©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tyrosine Phosphorylation and Translocation of the c-Cbl Protein after Activation of Tyrosine Kinase Signaling Pathways (*)

Sakae Tanaka (§) , Lynn Neff , Roland Baron , Joan B. Levy (¶)

From the (1)Departments of Cell Biology and Orthopedics, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The c-cbl protooncogene product (c-Cbl) is a 120-kDa protein that has been shown to bind to the Src homology 3 domains of various proteins, suggesting its involvement in signal transduction pathways. We identified one of the most prominent tyrosine-phosphorylated proteins in Fc receptor (FcR)-stimulated macrophages to be c-Cbl. Tyrosine phosphorylation of c-Cbl occurred within 20 s after stimulation and reached maximum levels within 3-5 min. c-Cbl was also tyrosine-phosphorylated in epidermal growth factor (EGF) receptor-overexpressing cells upon EGF stimulation, in macrophages in response to CSF-1 treatment, and in v-src transformed cells. Furthermore, we found that c-Cbl associated with these kinases in vivo. In vitro, c-Cbl bound to the Src homology 3 domains of Src, Fyn, and Lyn in both unstimulated and FcR-stimulated macrophages. Examination of cells by immunofluorescence revealed that c-Cbl is diffusely distributed in the cytoplasm in both unstimulated macrophages and EGF receptor-overexpressing cells and translocated to a more specific compartment of the cell, consistent with the trans-Golgi region, following FcR clustering and EGF stimulation, respectively. These results suggest that c-Cbl is involved in the signaling pathways utilized by different types of tyrosine kinases.


INTRODUCTION

The c-cbl protooncogene product (c-Cbl) was first identified as the cellular homologue of the viral transforming protein of the murine Cas NS-1 retrovirus(1, 2) . This retrovirus induces pre-B cell lymphomas and myeloid leukemia in infected newborn and adult mice and transforms different fibroblast cell lines(1, 2) . The v-cbl oncogene product (v-Cbl) represents a truncated form of its cellular homologue containing only the N-terminal 355 amino acids of c-Cbl. Structurally it possesses a potential nuclear localization signal, and in subcellular fractionation analysis it has been reported to localize to both nuclear and cytoplasmic fractions (3). The protooncogene product, c-Cbl, is a 120-kDa protein that contains additional structural features including a high proportion of basic amino acids, a putative leucine zipper at the C terminus, and a zinc finger-like motif, indicating that it may act as a transcription factor(2) . However, unlike v-Cbl, c-Cbl has been exclusively localized to the cytoplasm, and immunohistochemical studies suggest that it may be associated with the cytoskeleton(3) . Both in vivo and in vitro evidence demonstrates that c-Cbl is able to bind to the Src homology 3 (SH3)()domain of Nck, an adaptor protein that mediates specific protein-protein interactions in tyrosine kinase signaling pathways(4) .

We report here that c-Cbl becomes tyrosine-phosphorylated in response to the activation of different tyrosine kinase signaling pathways including stimulation by antibody clustering of the FcII/III receptors in murine macrophages, mitogen stimulation of fibroblasts and macrophages by the growth factors EGF and CSF-1, respectively, and v-src transformation in fibroblasts. Immunofluorescence analysis revealed that FcR clustering and EGF stimulation also induce the translocation of c-Cbl from its diffuse and reticular cytoplasmic localization in unstimulated cells to a more specific region of the cell, consistent with the trans-Golgi region. Overall, these results suggest the possibility that c-Cbl is recruited in the signal transduction pathways utilized by a variety of tyrosine kinases.


EXPERIMENTAL PROCEDURES

Cells and Antibodies

To prepare peritoneal macrophages, CD-1 mice received intraperitoneal injections of thioglycolate broth as previously reported(5) . Three days following these injections peritoneal exudate cells were isolated by washing the peritoneal cavity with phosphate-buffered saline. More than 90% of the cells were macrophages(5) . The murine macrophage cell line P388D1 (American Type Culture Collection, Bethesda, MD, TIB63) was maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum (Sigma). HER14 cells, an NIH 3T3 stable cell line overexpressing the human EGF receptor (EGFR), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated calf serum. NIH 3T3 cells overexpressing c-src (c-src 3T3 cells) and Balb/c3T3 cells transformed by v-src (SRD cells) were cultured in DMEM supplemented with 5% calf serum. Anti-phosphotyrosine antibody, clone 4G10, and anti-CSF-1 receptor (CSF-1R) antibody were obtained from UBI (Lake Placid, NY). Anti-murine FcRII/III monoclonal antibody (2.4G2) was purchased from PharMingen (San Diego, CA). F(ab`) fragments of goat anti-rat IgG were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Rabbit polyclonal anti-c-Cbl antibody (C-15) and anti-EGFR antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Stimulation and Lysis

