©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Proto-oncogene Product c-Cbl Becomes Tyrosine Phosphorylated by Stimulation with GM-CSF or Epo and Constitutively Binds to the SH3 Domain of Grb2/Ash in Human Hematopoietic Cells (*)

Hideharu Odai , Ko Sasaki , Akihiro Iwamatsu (1), Yutaka Hanazono , Tomoyuki Tanaka , Kinuko Mitani , Yoshio Yazaki , Hisamaru Hirai (§)

From the (1) Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 and Kirin Brewery Co. Ltd, Central Laboratory for Key Technology, 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 showed that the pp130 was identical to the human c- cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling pathway different from the Ras signaling pathway.


INTRODUCTION

Growth factors elicit and control a cascade of biochemical events including activation of Ras, Raf-1, and mitogen-activated protein kinase in a tyrosine kinase-dependent manner, and the earliest of them is tyrosine phosphorylation of several cellular proteins through the activation of receptor-type and nonreceptor-type tyrosine kinases (1, 2) . Hematopoietic growth factors such as GM-CSF and Epo also induce tyrosine phosphorylation of some cellular proteins including the common chain of GM-CSF/interleukin-3 (IL-3)() /IL-5 receptors, Shc, Vav, Raf, and mitogen-activated protein kinase (3) . Therefore, certain nonreceptor-type tyrosine kinases have to be activated by GM-CSF and Epo because the GM-CSF and Epo receptors has no intrinsic activity of tyrosine kinase.

It is considered that a primary role of such tyrosine kinases is to generate tyrosine-phosphorylated recognition motifs for binding to SH2 domains and to form signal transduction complexes (4, 5) . Several SH2-containing proteins have also catalytic or functional motifs as phospholipase C or GTPase-activating protein for Ras. However, other SH2-containing proteins may serve simply as adapter molecules to form a complex with signaling molecules such as Grb2/Ash, which binds to Sos leading to the Ras activation, and p85, which binds to the catalytic p110 subunit of the phosphatidylinositol 3-kinase (6) .

The Grb2/Ash is a 25-kDa protein composed of only SH2 and SH3 domains in the order of SH3-SH2-SH3 (7, 8) . The SH2 domain of Grb2/Ash binds to tyrosine-phosphorylated proteins such as epidermal growth factor receptor, Shc, IRS-1, and Syp (9, 10, 11, 12, 13, 14, 15) . On the other hand, the SH3 domains of Grb2/Ash bind to the proline-rich domain of Sos (16, 17) , dynamin (18, 19) , and C3G (20) , which regulate Ras or Ras-related proteins. As a result of these associations, the Grb2/Ash seems to be involved in coupling tyrosine kinases to the Ras regulators. Hematopoietic growth factors induce the tyrosine phosphorylation of Shc and form the complex of Shc and Grb2/Ash, and this complex has a key role in Ras activation that is critical for the proliferation (14, 21, 22, 23, 24) . These lines of evidence motivated us to search for signaling molecules that interact with Grb2/Ash and participate in the signal transduction of hematopoietic growth factors.

In this work, we have demonstrated from the in vitro experiments that Grb2/Ash binds to a number of cytosolic proteins that are tyrosine phosphorylated in response to the GM-CSF stimulation in a human leukemia cell line, UT-7. Among these proteins, we have purified pp130 and identified it as the product of the c- cbl proto-oncogene. c-Cbl has been shown to be a signaling molecule that binds to Grb2/Ash adapter protein through the SH3 domain and is tyrosine phosphorylated by GM-CSF or Epo stimulation. Recently, it was reported that c-Cbl was implicated in T cell receptor signaling system (25) , and these findings suggest that c-Cbl is implicated in the signaling pathways triggered by GM-CSF or Epo in hematopoietic cells and by stimulation of T cell receptor in T cells.


