in our laboratory to understand the role of CBL and CBL- GRB2 interaction in the regulation of RAS in myeloid cells.

Acknowledgments We thank Drs. Larry R. Rohrschneider and Gary M. Myles, Fred Hutchin- ␥ FIGURE 8. Fc RI signaling to RAS in myeloid cells. Ligand (IgG) son Cancer Research Center (Seattle, WA), for providing the pGEX-GRB2 ␥ ␣ binds to the Fc RI subunit, resulting in a conformation change in the constructs; and Drs. Yashwant Deo (Medarex, Annandale, NJ) and J. ␥ homodimeric ITAM or subunits. This change induces the activation of Kochan (Hoffmann-La Roche), for generously providing mAbs against HCK kinase activity, which results in the tyrosine phosphorylation of the Fc␥RI␣ and Fc␥RI␥ subunits, respectively. We thank Dr. Anat Epstein for ␥ ␥ ␥ ␥ ITAM motif of Fc RI . Phosphorylation of Fc RI recruits the binding careful reading of the manuscript before submission. and activation of SYK kinase. Other proteins are tyrosine phosphorylated, Downloaded from including the CBL and SHC adapter protein. The tyrosine phosphorylation of SHC is noted to bind to GRB2 (not shown) and the SOS nucleotide References exchange protein, thus activating small GTPases in the cell through the 1. Langdon, W. Y., J. W. Hartley, S. P. Klinken, S. K. Ruscetti, and H. C. Morse conversion of GDP-ras to GTP-ras. GTP-ras activates downstream cas- III. 1989. v-cbl, an oncogene from a dual recombinant murine retrovirus that cades, including RAF-1 and MAP kinase. CBL contains a PTB domain induces early B-lineage lymphomas. Proc. Natl. Acad. Sci. USA 86:1168. 2. Andoniou, C. E., C. B. Thien, and W. Y. Langdon. 1994. Tumour induction by putative binding site for tyrosine-phosphorylated SYK that most likely pro-

activated abl involves tyrosine phosphorylation of the product of the cbl onco- http://www.jimmunol.org/ motes the phosphorylation of CBL by SYK. The CBL-GRB2 and GRB2- gene. EMBO J. 13:4515. SOS interactions are distinct in our model, suggesting that CBL may mod- 3. Yoon, C. H., J. Lee, G. D. Jongeward, and P. W. Sternberg. 1995. Similarity of ulate the interaction between GRB2 and SOS. The model predicts that CBL sli-1, a regulator of vulval development in C. elegans, to the mammalian proto- ␥ oncogene c-cbl. Science 269:1102. regulates GRB2 interaction with downstream target, SOS, during Fc RI 4. Thaon, S., J. F. Quaranta, and D. Regnier. 1994. Genetic expression of the c-cbl stimulation, and hence regulates the small GTPase, RAS. Abbreviations: proto-oncogene in human thymocytes. Adv. Exp. Med. Biol. 355:9. Fc␥RI␣, ␣ subunit of high afﬁnity Fc receptor for IgG; ␥,Fc␥RI␥ subunit; 5. Meisner, H., and M. P. Czech. 1995. Coupling of the proto-oncogene product SH2, src homology 2 domain; and SH3, src homology 3 domain. c-Cbl to the epidermal growth factor receptor. J. Biol. Chem. 270:25332. 6. Galisteo, M. L., I. Dikic, A. G. Batzer, W. Y. Langdon, and J. Schlessinger. 1995. Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation.

