©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Major Tyrosine Kinase Substrate in Signaling Complexes Formed after Engagement of Fc Receptors (*)

Antonio Marcilla (§) , Octavio M. Rivero-Lezcano , Alka Agarwal , Keith C. Robbins (¶)

From the (1) Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4330

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently identified the protein product of the c- cbl proto-oncogene as an SH3 binding protein expressed in macrophages. To investigate the possibility that p120is involved in signaling pathways initiated by cell surface receptors for IgG (FcR), lysates of HL60 cells were examined for tyrosine phosphorylation of p120upon FcR engagement. Our findings demonstrate that p120is tyrosine-phosphorylated upon FcR engagement and that this molecule represents the major tyrosine kinase substrate in this signaling pathway. Protein complexes containing p120, p72, and p56 were observed either in resting or activated cells. In vitro studies showed that the direct association between p120and p56 was mediated by the SH3 domain of p56.


INTRODUCTION

Cellular receptors for the Fc domain of immunoglobulins (FcRs)() are widely expressed on the surface of immune cells. FcRs belong to the immunoglobulin superfamily and include high affinity receptors for IgE (FcRI), IgG (FcRI), and IgA as well as low affinity IgG receptors (FcRII and RIII) (1) . In the presence of antigen-antibody complexes, these receptors initiate a variety of immune responses such as exocytosis of inflammatory mediators and phagocytosis (2) . Recent studies of a murine strain deficient in FcRs have shown a markedly attenuated inflammatory response to immune complexes, directly demonstrating a requirement for Fc receptors in this process (3) .

Previous studies have shown that biological responses mediated by FcRs require tyrosine phosphorylation (4) . Because FcRs lack intrinsic kinase activity, substrate phosphorylation must result from activation of coupled kinases. Two members of the Src family of protein tyrosine kinases, p56 and p62, have been implicated in FcR signal transduction by their physical association with receptors in rat and mouse cell lines (5) . p56 has been found in association with the subunit of FcRI and p72, also a protein-tyrosine kinase, with its phosphorylated subunit (6, 7) . Recent studies have shown the physical association of p72 with activated FcRII in platelets (8) . According to a current model for signaling through FcRI, p56 associates with the subunit of the receptor and becomes active when the receptor is engaged. Activated p56 kinase then phosphorylates the subunit of the receptor, which in turn serves as a docking motif for the SH2 domains of p72 (6) . This model has also been suggested for the FcR in modulating B-cell activation triggered by the surface immunoglobulin complex (9) . The contribution of p72 kinase to FcR signaling is not well understood, but it has been shown to associate with tyrosine-phosphorylated FcRIIA acting as a catalyst in early events of platelet activation (8) . Furthermore, p72 coprecipitates with a 120-kDa protein which has been suggested to be a possible substrate in myelomonocytic cells (10) .

The cbl gene was initially discovered as the transforming component of Cas NS-1, a tumorigenic murine retrovirus (11) . The cbl proto-oncogene is mainly expressed in hematopoietic cells (11, 12) , and its protein product, p120, has been recently isolated from murine macrophages by virtue of its ability to bind SH3 elements (13) . Other recent studies have shown that conversion of c- cbl to a transforming gene involves tyrosine phosphorylation of its protein product (14) . These and other observations prompted us to examine p120as a potential player in the Fc receptor signaling pathway.


MATERIALS AND METHODS

Cells and Antibodies

The human promyelomonocytic cell line HL60 (15) was maintained as described previously (10) . Goat anti-mouse and goat anti-rabbit antibodies coupled to horseradish peroxidase, human IgG, and goat anti-human IgG F(ab)`antibodies were obtained from Cappel Organon Teknika Corp. Monoclonal anti-phosphotyrosine (4G10) coupled to agarose beads and polyclonal anti-p56 antibodies were purchased from Upstate Biotechnology Inc. Polyclonal anti-p120(C15) and polyclonal anti-p72 (SC 573) antibodies were purchased from Santa Cruz Biotechnology. Monoclonal anti-phosphotyrosine (PY20) antibody coupled to horseradish peroxidase was obtained from ICN. Anti-p72 serum was obtained by immunizing rabbits with a peptide representing residues 622-635 of p72.