MATERIALS AND METHODS

Cell Lines and Growth Factors

UT-7 cells were maintained in RPMI 1640 medium containing 8% bovine serum and 10 ng/ml GM-CSF. Recombinant human GM-CSF was supplied by Kirin Brewery Co. Ltd. (Tokyo), and recombinant human Epo was delivered by Chugai Pharmacy Co. Ltd. (Tokyo).

Antibodies

Polyclonal rabbit anti-c-Cbl antibody (c-15), purchased from Santa Cruz Biotechnology (Santa Cruz, CA), was used for the immunoprecipitation and the immunoblotting of c-Cbl. Polyclonal rabbit antibody to Grb2/Ash (c-23) purchased from Santa Cruz Biotechnology and monoclonal antibody to Grb2/Ash purchased from MBL (Nagoya, Japan) were used for the immunoprecipitation and the immunoblotting of Grb2/Ash, respectively. Protein A-purified polyclonal rabbit anti-Sos antibody was generously provided by Dr. Y. Kaburagi (University of Tokyo) and used for the immunoprecipitation and the immunoblotting of Sos. A mouse monoclonal anti-phosphotyrosine antibody (anti-Ptyr) 4G10 was used for the immunoblotting of phosphotyrosine-containing proteins.

GST Fusion Proteins

The bacterial expression plasmids coding GST fusion proteins containing the full-length and the N-terminal SH3 domain or the SH2 domain of Grb2/Ash (19) were generously provided by Dr. T. Takenawa (University of Tokyo). These plasmids were transformed into XL I-Blue strain of Escherichia coli, and the resulting transformants were induced with isopropyl-1-thio--D-galactopyranoside to produce GST fusion proteins. The bacteria were collected by centrifugation and resuspended in the E. coli lysis buffer containing 40 mM Tris/HCl (pH 7.5), 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. Vigorous sonication was performed before centrifugation at 25,000 g for 20 min. The resulting supernatants were saved as crude extracts containing GST fusion proteins.

Preparation of Cell Lysates

UT-7 cells were incubated in RPMI 1640 medium containing 0.1% bovine serum albumin without serum or growth factors for 8-15 h prior to stimulation with growth factors and then resuspended in RPMI 1640 medium containing 100 µM NaVO. The cells were treated with 10 ng/ml GM-CSF or 20 units/ml Epo for 5 min at 37 °C and then lysed at 4 °C in the lysis buffer containing 20 mM Tris/HCl (pH 8.0), 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 500 units/ml aprotinin, 2 mM EDTA, 50 mM NaF, and 1 mM NaVO. Unsolubilized materials were removed by centrifugation at 15,000 g at 4 °C for 10 min.

Binding of Cellular Proteins to GST Fusion Proteins

Lysates from 1 10 cells were mixed with 40 µg of the fusion protein noncovalently coupled to glutathione-agarose beads (Sigma) for 3 h at 4 °C. Beads were washed with the lysis buffer before resuspension in Laemmli's sample buffer.

Immunoprecipitation

To immunoprecipitate c-Cbl, lysates from 1 10 cells were mixed with polyclonal anti-c-Cbl antibody for 3 h at 4 °C The immunoprecipitates were collected with protein A-Sepharose (Sigma). All the immunoprecipitates were intensively washed with the lysis buffer before resuspension in Laemmli's sample buffer.

Immunoblotting

Samples were subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride filters (Millipore). Filters were blocked with the buffer containing 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 5% bovine serum albumin, and 0.05% Triton X-100. For the immunoblotting of phosphotyrosine-containing proteins or Grb2/Ash, filters were incubated with 4G10 or monoclonal anti-Grb2/Ash antibody, respectively, and then with goat alkaline phosphatase-conjugated anti-mouse IgG (Fc) antibody (Promega). For the immunoblotting of c-Cbl, filters were sequentially incubated with the polyclonal anti-c-Cbl antibody and with goat alkaline phosphatase-conjugated anti-rabbit IgG (Fc) antibody (Promega). After each incubation, filters were washed three times in the buffer containing 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, and 0.05% Triton X-100. Color reaction was performed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Promega).