J. Biol. Chem. 270:20242. by guest on September 26, 2021 CBL C terminus is composed of a proline-rich region containing 7. Husson, H., B. Mograbi, H. Schmid Antomarchi, S. Fischer, and B. Rossi. 1997. 11 PXXP motifs that bind to SH3 domain-containing proteins. Our CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2. Oncogene 14:2331. GST fusion protein data demonstrate that the constitutive interac- 8. Cory, G. O., R. C. Lovering, S. Hinshelwood, L. MacCarthy Morrogh, tion of CBL with GRB2 and CRKL is mediated via two discrete R. J. Levinsky, and C. Kinnon. 1995. The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 regions of the CBL C terminus. The proximal C terminus (residues domain of Bruton’s tyrosine kinase. J. Exp. Med. 182:611. 461–670) binds to GRB2 constitutively, and under conditions of 9. Marcilla, A., O. M. Rivero Lezcano, A. Agarwal, and K. C. Robbins. 1995. receptor activation binds to the tyrosine-phosphorylated SHC Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fc␥ receptors. J. Biol. Chem. 270:9115. adapter molecule (Fig. 7, lanes 5 and 6). The distal C terminus of 10. Donovan, J. A., R. L. Wange, W. Y. Langdon, and L. E. Samelson. 1994. The CBL (residues 671–906) binds the CRKL adapter protein and en- protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphory- dogenous CBL (Fig. 7, lanes 7 and 8). CBL contains 22 potential lated protein in Jurkat cells activated via the T cell antigen receptor. J. Biol. Chem. 269:22921. tyrosine phosphorylation sites, 14 in the N terminus and 8 within 11. Rivero Lezcano, O. M., J. H. Sameshima, A. Marcilla, and K. C. Robbins. 1994. the C terminus (1, 43). Based on our data and the data of Buday et Physical association between Src homology 3 elements and the protein product of al. and Meisner et al., we suggest that CBL may serve an exchange the c-cbl proto-oncogene. J. Biol. Chem. 269:17363. 12. De Jong, R., J. ten Hoeve, N. Heisterkamp, and J. Groffen. 1995. Crkl is com- function as an adapter shield in resting cells, modulating the on- plexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia. J. Biol. Chem. loading of SOS to GRB2 (Fig. 8) (5, 25). Cell fractionation ex- 270:21468. 13. Odai, H., K. Sasaki, A. Iwamatsu, Y. Hanazono, T. Tanaka, K. Mitani, Y. Yazaki, periments from our laboratory support this hypothesis in that we and H. Hirai. 1995. The proto-oncogene product c-Cbl becomes tyrosine phos- observe a 6- to 10-fold recruitment of GRB2 and SOS to form a phorylated by stimulation with GM-CSF or Epo and constitutively binds to the complex in membrane fractions prepared under conditions of SH3 domain of Grb2/Ash in human hematopoietic cells. J. Biol. Chem. 270: ␥ 10800. Fc RI stimulation (unpublished results). Similar data suggest that 14. Matsuo, T., K. Hazeki, O. Hazeki, T. Katada, and M. Ui. 1996. Specific associ- 90 to 100% of GRB2 complexed with CBL is present within these ation of phosphatidylinositol 3-kinase with the protooncogene product Cbl in Fc␥ membrane fractions. The existence of discrete complexes contain- receptor signaling. FEBS Lett. 382:11. 15. Tanaka, S., L. Neff, R. Baron, and J. B. Levy. 1995. Tyrosine phosphorylation ing either CBL-GRB2 or GRB2-SOS (Fig. 6), as also observed by and translocation of the c-cbl protein after activation of tyrosine kinase signaling Meisner et al. and Donovan et al., in T cells (5, 10) is consistent pathways. J. Biol. Chem. 270:14347. with an exchange function for the CBL-GRB2 complex in the 16. Durden, D. L., H. Rosen, and J. A. Cooper. 1994. Serine/threonine phosphorylation of the ␥-subunit after activation of the high-affinity Fc receptor for immu- regulation of RAS in myeloid cells. The decrease in GRB2-SH3- noglobulin G. Biochem. J. 299:569. CBL interaction could promote the binding of GRB2-SH3 to SOS 17. Durden, D. L., H. Rosen, B. R. Michel, and J. A. Cooper. 1994. Protein tyrosine phosphatase inhibitors block myeloid signal transduction through the Fc␥RI re- within the same signaling complex. To date, an exchange of SOS ceptor. Exp. Cell Res. 211:150. for CBL in a receptor complex containing GRB2 has not been 18. Schoeneich, J. T., V. L. Wilkinson, H. Kado Fong, D. H. Presky, and demonstrated. Our results (Fig. 6) and other data from our lab, J. P. Kochan. 1992. Association of the human Fc⑀RI ␥ subunit with novel cell surface polypeptides. J. Immunol. 148:2181. including CBL and SOS immunodepletion experiments, do not 19. Durden, D. L., H. M. Kim, B. Calore, and Y. Liu. 1995. The Fc␥RI receptor support the existence of a trimolecular protein complex containing signals through the activation of HCK and MAP kinase. J. Immunol. 154:4039. The Journal of Immunology 5027