FcR clustering in peritoneal macrophages and the macrophage cell line P388D1 cells was performed as previously reported(5) . The cells were pelleted at 14,000 g and lysed in ice-cold TNE lysis buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 2 mM NaVO, 10 mM NaF, and 20 µg/ml aprotinin). In some experiments, P388D1 cells were stimulated with CSF-1 (25 nM) for 30 s at room temperature and lysed with TNE buffer. HER14 cells were stimulated with EGF (275 ng/ml) for 5 min at 37 °C and lysed with HNTG buffer (30 mM HEPES, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mg/ml bovine serum albumin, 2 mM NaVO, 10 mM NaF, and 20 µg/ml aprotinin) as previously reported(6) . SRD cells were lysed with either TNE buffer or CHAPS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 8 mM CHAPS, 2 mM EDTA, 2 mM NaVO, 10 mM NaF, and 20 µg/ml aprotinin).

Western Blotting and Immunoprecipitation

Western blotting was performed using ECL Western blotting detection reagents (Amersham Corp.) according to the conditions recommended by the supplier. For immunoprecipitation of c-Cbl, cell lysates were precleared with protein A-Sepharose (Sigma) for 1 h and then incubated with 1 µg of anti-c-Cbl antibody for 1 h. Immune complexes were recovered with protein A-Sepharose and extensively washed 3 times with TNE buffer. The protein was eluted by boiling in 1 sample buffer (62.5 mM Tris-HCl, pH 6.9, 2 mM EDTA, 3% SDS, 3.75% glycerol, and 180 mM -mercaptoethanol) for 2 min.

SH3-binding Proteins

Two hundred µg of the cell lysate prepared in TNE buffer was precleared by incubating with GST protein non-covalently bound to glutathione-Sepharose (Pharmacia Biotech Inc.) for 2 h at 4 °C. After removing the GST-Sepharose beads by centrifugation, the supernatants were incubated with 15 µg of the GST-SH3 fusion proteins and 40 µl of a 1:1 slurry of glutathione-Sepharose for 1 h at 4 °C. Cellular proteins bound to the GST-SH3 fusion proteins were washed 3 times with lysis buffer and eluted by boiling in 1 sample buffer for 2 min.

Confocal Microscopy

For immunofluorescence analysis, cells were plated on glass coverslips and cultured overnight at 37 °C. After the indicated treatments, cells were fixed in phosphate-buffered saline containing 3.7% formaldehyde for 10 min at room temperature and stained with anti-c-Cbl antibody following the method previously reported(7) . Microscopy was performed using a confocal microscope (model MRC 600, Bio-Rad) with krypton/argon lasers.


RESULTS

Fc Receptor-mediated Tyrosine Phosphorylation of c-Cbl

Fig. 1indicates that stimulation of the FcR by antibody clustering in peritoneal macrophages (A) and the murine macrophage cell line P388D1, which is known to have FcR (B), induced tyrosine phosphorylation of several proteins, which is consistent with previous reports(5) . Enhanced tyrosine phosphorylation of the majority of these proteins was apparent within 20 s after the addition of the secondary antibody, reached a maximum level within 3-5 min, and significantly diminished by 30 min (Fig. 1A). Due to the appearance of a 120-kDa protein that was rapidly tyrosine-phosphorylated upon FcR stimulation, we determined whether the c-Cbl protein became tyrosine-phosphorylated in response to FcR stimulation of macrophages. c-Cbl was immunoprecipitated from unstimulated and stimulated peritoneal macrophages, and the immunoprecipitated material was analyzed by anti-phosphotyrosine immunoblotting. As shown in Fig. 1C (Anti-PY blot), FcR clustering rapidly induced the tyrosine phosphorylation of c-Cbl. This increase in tyrosine phosphorylation was not due to changes in the level of the c-Cbl protein upon FcR stimulation (Fig. 1C; Anti-Cbl blot). Similar results were obtained in FcR-stimulated P388D1 cells (data not shown).