Purification of pp130

pp130 was purified from UT-7 cell lysates by affinity columns. After pretreatment of lysates from 1 10 cells with GST protein noncovalently coupled to glutathione-agarose beads, lysates were mixed with GST-Grb2/Ash fusion protein noncovalently coupled to glutathione-agarose beads. Beads were washed with the lysis buffer and eluted with 50 mM Tris/HCl (pH 9.6) containing 20 mM reduced glutathione. The fractions containing pp130 were subjected to SDS-PAGE and blotted onto a ProBlott membrane (Applied Biosystems, Foster City, CA). After being visualized by Ponceau S, an isolated 130-kDa band (5 pmol) was cut out and in situ digested with Achromobactor protease I (lysylendopeptidase; Wako, Tokyo). Sequences of three digested fragments were determined by the amino acid sequence analysis. Homology search was carried out using the Entrez program of NCBI, which includes Genbank, EMBL, DDBJ, PIR, and SWISS-PROT data bases.


RESULTS

pp130 Binds to Grb2/Ash and GM-CSF Induces Tyrosine Phosphorylation of pp130

As shown in Fig. 1A, several tyrosine-phosphorylated proteins from UT-7 cell lysates bound to Grb2/Ash in vitro, and this phosphorylation was depending upon GM-CSF stimulation. Using anti-Shc polyclonal antibody, the 52-kDa (pp52) and 66-kDa proteins (pp66) were identified as Shc, which is one of the well known molecules that bind to Grb2/Ash and has an important role in the signal transduction of epidermal growth factor, Epo, and IL-3 (14, 26) (data not shown). However, it is shown that neither pp130 nor pp135 was Sos, dynamin, or C3G, which is reported to bind to Grb2/Ash, from the results of experiments using the specific antibodies to each protein (data not shown). In addition, pp130 and pp135 was not the chain of the GM-CSF receptor or phospholipase C. We then have determined which domain of Grb2/Ash bound to pp130 or pp135 using GST fusion proteins with the SH2 or the N-terminal SH3 of Grb2/Ash. As shown in Fig. 1B, both pp130 and pp135 were associated with the N-terminal SH3 domain but not with the SH2 domain of Grb2/Ash. Conversely, Shc (pp66 and pp52) was shown to bind to the SH2 domain of Grb2/Ash as described before (14, 26) . When pp130 and pp135 bound not only to GST-full-length Grb2/Ash fusion protein but also to the N-terminal SH3 domain of Grb2/Ash, tyrosine phosphorylation of these proteins was dependent on the treatment with GM-CSF. In another human leukemia cell line, F36E, which proliferates in an Epo-dependent manner (27) , Shc, pp130, and pp135 were also associated with the Grb2/Ash and became tyrosine phosphorylated by the treatment with Epo (data not shown).


Figure 1: A, proteins associated with Grb2/Ash in vitro and their tyrosine phosphorylation. The lysates from UT-7 cells unstimulated ( lane2) or stimulated with GM-CSF ( lane1) were mixed with the GST fusion protein including the full-length Grb2/Ash noncovalently coupled to glutathione-agarose beads. Beads were resuspended in Laemmli's sample buffer, subjected to SDS-PAGE, and immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated at the left, are given in kDa. The arrows indicate the positions of pp52, pp66, pp130, and pp135. B, in vitro association of pp130 with the SH3 domain of Grb2/Ash and tyrosine phosphorylation of pp130. The lysates from UT-7 cells unstimulated ( lanes1 and 3) or stimulated with GM-CSF ( lanes2 and 4) were mixed with GST fusion protein including the SH2 domain ( lanes1 and 2) or the N-terminal SH3 domain ( lanes3 and 4) of Grb2/Ash noncovalently coupled to glutathione-agarose beads. The resulting precipitates were resuspended in Laemmli's sample buffer, subjected to SDS-PAGE, and immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated at the left, are given in kDa. The arrows indicate the positions of pp52, pp66, pp130, and pp135.