Expression of Glutathione S-Transferase Fusion Proteins

A fragment containing the SH3 domain of lyn (amino acids 60-104) was amplified by polymerase chain reaction from a full-length human lyn cDNA (16) , kindly provided by Joseph Bolen, and ligated into the BamHI and EcoRI sites of pGEX-4T-3 (Pharmacia Biotech Inc.). The MscI- NcoI (Klenow end-filled) fragment of lyn containing its SH2 domain (amino acids 105-226) was subcloned into SmaI-cut pGEX-4T-3. Purification of GST fusion proteins has been described previously (17) .

FcR Cross-linking

Receptors were engaged as described (10) . Briefly, cells were suspended at a concentration of 1 10/ml in serum-free RPMI 1640 containing 0.1% bovine serum albumin and 1 m M sodium vanadate. Cells were treated with 10 µg/ml human IgG on ice for 10 min and washed once with phosphate-buffered saline. Cells were then resuspended in serum-free RPMI medium prewarmed to 37 °C and incubated with 20 µg/ml goat anti-human IgG F(ab)`for periods indicated. After washing with cold phosphate-buffered saline, cells were lysed at 4 °C for 15 min in a buffer containing 50 m M Tris-HCl, pH 7.6, 5 m M EDTA, 150 m M NaCl, 1% Nonident P-40, 1 m M sodium vanadate, and a mixture of protease inhibitors which included 2 m M diisopropylfluorophosphate, 1 m M phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 0.5 m M 4-(2-aminoethyl)benzenesulfonyl fluoride, and 2 m M EDTA. Lysates were clarified at 100,000 g for 20 min at 4 °C, and their protein concentrations were determined by the method of Bradford using commercially available reagents (Bio-Rad). Supernatants were processed for either sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting or for immunoprecipitation.

Immunoprecipitation and Western Blotting Analysis

For immunoprecipitation cell lysates were incubated 1 h on ice with indicated antibodies and for an additional 30 min with 10 µl of GammaBind G-Sepharose beads (Pharmacia Biotech Inc.), washed twice with cold lysis buffer and once with lysis buffer containing 1 M NaCl (high salt buffer). Total cell lysates and immunoprecipitates were fractionated by SDS-PAGE, and proteins were transferred to PVDF membranes (Immobilon-P, Millipore). Filters were blocked for 1 h at 37 °C in 10 m M Tris-HCl, pH 7.4, NaCl 150 m M (TNB), containing 4% bovine serum albumin and blotted for 1 h at room temperature with TNB containing the antibodies indicated. After exposure to secondary antibodies coupled to horseradish peroxidase, proteins were visualized with the aid of an enhanced chemilumiscence kit (Amersham Corp.).

Adsorption of Proteins with GST Fusion Proteins

5 µg of each GST fusion protein bound to GSH-agarose beads (Sigma) were incubated for 60 min at 4 °C with 5 mg of cell lysates. After two washes with cold lysis buffer and one with lysis buffer containing 1 M NaCl (high salt buffer), proteins were eluted from the beads by boiling in sample buffer and processed for SDS-PAGE as described above.


RESULTS

p120 Is Tyrosine-phosphorylated upon Engagement of Fc Receptors

Several lines of evidence have raised the possibility that p120might be involved in Fc receptor signaling. To test this hypothesis, lysates from cells treated sequentially with human IgG and goat anti-human IgG F(ab)`to cross-link IgG-bound Fc receptors were analyzed by immunoblotting with anti-phosphotyrosine antibodies (anti-PY). As expected (10) , proteins of 120, 72, and 55-60 kDa were tyrosine-phosphorylated in activated but not untreated HL60 cells, demonstrating successful stimulation of the FcR signaling pathway (Fig. 1 A). When the same lysates were first immunoprecipitated with a previously characterized antibody capable of recognizing p120(13) , the protein was detected as a highly tyrosine-phosphorylated species in lysates from activated but not control cells. Analysis of these same immunoprecipitates and cell lysates with anti-p120antibody demonstrated that similar amounts of p120were present in activated and resting cells (Fig. 1 B). Specific and independent stimulation of FcRI and II (undifferentiated HL60 cells do not express FcRIII) resulted in tyrosine phosphorylation of p120, demonstrating that both receptors participate in the event after stimulation of the cells with IgG (data not shown). Thus, p120is highly phosphorylated on tyrosine in response to FcR stimulation.