Figure 1: FcR clustering induces tyrosine phosphorylation of c-Cbl in macrophages. A and B, FcR clustering induces tyrosine phosphorylation in macrophages. Peritoneal macrophages (A) and the murine macrophage cell line P388D1 (B) were incubated with anti-FcRII/III antibody for 30 min at 4 °C. Thereafter, the cells were treated with goat F(ab`) anti-rat IgG for the indicated times at 37 °C. Cells were lysed and immunoblotted with anti-phosphotyrosine antibody, clone 4G10. The mobility of the size markers is indicated. C, FcR clustering induces tyrosine phosphorylation of c-Cbl in macrophages. c-Cbl was immunoprecipitated from cell lysates and immunoblotted with anti-phosphotyrosine antibody (Anti-PY). The anti-phosphotyrosine blot was stripped of antibody and reprobed with anti-c-Cbl antibody (Anti-Cbl). The position of c-Cbl is indicated by the arrow.



c-Cbl Binds to the SH3 Domain of Src Family Tyrosine Kinases

Members of the Src family of tyrosine kinases are activated upon signaling via Fc receptors (8, 9, 10) and possess SH3 domains that mediate interactions with various cellular proteins(11) . Since c-Cbl has been shown to bind to the SH3 domain of Nck(4) , we then determined whether the c-Cbl protein was able to bind to the SH3 domain of different Src family members. The SH3 domains of Src, Fyn, and Lyn were expressed as GST fusion proteins and purified by glutathione-Sepharose chromatography. Lysates prepared from unstimulated P388D1 cells or FcR-stimulated cells were incubated with glutathione-Sepharose beads containing either GST alone or the GST-Src-SH3, GST-Fyn-SH3, or GST-Lyn-SH3 fusion proteins. Cellular proteins bound to the different GST fusion proteins were fractionated by 8% SDS-PAGE and immunoblotted with anti-c-Cbl antibody. Anti-c-Cbl antibody reacted with a Src, Fyn, and Lyn SH3-binding protein in both unstimulated and stimulated cells (Fig. 2B). In both unstimulated and FcR-stimulated cells, the GST fusion proteins containing the Src, Fyn, and Lyn SH3 domains bound to a 120-kDa protein that reacted with anti-c-Cbl antibody on Western blots (Fig. 2B). Even though FcR stimulation led to enhanced tyrosine phosphorylation of c-Cbl over the low, basal level of phosphorylation observed in unstimulated cells (Fig. 2A), binding of c-Cbl to the SH3 domains of Src, Fyn, and Lyn appeared to be similar in unstimulated or stimulated cells. These results suggest that binding of c-Cbl to the SH3 domains is not dependent on the tyrosine phosphorylation induced by FcR stimulation.


Figure 2: c-Cbl associates with the SH3 domains of three different Src family members in vitro. A, TCL and c-Cbl immunoprecipitates (c-Cbl IP) from unstimulated and FcR-stimulated P388D1 cells (3 min of stimulation) were immunoblotted with anti-phosphotyrosine antibody. B, cell lysates from unstimulated and FcR-stimulated P388D1 cells were incubated with glutathione-Sepharose beads containing GST, GST-Src SH3, GST-Fyn SH3, and GST-Lyn SH3 fusion proteins. After extensively washing the beads, the bound proteins were eluted in SDS-sample buffer, separated on SDS-PAGE, and transferred to nitrocellulose. Filters were immunoblotted with anti-c-Cbl antibody. c-Cbl is indicated by the arrow.



Activation of Other Types of Tyrosine Kinases Also Induces Tyrosine Phosphorylation of c-Cbl

We then examined whether tyrosine phosphorylation of c-Cbl was specific to FcR-mediated signaling or whether it could also be induced in other tyrosine kinase signaling pathways. We first tested the ability of c-Cbl to become tyrosine-phosphorylated in response to stimulation of growth factor receptors that possess intrinsic tyrosine kinase activity. For this purpose, a cell line, HER14, that expresses high levels of the EGFR was stimulated with EGF, and the macrophage cell line, P388D1, was stimulated with CSF-1. In total cell lysates (TCL), increased tyrosine phosphorylation of a protein with an electrophoretic mobility similar to c-Cbl was observed in response to both EGF and CSF-1 stimulation (Fig. 3, A and B). c-Cbl was then immunoprecipitated from cell lysates of unstimulated and stimulated cells and immunoblotted with anti-phosphotyrosine antibody as before. As shown in Fig. 3, A and B, both EGF and CSF-1 induced the tyrosine phosphorylation of immunoprecipitated c-Cbl in HER14 and P388D1 cells, respectively (anti-PY blot). The increase in tyrosine phosphorylation of c-Cbl in EGF and CSF-1-stimulated cells was not due to an increase in the amount of protein in the stimulated cells (Fig. 3, A and B, anti-Cbl blot). Hence, tyrosine phosphorylation of c-Cbl is induced not only after FcR clustering but also in the signal transduction pathways of various receptor-type tyrosine kinases.