Identification of pp130 as the c-cbl Proto-oncogene Product

To identify the Grb2/Ash-binding protein, pp130, a large scale affinity purification was carried out using GST-Grb2/Ash fusion protein. After the lysates from 1 10 UT-7 cells were pretreated with GST protein to preclear proteins associated with GST, they were mixed with the GST-Grb2/Ash fusion protein noncovalently coupled to glutathione-agarose beads. Successively, the beads were washed and eluted with the elution buffer containing reduced glutathione. The fractions containing pp130 were subjected to SDS-PAGE and blotted onto the polyvinylidene difluoride membrane. After being visualized by Ponceau S, an isolated 130-kDa band (5 pmol) was cut out and in situ digested with lysylendopeptidase Achromobactor protease I. The sequences of the three obtained fragments determined by amino acid sequence analysis were (K)TIVPWK (P1), (K)ALVIAQNNIEMA (P2), and (K)NILREFVS (P3). These sequences were used to search for homology with other known proteins, and all three peptides were shown to be identical to the equivalent sequence of the c- cbl proto-oncogene product (P1 = aa 197-203, P2 = aa 876-888, and P3 = aa 889-897) (28) . Therefore, we concluded that the Grb2/Ash-binding protein, pp130, is the human c- cbl proto-oncogene product.

Constitutive Binding of the c-cbl Proto-oncogene Product and Grb2/Ash through the SH3 Domain

We then tried to confirm the association between the c- cbl proto-oncogene product and Grb2/Ash. As shown in Fig. 2 A, Western blotting analysis revealed that anti-c-Cbl antibody recognized a 130-kDa molecule both in the UT-7 cell lysates and in immunoprecipitates with anti-c-Cbl antibody. The c-Cbl was detected after incubating the lysates with GST-full-length Grb2/Ash fusion protein but not detected when GST protein was used instead of GST-Grb2/Ash. Moreover, as shown in Fig. 2 B, the GST fusion protein including the N-terminal SH3 domain of Grb2/Ash also bound to c-Cbl, but GST protein and the GST-SH2 domain of Grb2/Ash fusion protein did not. The association of c-Cbl with the full-length or the N-terminal SH3 domain of Grb2/Ash was independent on GM-CSF stimulation. These results show that the association of the c- cbl proto-oncogene product and Grb2/Ash is constitutive through the N-terminal SH3 domain of Grb2/Ash. The c-Cbl has a proline-rich domain (28) , and we could find in this domain two sequences that resemble the proposed consensus sequence for the Abl SH3-binding site (29) and the Grb2/Ash-binding sites on mouse Sos1 (30) (Fig. 2 C). Therefore, it is considered that c-Cbl binds to the SH3 domain of Grb2/Ash through these two proline-rich sequences.


Figure 2: A, in vitro association of c-Cbl and Grb2/Ash. The lysates from UT-7 cells unstimulated ( lanes1-4) or stimulated with GM-CSF ( lane5) were prepared. After the lysates were mixed with anti-c-Cbl ( lane2), GST protein ( lane3), and GST fusion protein including full-length Grb2/Ash ( lanes4 and 5), the resulting precipitates and the total UT-7 cell lysate ( TCL, lane1) were subjected to SDS-PAGE and immunoblotted with anti-c-Cbl. Molecular mass markers, indicated at the left, are given in kDa. The arrow indicates the position of c-Cbl. B, in vitro association of c-Cbl with the SH3 domain of Grb2/Ash. The lysates from UT-7 cells unstimulated ( lanes3, 5, and 6) or stimulated with GM-CSF ( lanes2 and 4) were mixed with GST fusion protein including the N-terminal SH3 domain ( lanes2 and 3), the SH2 domain ( lanes4 and 5) of Grb2/Ash, and GST ( lane6). The resulting precipitates and the total UT-7 cell lysate ( TCL, lane1) were subjected to SDS-PAGE and immunoblotted with anti-c-Cbl. Molecular mass markers, indicated at the left, are given in kDa. The arrow indicates the position of c-Cbl. C, alignment of the proline-rich sequences of c-Cbl with the consensus sequence for Abl SH3-binding region and four proline-rich GRB2/ASH binding sequences of mouse ( m) Sos1. The amino acid sequences shown as Cbl 1 and Cbl 2 are from aa 489-498 and aa 528-537, respectively. and X in the consensus sequence represent hydrophobic and nonconserved amino acid residues, respectively.