Figure 1: p120 is tyrosine phosphorylated upon engagement of FcRs in HL60 cells. Protein extracts (5 mg) from untreated (-) or stimulated (+) HL60 cells were immunoprecipitated with anti-p120antibodies, fractionated by 10% SDS-PAGE, and blotted onto PVDF membranes. Total cell lysate (60 µg/lane) was similarly electrophoresed and blotted. Filters were treated with anti-phosphotyrosine ( Panel A) or anti-p120( Panel B) antibodies. Proteins were visualized by chemiluminescence. The positions of molecular mass markers in kilodaltons and p120are indicated.



p120 Is the Major Protein Tyrosine-phosphorylated in Response to FcR Engagement

To determine what portion of the 120-kDa band detected by anti-PY was represented by p120, this latter molecule was first depleted from lysates of stimulated cells by immunoprecipitation. As shown in Fig. 2, anti-p120antibodies did not detect the protein after three rounds of depletion by immunoprecipitation (Fig. 2 A, compare lanes 2 and 3). When blots containing lysates devoid of p120were incubated with anti-PY, the intensity of the p120 band was notably decreased (Fig. 2 B, compare lanes 2 and 3). Depleted lysates were also examined by immunoprecipitation with anti-PY. Under these conditions the intensity of p120 was decreased whereas the intensity of p72 was not appreciably affected (Fig. 2 B, compare lanes 4 and 5). Thus, depletion of p120was selective. We conclude from these results that p120represents a major portion of the 120-kDa proteins tyrosine-phosphorylated upon FcR engagement.


Figure 2: Immunodepletion of p120 decreases detectability of the phosphoprotein p120. A, protein extracts (1 mg) from activated HL60 cells were subjected to one ( lane 2) or three ( lane 3) rounds of immunoprecipitation with anti-p120antibody, fractionated by 10% SDS-PAGE, and immunoblotted with anti-p120antibody. Immunoprecipitates using a nonreactive serum served as a control ( lane 1). B, identical samples subjected to one ( lanes 2 and 4) or three ( lanes 3 and 5) rounds of immunoprecipitation were either blotted directly onto filters ( lanes 2 and 3) or reimmunoprecipitated with anti-PY ( lanes 4 and 5). Immunoprecipitates using nonreactive serum ( lane 1) served as a control. After fractionation and blotting, filters were probed with anti-PY, and proteins were visualized by chemiluminescence. The positions of molecular mass markers in kilodaltons, p120, and p72 are indicated.



p120 Associates with p56and p72Tyrosine Kinases

To determine whether p56 or p72, two protein tyrosine kinases involved in the FcR signaling pathway, might be responsible for phosphorylation of p120, lysates from resting or activated cells were immunoprecipitated with either anti-p56 or anti-p72 and examined for the presence of p120. As shown in Fig. 3, p120was detected clearly associated with p56 ( lanes 5 and 6) and in less amount with p72 ( lanes 7 and 8). The formation of this complex was not dependent upon FcR cross-linking, suggesting that the associations between p56, p72, and p120are not mediated by tyrosine phosphorylation. When the same blots were stripped and reblotted with anti-p72 antibodies, very little p72 was detected in anti-p120immunoprecipitates, and no p72 was present in anti-p56 immunoprecipitates (data not shown). In reciprocal experiments, p56 was detected in anti-p120immunoprecipitates. When the kinetics of p56-p120association was analyzed, a slight increase in the intensity of p56 was observed 10 min after FcR activation. By 60 min, p56 was less apparent (Fig. 4). We conclude that a portion of p56 is physically associated with p120in resting cells and that by 60 min after activation, the number of p56 molecules complexed with p120was reduced.


Figure 3: p120 associates with p56 and p72. Protein extracts (10 mg) from resting (-), or activated (+) HL60 cells were immunoprecipitated with either a non reactive serum ( NRS), anti-p120( Anti-Cbl), anti-p56 ( Anti-Lyn), or anti-p72 ( Anti-Syk) antibodies. Immunoprecipitates were fractionated by 10% SDS-PAGE, blotted onto filters and immunodetected with anti-Cbl antibody. The positions of molecular mass markers in kDa, p120and immunoglobulin heavy chain ( IgG H) are indicated.