Figure 3: c-Cbl is tyrosine-phosphorylated in response to mitogen stimulation and in fibroblasts transformed by v-src. A and B, mitogen stimulation induces tyrosine phosphorylation of c-Cbl. Total cell lysates prepared from unstimulated NIH3T3 cells (A) overexpressing the human EGFR (HER14) and HER14 cells stimulated with 275 ng/ml EGF for 5 min at 37 °C and murine macrophage cells (B) (P388D1) unstimulated or treated with 25 nM human recombinant CSF-1 for 30 s at 37 °C were fractionated on 8% SDS-PAGE and blotted with anti-phosphotyrosine antibodies (TCL anti-PY). Utilizing 300 µg of lysate prepared from unstimulated and stimulated cells in A and B, c-Cbl was immunoprecipitated and Western blotted with anti-phosphotyrosine antibody (c-Cbl IP; anti-PY). The c-Cbl IP blot was then stripped of antibody and reprobed with anti-c-Cbl antibody (c-Cbl IP; anti-Cbl). The migration of c-Cbl is indicated by the arrows, and the 170-kDa band that is tyrosine-phosphorylated in response to EGF and has a similar mobility to the EGFR is indicated by an arrowhead. C, c-Cbl is tyrosine-phosphorylated in v-src-transformed cells. c-Cbl was immunoprecipitated from cell lysates of normal lung fibroblasts (LungFB), NIH3T3 cells overexpressing c-src (c-src3T3), and Balb/c3T3 cells transformed by v-src (SRD), and the immunoprecipitated material was blotted with anti-phosphotyrosine antibodies (c-Cbl IP; anti-PY). Antibody was removed from these blots and reprobed with anti-c-Cbl antibody (c-Cbl IP; anti-Cbl).



We next determined whether the non-receptor tyrosine kinase, Src, induces tyrosine phosphorylation of c-Cbl. As shown in Fig. 3C (anti-PY), c-Cbl does not appear to be tyrosine-phosphorylated in lung fibroblasts that possess endogenous c-Src or in NIH3T3 cells that overexpress the c-Src protein. In contrast, c-Cbl is heavily tyrosine-phosphorylated in v-src-transformed fibroblasts (SRD cells). The level of c-Cbl does not, however, appear to differ in the three types of cultures examined (Fig. 3C, anti-Cbl). Therefore, c-Cbl appears to be tyrosine-phosphorylated by activated non-receptor tyrosine kinases such as the viral Src protein.

c-Cbl Associates with Tyrosine Kinases in Vivo

We then examined the physical association of c-Cbl with various tyrosine kinases. EGFR was immunoprecipitated from HER14 lysates with or without EGF stimulation and immunoblotted with anti-c-Cbl antibody. As shown in Fig. 4A, c-Cbl co-immunoprecipitated with EGFR in stimulated HER14 cells but not in unstimulated cells (Fig. 4A, anti-Cbl). c-Cbl was also physically associated with CSF-1R in P388D1 cells, and the association was enhanced by stimulating the cells with CSF-1 (Fig. 4B, anti-c-Cbl). In both experiments, the amount of the immunoprecipitated receptors did not change upon stimulation with the appropriate ligand (Fig. 4, A and B, anti-EGFR and anti-CSF-1R, respectively). c-Cbl was co-immunoprecipitated with v-Src in SRD cells when cells were lysed in CHAPS buffer (Fig. 4C, C), but the co-immunoprecipitation was not observed when cells were lysed in TNE buffer (Fig. 4C, N). Co-immunoprecipitation of c-Cbl with Src, Fyn, Lyn, or Syk was not observed in macrophages with or without FcR stimulation (data not shown).