In Vivo Association between the c-cbl Proto-oncogene Product and Grb2/Ash

It is important to know whether c-Cbl is associated with Grb2/Ash in vivo. Anti-c-Cbl immunoprecipitates from lysates of the UT-7 cells unstimulated or stimulated with GM-CSF were subjected to SDS-PAGE and immunoblotted with anti-Grb2/Ash antibody (Fig. 3 A). Grb2/Ash was shown to be co-immunoprecipitated with c-Cbl from both lysates of GM-CSF-stimulated or unstimulated cells. Conversely, c-Cbl has not been detected in immunoprecipitates with anti-Grb2/Ash antibody probably because the antibody we used could immunoprecipitate Grb2/Ash and might inhibit the association of Grb2/Ash and c-Cbl. Moreover, the amounts of c-Cbl expression and Grb2/Ash bound to c-Cbl were not affected by GM-CSF stimulation (Fig. 3 A). Sos is a well known protein that binds to the SH3 domain of Grb2/Ash and regulates the activation of Ras (17) . Similarly to c-Cbl, Sos was constitutively associated with Grb2/Ash, and the amounts of Sos expression, Sos bound to Grb2/Ash, and Grb2/Ash bound to Sos were not affected by GM-CSF stimulation (Fig. 3 B). These results suggest that the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation.


Figure 3: A, in vivo association of c-Cbl with Grb2/Ash. The lysates from UT-7 cells stimulated with GM-CSF ( lanes3 and 6) or unstimulated ( lanes4, 5, and 7) were mixed with anti-c-Cbl ( lanes3, 4, 6, and 7) or normal rabbit serum ( NRS, lane5). The resulting precipitates were subjected to SDS-PAGE and immunoblotted with anti-Grb2/Ash ( lanes3-5) or anti-Cbl ( lanes6 and 7). The total UT-7 cell lysate ( TCL, lane1) and the immunoprecipitates with anti-Grb2/Ash ( lane2) from UT-7 cell lysates were also applied for reference. Molecular mass markers, indicated at the left, are given in kDa. The arrow indicates the position of Grb2/Ash or c-Cbl. B, in vivo association of Sos with Grb2/Ash. The lysates from UT-7 cells stimulated with GM-CSF ( lanes1, 3, and 5) or unstimulated ( lanes2, 4, and 6) were mixed with anti-Grb2/Ash ( lanes1 and 2) or with anti-Sos ( lanes3-6). The resulting precipitates were subjected to SDS-PAGE and immunoblotted with anti-Sos ( lanes1-4) or anti-Grb2/Ash ( lanes5 and 6). The arrow indicates the position of Sos or Grb2/Ash.



GM-CSF and Epo Stimulation Induce Tyrosine Phosphorylation of c-Cbl

Next, we examined whether the tyrosine residues of c-Cbl were phosphorylated and whether the phosphorylation was dependent on the factor stimulation. Lysates from UT-7 cells with or without the stimulation with GM-CSF or Epo were immunoprecipitated with anti-c-Cbl antibody, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (4G10). As shown in Fig. 4 A, c-Cbl was tyrosine phosphorylated, and this phosphorylation was induced by the stimulation with GM-CSF or Epo. As shown in Fig. 4 B, c-Cbl was tyrosine phosphorylated within 2 min after GM-CSF stimulation of the cells at 37 °C, attained the maximum level of phosphorylation at 5 min, and was dephosphorylated within 30 min.