Figure 4: Kinetics of the p120-p56 association after FcR engagement. Protein extracts (5 mg) from HL60 cells stimulated for the times indicated, were immunoprecipitated with anti-p120( Anti-Cbl) antibody, or with a nonreactive serum ( NRS). Immunoprecipitates as well as cell lysates ( None) were fractionated by electrophoresis, blotted onto filters and probed with anti-p56 ( Anti-Lyn) antibody. The positions of molecular mass markers in kilodaltons and p56 are indicated.



To study the tyrosine phosphorylation state of p120complexed with p56 and p72, immunoprecipitates from lysates of resting and activated HL60 cells were examined by anti-PY immunoblotting. As shown in Fig. 5, tyrosine phosphorylated p120was detected in either anti-Lyn or anti-Syk immunoprecipitates, as well as in lysates, from activated cells. These findings demonstrated that the population of p120who was associated with p56 or p72 was tyrosine-phosphorylated upon FcR engagement.


Figure 5: p120 coprecipitated with p56 and p72 is tyrosine phosphorylated upon FcR engagement. Protein extracts (5 mg) from resting (-) or activated (+) HL60 cells were immunoprecipitated with anti-p56 ( Anti-Lyn), or anti-p72 ( Anti-Syk) antibodies, and fractionated by electrophoresis. Immunoprecipitates and total cell lysates ( None) similarly fractionated were blotted onto filters, and probed with anti-phosphotyrosine (anti-PY) antibody. The positions of molecular mass markers in kilodaltons, p120, p72, and p56 are indicated.



To determine whether p56 or p72 kinases might phosphorylate p120in vitro, lysates from resting or activated cells were immunoprecipitated with anti-p56, anti-p72, or anti-p120antibodies and assayed in vitro for kinase activity, but no phosphorylation in vitro was detected (data not shown). These results provided no direct evidence that p120was a substrate for either p56 or p72 kinases.

p120 Associates with the SH3 Domain of p56

To further examine the nature of the interaction between p56 and p120, we tested the ability of the SH2 and SH3 domains of p56 to bind p120in vitro. GST fusion proteins containing these domains were attached to glutathione agarose beads and incubated in the presence of lysates from resting or activated HL60 cells. The associated proteins were then fractionated by SDS-PAGE and immunoblotted with anti-p120antibody. p120specifically associated with the SH3 domain of p56 (Fig. 6, lanes 5 and 6), but not with GST ( lanes 1 and 2) or the SH2 domain of p56 ( lanes 3 and 4). Moreover, the amount of p120that bound to Lyn-SH3 did not vary after 10 min of FcR cross-linking ( lane 6), consistent with the level of association observed in assays in vivo (Figs. 3 and 4).


Figure 6: p120 binds in vitro to the SH3 domain of p56. Protein extracts (5 mg) from resting (-) or activated (+) HL60 cells were incubated with 5 µg of the indicated GST fusion proteins coupled to glutathione-agarose beads (Sigma). GST protein ( GST) and anti-p120(anti-() Cbl) immunoprecipitate were included as controls. Samples were fractionated in 10% SDS-PAGE and analyzed by Western blotting using anti-p120as a probe. The positions of molecular mass markers in kilodaltons and p120are indicated.



Phosphorylation of p120 and Its Associated Proteins in Vivo

To gain information regarding the sequence of association and tyrosine phosphorylation events among p120, p72, and p56, we performed a series of time course experiments. Each of these molecules was immunoprecipitated and examined by Western blotting using anti-PY as a probe. As shown in Fig. 7B, tyrosine phosphorylation of p56 varied by 2-3-fold during the time course, reaching a maximum at 5-10 min. Associated proteins of 72 and 120 kDa were also observed, their intensities peaking at 5-10 min after activation (Fig. 7 B). A molecule of 120 kDa (likely p120), also associated with p72, is seen peaking in its detectability at 20 min (Fig. 7 C). Phosphorylation of p120(Fig. 7 D) was also maximal at 20 min and closely followed the pattern observed for p120 in total cell lysates (Fig. 7 A). These experiments demonstrate transient tyrosine phosphorylation of p56, p72, and p120upon FcR activation. The data also confirm the formation of a complex consisting of p56, p72, and p120.