Figure 4: c-Cbl associates with tyrosine kinases in vivo. A, EGFR was immunoprecipitated from HER14 cells untreated or treated with 275 ng/ml EGF for 5 min at 37 °C. B, CSF-1R was immunoprecipitated from P388D1 cells untreated or treated with 25 nM CSF-1 for 30 s. C, v-Src was immunoprecipitated from SRD cells lysed in either TNE buffer (N) or CHAP buffer (C). Immunoprecipitated material was Western blotted with anti-c-Cbl antibody (anti-c-Cbl). Antibody was removed from these blots and reprobed with anti-EGFR antibody (A, anti-EGFR), anti-CSF-1R antibody (B, anti-CSF-1R), and anti-Src antibody (C, anti-Src), respectively. TCL were included from unstimulated cells, cells stimulated with EGF (A) and CSF-1 (B), and SRD cells prepared in TNE lysis buffer (C) and were included in immunoblot analysis with anti-c-Cbl antibody.



Stimulation Induces Translocation of c-Cbl

Finally, we determined whether the subcellular localization of c-Cbl was altered after stimulation of the different receptors described in the above studies. Utilizing immunocytochemical techniques with anti-c-Cbl antibody in conjunction with confocal microscopy, diffuse staining in the cytoplasm was observed in unstimulated macrophages and in HER14 cells (Fig. 5, A and C, respectively). Whether we stimulated macrophages by antibody clustering of the FcR or HER14 cells by EGF treatment, c-Cbl was found to translocate similarly upon stimulation and to concentrate in a more specific region in the Golgi area of the cell (Fig. 5, B and D, arrows). In both P388D1 cells and HER14 cells, the translocation began at 3 min after stimulation (data not shown) and was complete after 30 min (Fig. 5, B and D). These results suggest that tyrosine phosphorylation of c-Cbl resulting from various types of stimulation similarly alters its subcellular localization.


Figure 5: Stimulation induces a change in the subcellular localization of c-Cbl. Cells were plated on coverslips and cultured for overnight at 37 °C. A and B, P388D1 cells. C and D, HER14 cells. P388D1 cells were unstimulated (A) or stimulated by FcR clustering for 30 min at 37 °C (B). HER14 cells were further cultured in DMEM with 0.5% calf serum for 16 h and then unstimulated (C) or stimulated with 275 ng/ml EGF for 30 min (D). Cells were fixed with 3.7% formaldehyde for 10 min at room temperature and stained with anti-c-Cbl antibody. Bars = 5 µm.




DISCUSSION

In this report we identify a protein, c-Cbl, that becomes tyrosine-phosphorylated in response to a variety of tyrosine kinase signaling events, including activation of receptors that possess intrinsic tyrosine kinase activity (EGFR and CSF-1R), activation of receptors that recruit cytoplasmic tyrosine kinases (FcR), and intracellular tyrosine kinases that are constitutively activated (v-Src).

In macrophages, rapid tyrosine phosphorylation of c-Cbl was observed in response to FcR stimulation. Though Fc receptors have no intrinsic tyrosine kinase or tyrosine phosphatase activity, two classes of cytoplasmic tyrosine kinases, the Src family and the Syk/ZAP70 family, have been shown to be implicated in signaling via Fc receptors (for review see Ref. 10). With respect to the Src family, it has been reported that Fc receptor stimulation activates Src family tyrosine kinases in various cell systems(8, 9, 10) . In this study, we report that c-Cbl is able to bind to the SH3 domains of Src, Fyn, and Lyn at least in vitro in both unstimulated and stimulated cells via an association that may be related to the presence of a proline-rich domain in the C terminus of c-Cbl(2) . In spite of the rapid tyrosine phosphorylation of c-Cbl in FcR-stimulated macrophages and the in vitro binding of c-Cbl to the SH3 domains of Src family tyrosine kinases, we failed to demonstrate the physical association of c-Cbl with Src, Fyn, or Lyn in FcR-stimulated macrophages. In addition we could not demonstrate the physical association of c-Cbl with Syk. This may either be due to an unstable or very transient association of the kinases tested with c-Cbl in vivo or to the involvement of other kinases in the tyrosine phosphorylation of c-Cbl.