Figure 4: A, GM-CSF and Epo induce tyrosine phosphorylation of c-Cbl. The lysates from UT-7 cells stimulated with GM-CSF ( lane3), Epo ( lane2), or unstimulated ( lane1) were mixed with anti-c-Cbl, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated at the left, are given in kDa. The arrow indicates the position of c-Cbl. B, time course of tyrosine phosphorylation of c-Cbl. UT-7 cells were stimulated with GM-CSF for indicated times at 37 °C. The cell lysates were mixed with anti-c-Cbl, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-Ptyr (4G10). Molecular mass markers, indicated at the left, are given in kDa. The arrow indicates the position of c-Cbl.




DISCUSSION

The c- cbl gene was cloned as the cellular homolog of the v- cbl oncogene, which is the transforming component of a murine tumorigenic retrovirus, CAS NS-1 (31) . The v- cbl oncogene is transforming in early B-lineage, and myeloid cells and Gag-v-Cbl transforming protein lacks the C-terminal 62% of c-Cbl containing the proline-rich region and leucine zipper motif but still possesses high proportion of basic amino acids (28) . However, the biological roles of c-Cbl remain to be elucidated. In this work, we have found that pp130 is constitutively associated with Grb2/Ash and becomes tyrosine phosphorylated depending on GM-CSF or Epo stimulation of UT-7 cells. Therefore, we purified the pp130 and identified it as the proto-oncogene product of c- cbl.

We have shown that the c-Cbl (pp130) is constitutively associated with Grb2/Ash through the SH3 domain of Grb2/Ash in vitro and in vivo. This association is supported from the observation that c-Cbl has proline-rich sequences resembling the proposed consensus sequence for the Abl SH3-binding region and the Grb2/Ash binding sites on mouse Sos 1 (Fig. 2 C). Moreover, it has been recently reported that Nck, an adapter protein that has three SH3 domains, binds to the product of proto-oncogene c- cbl in HL-60 promyelocytic leukemia cell (32) and that c-Cbl binds to the GST-Grb2/Ash fusion protein in Jurkat T cell (25) . The SH3 domain of Grb2/Ash binds to the proline-rich domains of dynamin (18, 19) , C3G (20) , and Sos (17) , which regulate the small G-proteins. By forming these complexes, Grb2/Ash plays important roles in Ras activation elicited by growth factors. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating protein in c-Cbl, as described by Blake (28) , unlike C3G and Sos. Moreover, tyrosine phosphorylation of dynamin, C3G, and Sos was not reported, but c-Cbl was tyrosine phosphorylated by GM-CSF or Epo stimulation, and, as shown in Fig. 3, the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Therefore, it is likely that by the association with c-Cbl, Grb2/Ash might also link a different signaling pathway from the pathway regulating G-proteins and that these two different pathways are mutually independent. By the association of Grb2/Ash with Sos, Sos was translocated to the plasma membrane, where Ras was anchored, and worked as a guanine nucleotide exchange factor (33) . It is also possible that the localization of c-Cbl is defined by its binding to Grb2/Ash. The product of gag-v- cbl lacks the proline-rich region of c-Cbl (28) ; therefore, it cannot bind to Grb2/Ash, resulting in the improper localization of the oncogene product. This may contribute to its tumorigenicity.