20. Lioubin, M. N., G. M. Myles, K. Carlberg, D. Bowtell, and L. R. Rohrschneider. receptor I (Fc␥RI) and receptor II (Fc␥RII) on monocytic cells activates a signal 1995. Shc, Grb2, Sos1, and a 150-kilodalton tyrosine-phosphorylated protein transduction pathway common to both Fc receptors that involves the stimulation form complexes with Fms in hematopoietic cells. Mol. Cell. Biol. 14:5682. of p72 Syk protein tyrosine kinase. J. Biol. Chem. 268:24442. 21. Park, R. K., Y. B. Liu, and D. L. Durden. 1996. A role for Shc, Grb2, and Raf-1 34. Lupher, M. L., K. A. Reedquist, S. Miyake, W. Y. Langdon, and H. Band. 1996. in Fc␥RI signal relay. J. Biol. Chem. 271:13342. A novel phosphotyrosine-binding domain in the N-terminal transforming region syk 22. Durden, D. L., and Y. B. Liu. 1994. Protein-tyrosine kinase p72 in Fc␥RI of Cbl interacts directly and selectively with ZAP-70 in T cells. J. Biol. Chem. receptor signaling. Blood 84:2102. 271:24063. 23. Ravetch, J. V., and J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457. ␥ ␨ 35. Fournel, M., D. Davidson, R. Weil, and A. Veillette. 1996. Association of ty- 24. Kinet, J. P. 1992. The - dimers of Fc receptors as connectors to signal trans- rosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T duction. Curr. Opin. Immunol. 4:43. lymphocytes. J. Exp. Med. 183:301. 25. Buday, L., A. Khwaja, S. Sipeki, A. Farago, and J. Downward. 1996. Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation. J. Biol. 36. Downward, J. 1992. Ras regulation: putting back the GTP. Curr. Biol. 2:329. Chem. 271:6159. 37. Buday, L., S. E. Egan, P. Rodriguez Viciana, D. A. Cantrell, and J. Downward. 26. Liu, Y. C., C. Elly, H. Yoshida, N. Bonnefoy Berard, and A. Altman. 1996. 1994. A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa Activation-modulated association of 14-3-3 proteins with Cbl in T cells. J. Biol. membrane-bound tyrosine phosphoprotein is implicated in ras activation in T Chem. 271:14591. cells. J. Biol. Chem. 269:9019. 27. Ota, Y., and L. E. Samelson. 1997. The product of the protooncogene c-cbl a 38. Buday, L., and J. Downward. 1993. Epidermal growth factor regulates p21ras negative regulator of the syk tyrosine kinase. Science 276:418. through the formation of a complex of receptor, Grb2 adapter protein, and Sos 28. Ojaniemi, M., S. S. Martin, F. Dolﬁ, J. M. Olefsky, and K. Vuori. 1997. The nucleotide exchange factor. Cell 73:611. ( proto-oncogene product p120 cbl) links c-Src and phosphatidylinositol 3Ј-kinase 39. Tuosto, L., F. Michel, and O. Acuto. 1996. p95vav associates with tyrosine-phos- to the integrin signaling pathway. J. Biol. Chem. 272:3780. phorylated SLP-76 in antigen-stimulated T cells. J. Exp. Med. 184:1161. 29. Yamada, T., T. Taniguchi, K. Nagai, H. Saitoh, and H. Yamamura. 1991. The 40. Jackman, J. K., D. G. Motto, Q. Sun, T. Tanemoto, C. W. Turck, G. A. Peltz, lectin wheat germ agglutinin stimulates a protein-tyrosine kinase activity of syk G. A. Koretzky, and P. R. Findell. 1996. Molecular cloning of SLP-76, a 76-kDa p72 in porcine splenocytes. Biochem. Biophys. Res. Commun. 180:1325. tyrosine phosphoprotein associated with Grb2 in T cells. J. Biol. Chem. 30. Iwashima, M., B. A. Irving, N. S. C. van Oers, A. C. Chan, and A. Weiss. 1994. 270(Suppl. 13):7029. Downloaded from Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263:1136. 41. Huang, X., Y. Li, K. Tanaka, K. G. Moore, and J. I. Hayashi. 1995. Cloning and 31. Chacko, G. W., J. T. Brandt, K. M. Coggeshall, and C. L. Anderson. 1996. characterization of Lnk, a signal transduction protein that links T-cell receptor Phosphoinositide 3-kinase and p72syk noncovalently associate with the low af- activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol ﬁnity Fc␥ receptor on human platelets through an immunoreceptor tyrosine-based 3-kinase. Proc. Natl. Acad. Sci. USA 92:11618. activation motif. J. Biol. Chem. 271:10775. 42. Smit, L., G. van der Horst, and J. Borst. 1996. Sos, Vav, and C3G participate in 32. Hutchcroft, J. E., M. L. Harrison, and R. L. Geahlen. 1992. Association of the B cell receptor-induced signaling pathways and differentially associate with Shc- 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor. J. Biol. Grb2, Crk, and Crkl adaptors. J. Biol. Chem. 271:8564. Chem. 267:8613. 43. Andoniou, C. E., C. B. Thien, and W. Y. Langdon. 1996. The two major sites of http://www.jimmunol.org/ 33. Kiener, P. A., B. M. Rankin, A. L. Burkhardt, G. L. Schieven, L. K. Gilliland, cbl tyrosine phosphorylation in abl-transformed cells select the crkl SH2 domain. R. B. Rowley, J. B. Bolen, and J. A. Ledbetter. 1993. Cross-linking of Fc␥ Oncogene 12:1981. by guest on September 26, 2021