In contrast, when tyrosine phosphorylation of c-Cbl was induced by EGF stimulation of fibroblasts overexpressing EGFR and by CSF-1 stimulation of macrophages, co-immunoprecipitation experiments revealed that c-Cbl was physically associated with activated EGFR and CSF-1R. Since the EGFR and CSF-1R possess intrinsic tyrosine kinase activity(12) , our results indicate that tyrosine phosphorylation of c-Cbl is induced in the signaling pathways utilized by receptor-type tyrosine kinases as well. Because c-Cbl has no putative binding sites for activated tyrosine kinase receptors such as SH2 domains(2, 13) , association of c-Cbl to the receptors may occur indirectly via adaptor molecules such as Grb2, Nck, and c-Crk, which are known to bind to activated EGFR (14-18), and, for Nck, to also bind to c-Cbl(4) .

Fibroblasts transformed by the v-src oncogene also displayed enhanced tyrosine phosphorylation of c-Cbl, and under certain lysis conditions, the v-Src and c-Cbl proteins associated in vivo. On the other hand, c-Cbl was not markedly tyrosine-phosphorylated in normal lung fibroblasts and in a 3T3 cell line that was engineered to overexpress the c-Src protein. Enhanced tyrosine phosphorylation of c-Cbl may reflect the fact that the Src protein in v-src-transformed fibroblasts has a higher tyrosine kinase activity than the Src protein in c-src-overexpressing fibroblasts(19) . Therefore, tyrosine phosphorylation of c-Cbl may depend on Src kinase activation.

Immunofluorescence analysis has revealed that c-Cbl was localized in the cytoplasm and showed a diffuse and/or reticular pattern in unstimulated macrophages or HER14 cells. FcR stimulation of macrophages and EGF stimulation of HER14 cells both induced the translocation of c-Cbl to a somewhat more restricted area of the cell, which, at least morphologically, is reminiscent of the trans-Golgi region. These results suggest that tyrosine phosphorylation of c-Cbl induced by various types of stimulation is associated with a change in the subcellular localization of c-Cbl. Although c-Cbl has putative motifs that have been identified in transcription factors and v-Cbl has been reported to have nuclear as well as cytoplasmic localization(2, 3) , we have not been able to detect c-Cbl in the nucleus of cells before or after stimulation with different agents. It is interesting to point out that v-Cbl, which localizes in the nucleus, lacks the C-terminal region of c-Cbl, which contains a proline-rich sequence that could be responsible for binding to SH3-containing proteins such as Nck (2, 4) and, as shown here, members of the Src family. It is therefore possible that the proline-rich domain of c-Cbl associates with the SH3-containing molecules such as Src family tyrosine kinases, adaptor molecules, or cytoskeletal proteins such as -spectrin, resulting in the retention of the c-Cbl protein in the cytoplasm. Further studies will be required to clarify the physiological role of the tyrosine phosphorylation of c-Cbl in the subcellular localization of the protein as well as its function.

While this report was being prepared, tyrosine phosphorylation of c-Cbl has also been reported in Jurkat cells activated via the T cell receptor(20) . Similar to what we show here for Src family members, these investigators also showed that c-Cbl associates with the SH3 domains of Grb2, at least in vitro. More recently, it was reported that the expression of v-abl induced the tyrosine phosphorylation of c-Cbl and that Abl and Cbl associated in vivo(21) . Hence, c-Cbl is tyrosine-phosphorylated in response to FcR clustering and CSF-1 binding in macrophages, to EGF binding in HER14 cells, and to v-src and v-abl transformation, as well as in response to T cell receptor activation. For FcR and EGFR stimulation, this is associated with the translocation of c-Cbl to the central region of the cell, in the trans-Golgi area. The fact that c-Cbl is tyrosine-phosphorylated and translocated in response to different agents may indicate that this protein is involved in a general cellular phenomenon, which follows activation of signal transduction pathways by various types of tyrosine kinases. Further study will be necessary to identify the putative biological role of c-Cbl in tyrosine kinase-mediated cell signaling.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AR42927 (to R. B.). 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.

§
To whom correspondence should be addressed: Depts. of Cell Biology and Orthopedics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-4150; Fax: 203-785-2744.

Recipient of National Research Service Award AR08261.

The abbreviations used are: SH3, Src homology 3; FcR, Fc receptor; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EGFR, EGF receptor; CSF-1, colony-stimulating factor-1; CSF-1R, CSF-1 receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; TCL, total cell lysates; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. J. S. Brugge for providing the GST-SH3 fusion proteins and the SRD cell line. We also thank Dr. J. Schlessinger for his generous gift of HER14 cells, Dr. D. Shalloway for the c-src 3T3 cell line, and Dr. W. C. Horne for his careful reading of the manuscript and helpful advice.


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