As shown in Figs. 1 and 4, c-Cbl (pp130) was tyrosine phosphorylated depending on GM-CSF or Epo stimulation. The comparison of the tyrosine-phosphorylated level of c-Cbl in c-Cbl immunoprecipitates in Fig. 4 with that in GST-Grb2/Ash precipitates in Fig. 1 suggests that nearly all of the tyrosine-phosphorylated c-Cbl seems to be complexed with Grb2/Ash. It is interesting what protein-tyrosine kinase is responsible for the phosphorylation of c-Cbl. One candidate is c-Fes because we reported that GM-CSF, IL-3, or Epo activates c-Fes in TF-1 human leukemia cells (34, 35) . Jak2, Lyn, and Tec were also candidates because these tyrosine kinases were tyrosine phosphorylated and activated by hematopoietic growth factors including Epo, GM-CSF, or IL-3 (36, 37, 38, 39) . However, we did not find the potential association of c-Cbl with these kinases. The role of tyrosine phosphorylation of c-Cbl is not clear, but several reports suggest the importance of tyrosine phosphorylation of c-Cbl on its tumorigenesis. In the 70Z/3 pre-B lymphoma, a mutant form of c-Cbl, which deleted 17 amino acids, became tyrosine phosphorylated and activated to have its tumorigenic potential. On the other hand, c-Cbl was tyrosine phosphorylated in tumor cells expressing the product of v- abl or bcr-abl and co-immunoprecipitated with anti-Abl (40) . These observations indicate that tyrosine phosphorylation of c-Cbl could activate the signaling pathway downstream of c-Cbl.

It is unique that c-Cbl is tyrosine phosphorylated by the stimulation with GM-CSF and Epo because other SH3-binding proteins including Sos, C3G, and dynamin are not tyrosine phosphorylated. Therefore, it is possible that c-Cbl binds to SH2-containing proteins by GM-CSF or Epo stimulation. Although we have not detected the association of c-Cbl with the SH2 domains of Grb2/Ash or Shc, c-Cbl has specific peptide sequences resembling the proposed sequences of binding sites for the SH2-containing proteins YEEN (aa 141-144) for the Src family SH2, YDEV (aa 274-277) for the Nck SH2, and NAIY (aa 671-674) for the Shc SH2 (5, 41) . This possibility was also suggested by the report that showed the product of the c- cbl proto-oncogene can bind to the GST-SH2 domain of Fyn, Lck, Blk, GTPase-activating protein, and phospholipase C fusion proteins after tyrosine phosphorylation of c-Cbl in T cell receptor-mediated activation (25) . In the report, it was demonstrated that c-Cbl constitutively binds to Grb2/Ash and becomes tyrosine phosphorylated by the T cell receptor-mediated activation. It was also mentioned that phosphorylation of c-Cbl by the activation might induce interaction with SH2-containing proteins and/or affect associations mediated by SH3 domains as suggested by the decrease in binding to the SH3 domain of Grb2/Ash (25) . In our studies, the stimulation with GM-CSF did not affect the association of c-Cbl and Grb2/Ash in vitro and in vivo, and this may be due to the difference in the associated molecules involved in the signal transduction pathways between the T-cell receptor and the GM-CSF/Epo receptor. These findings demonstrate that c-Cbl contributes to the signal transduction triggered by hematopoietic growth factors such as GM-CSF and Epo as well as T-cell receptor-mediated activation. It is important to search for signaling molecules downstream of c-Cbl and mechanisms of tumorigenesis by abnormalities of c-Cbl.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and from the Ministry of Health and Welfare of Japan. 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. Tel.: 81-3-3815-5411 (ext. 3116); Fax: 81-3-3815-8350.

The abbreviations used are: IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; Epo, erythropoietin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; anti-Ptyr, anti-phosphotyrosine antibody; aa, amino acid.


ACKNOWLEDGEMENTS

UT-7 was a kind gift from Dr. Norio Komatsu (Jichi Medical School, Tochigi, Japan). Polyclonal anti-Grb2/Ash antibody and 4G10 were generously provided by Dr. Tadaomi Takenawa (University of Tokyo) and by Dr. Deborah K Morrison (NCI, National Institutes of Health), respectively.


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