Figure 7: Tyrosine phosphorylation of p120 and its associated proteins. Protein extracts (5 mg) from HL60 cells stimulated for the times indicated were immunoprecipitated with anti-p56 ( Anti-Lyn) ( Panel B), anti-p72 ( Anti-Syk) ( Panel C), or anti-p120( Anti-Cbl) ( Panel D) and fractionated by electrophoresis. Immunoprecipitates as well as total cell lysates similarly fractionated ( Panel A), were blotted onto PVDF membranes, and analyzed with anti-PY antibody. Electrophoretic mobility of molecular mass markers in kilodaltons is indicated.




DISCUSSION

A number of substrates have been identified for protein-tyrosine kinases involved in the pathways initiated by FcR engagement. These include individual components of the multimeric Fc receptors (18, 19) as well as the kinases p56, p62 (5) , and p72 (8, 10, 20) . In the present study, we have shown that p120is phosphorylated on tyrosine in response to FcR engagement and is the most prominent of all tyrosine-phosphorylated proteins in this pathway. The lack of obvious p120 tyrosine phosphorylation upon FcRI activation in rat basophilic leukemia cells (4) () suggests that phosphorylation of p120might distinguish Fc from Fc receptor signaling pathways. Nevertheless, recent studies have shown tyrosine phosphorylation of p120in response to T cell-receptor activation as well (21) . Thus, p120emerges as an important player in immune recognition receptor signaling in hematopoietic cells.

The involvement of tyrosine phosphorylation in recruiting molecules containing SH2 domains to signaling complexes has been well documented (22) . For example, tyrosine phosphorylation of receptor activation motifs of Fc (6, 7) and probably Fc receptors (8) serves as the mechanism by which p72 binds activated receptor components. However, the involvement of SH3 domains in the formation of FcR signaling complexes has not been previously described. In this study we show that p120associates with the SH3 domain of p56, as it has been observed for other tyrosine kinases (13, 21) , and this association is not affected after FcR cross-linking. Furthermore, the p120-p56 complex is constitutive in vivo and is present in both resting and activated cells. The amount of p120coprecipitated with anti-p56 did not vary upon FcR cross-linking suggesting that Lyn-SH2 domain does not contribute to the stability of the complex. Consistent with this finding, Lyn-SH2 domain was unable to bind p120in vitro, even after activation of the cells. This result differs from the observed in T cells, in which the association of p59 and a phosphorylated p120, probably p120, requires both the SH3 and SH2 elements (23) . The small amount of p120associated with p72 in vivo would suggest a nondirect interaction between these two molecules. The steady state association of p120with p56 and p72, in combination with its tyrosine phosphorylation, might suggest that these kinases directly phosphorylate p120, but in vitro kinase assays failed to confirm this point. It is possible that the binding of p120to p56, a known membrane-attached protein, might carry this molecule to the proximity of an active tyrosine kinase located in the plasma membrane, where p120would became phosphorylated. Further studies will be required to identify the kinase responsible for p120phosphorylation.

Previous studies have shown that p120physically binds to p47 in vitro and in vivo (13) . Thus, p47 may also contribute fundamentally to the ability of p120to associate with the activated signaling complex. The role of p120in the FcR signaling complex is not clear. Its known associations with p47 and p56 are consistent with the idea that p120serves as a bridge together with p47 to position p56 for activation after FcR engagement. Alternatively, p120may be responsible for initiating biologic responses when phosphorylated. The ability of oncogenic forms of p120to subvert growth regulatory pathways in fibroblasts and hematopoietic cells (14, 21) attests to its biological potential, an area that warrants further investigation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a NATO Scientific Committee fellowship (Spain).

To whom correspondence should be addressed: Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, Bldg. 30, Rm. 211, 9000 Rockville Pike, Bethesda, MD 20892-4330. Tel.: 301-496-3303; Fax: 301-402-0823.

The abbreviations used are: FcR, receptors for the Fc domain of immunoglobulins; FcR, receptors for IgE; Fc, receptors for IgG; GST, glutathione S-transferase; SH, Src homology regions; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PY, phosphotyrosine.

A. Marcilla, unpublished observations.


ACKNOWLEDGEMENTS

We thank Jeanne H. Sameshima for her help in preparing the manuscript and Dr. Silvio Gutkind for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.